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Heitman, Joseph

Overview:

Signal transduction cascades regulating development and virulence of microorganisms

Our research focuses on how cells sense their environment and communicate with other cells. We employ genetic and biochemical approaches to study two divergent single-celled eukaryotic organisms, the yeast Saccharomyces cerevisiae and the pathogenic fungus Cryptococcus neoformans. These organisms both grow as budding yeasts and appear quite similar, yet they have been diverging over 500 million years of evolution such that one is now a harmless saprophyte and the other a virulent human pathogen that causes life threatening infections of the central nervous system in patients with compromised immunity. We are interested in what their comparison can teach us, both about conserved cellular principles and about the unique adaptations that have occured during the evolution of a human pathogen.

All organisms sense and respond to nutrients. We study how cells sense nutrients during filamentous differentiation in S. cerevisiae and mating and virulence in C. neoformans. In response to limiting nutrients, yeast cells differentiate, undergoing a dimorphic transition called pseudohyphal growth in which the cells elongate and grow in filaments extending away from the colony and into the growth medium to forage for nutrients. We have delineated nutrient sensing signal transduction cascades that regulate filamentous growth. One is regulated by an ammonium permease that we propose functions both to transport and to sense ammonium ions. Intriguingly, this cell surface sensor is structurally, and functionally, related to the Rh blood group antigens in mammals.

The second nutrient sensing pathway is controlled by a novel G protein-coupled receptor, Gpr1, which senses sugars and activates the coupled G alpha protein Gpa2 to stimulate cAMP production by adenylyl cyclase. cAMP then activates cAMP-dependent protein kinase, and the Tpk2 catalytic subunit of this enzyme activates filamentous growth, in part by regulating transcription factors that modulate expression of cell surface proteins involved in adhesion and invasion. The divergent PKA catalytic subunits Tpk1 and Tpk3 play a distinct role and inhibit filamentation.

Importantly, the Gpr1 receptor is the first fungal G-protein coupled receptor whose ligand is not a mating pheromone. Gpr1 homologs have been identified in fission yeast and the human fungal pathogen Candida albicans. Homologs of the coupled G alpha protein are expressed in a dozen different fungi, suggesting the pathway is broadly conserved. Because this family of novel fungal G-protein coupled receptors plays a conserved role in glucose sensing, these receptors could be related to the receptors involved in sweet taste transduction in drosophila and mammals.

Our studies in yeast have served as a model to elucidate signaling cascades that control mating, filamentation, and virulence in the pathogenic fungus C. neoformans. This organism is an ideal model pathogen, and has a defined sexual cycle, exogenous DNA can be introduced by transformation, genes are readily disrupted by homologous recombination, and animal models for studies of infection have been well developed. Genome sequencing projects are in progress for three related by diverged pathogenic forms of C. neoformans. We have capitalized upon these advances to analyze the molecular determinants of virulence. Much of this work is conducted in collaboration with the Duke University Mycology Research Unit, which includes the labs of John Perfect, Gary Cox, Andy Alspaugh, Fred Dietrich, Tom Mitchell, John McCusker, Rytas Vilgalys, and our group.

Our studies have revealed a conserved nutrient sensing pathway conrols virulence of C. neoformans. This pathway is composed of the G alpha protein Gpa1 (the homolog of yeast Gpa2), which signals via adenylyl cyclase, cAMP, and cAMP-dependent protein kinase to induce expression of virulence factors during infection. Mutants lacking the G alpha protein Gpa1, adenylyl cyclase, or the Pka1 catalytic subunit of protein kinase A are unable to produce two specialized virulence factors, the antioxidant pigment melanin and an antiphagocytic polysaccharide capsule, in response to host cues. Importantly, these mutant strains are attenuated in animals. In contrast, mutation of the protein kinase A regulatory subunit Pkr1 enhances capsule production and results in hypervirulence. Further studies aim to identify the receptors, transcription factors and target genes of this pathway.

Our complementary studies reveal a MAP kinase signaling cascade functions in parallel with the G protein-cAMP-PKA signaling pathway and senses pheromones during mating. We have synthesized the lipid modified peptide pheromone that activates this cascade. We developed a novel confrontation assay and find that the pheromones direct signaling between mating partners. In particular, the MFalpha pheromone produced by MAT alpha cells triggers confronting MAT alpha cells to filament and sporulate by a process known as haploid fruiting, which is thought to produce the highly infectious basidiospores that are inhaled into the alveoli of the lung. Our studies further suggest haploid fruiting is linked to the sexual cycle, and functions to enable distant mating partners to locate each other. Recent studies by others reveal mating pheromones also trigger filamentous differentiation of S. cerevisiae haploid cells. Together, these studies illustrate an ancient link between filamentation, the sexual cycle, and sporulation that has been coopted during evolution of a pathogen. Several conserved components of the MAP kinase pathway, including the Gbeta protein Gpb1, homologs of the Ste20 and Ste7 kinases, the MAP kinase homolog Cpk1, and transcription factors that function during mating and differentiation have also been identified.

Interestingly, mating type has been linked to physiology and virulence of C. neoformans. Strains of the MAT alpha mating type are more common in nature, most clinical isolates are MAT alpha, and MAT alpha strains are more virulent than congenic MAT alpha strains. Our studies reveal divergent alleles of the Ste20 kinase are encoded by the MAT alpha and MATa loci. Mutants lacking the Ste20a kinase are mating imparied and attenuated for virulence, providing a molecular link between the MATa locus, the MAP kinase cascade, and virulence. Recently we identified genes encoded by the corresponding MATa mating type locus. Using these mating type specific genes we have: 1) discovered and characterized unusual self-filamentous diploid strains of C. neoformans that arise following genetic crosses, 2) shown that unusual hybrid diploid strains also occur in nature and are infectious, and 3) discovered an unusual MAT alpha isolate of the serotype A lineage that was thought to be extinct. These studies have redefined central features of the life cycle and sexual cycle of this pathogenic fungus. Studies in progress aim to define the structure, evolution, and functions of the mating type loci.

In summary, these complementary studies in yeast and pathogenic fungi reveal two distinct signaling pathways that function coordinately to sense different environmental signals and give rise to appropriate developmental fates.

In parallel, we employ natural toxins as molecular probes to dissect signaling. We focus on the immunosuppressants cyclosporin, FK506, and rapamycin, which suppress the immune system by blocking signaling events required for T-cell activation. These agents are widely used to treat transplant rejection, all three are natural products of soil microorganisms that likely play a role in nature as toxins to inhibit competing microorganisms. Based on this hypothesis, we analyzed the mechanisms of drug action in S. cerevisiae and discovered the signaling cascades targeted by these drugs are highly conserved.

Each of these drugs diffuses into the cell and associates with a binding protein, cyclosporin with cyclophilin and FK506 and rapamycin with FKBP. Both drug binding proteins are enzymes that catalyze a rate limiting step in protein folding following synthesis by the ribosome. The drugs bind to and inhibit the enzyme active sites, but this is not how cell function is disrupted. For example, yeast and fungal cells missing the cyclophilin or FKBP proteins are viable and resistant to the toxic effects of these drugs. Thus, these compounds do not kill the cell by inhibiting the binding proteins, because cells lacking the proteins are still viable. Instead, the protein-drug complexes are the active agents, and these complexes bind to and inhibit signaling molecules. The target of the cyclophilin-cyclosporin and FKBP-FK506 complexes is calcineurin, a conserved calcium sensing protein phosphatase.

Our studies now address the normal cellular functions of these drug targets. We discovered that calcineurin is required for mating, filamentation, and virulence of C. neoformans and are identifying other elements of these pathways. A calcineurin binding protein identified in C. neoformans is conserved in yeast and humans and may represent a calcineurin effector or inhibitor. The human homolog is encoded by the first gene in the Down's syndrome critical region on human chromosome 21. Both calcineurin and the the human DSCR1 protein are highly expressed in the heart and the brain, two tissues prominently effected in Down's Syndrome patients, suggesting overexpression of DCSR1 and perturbations in calcineurin signaling could underlie some of the clinical manifestations of this common syndrome.

We found that the cyclophilin A enzyme has novel nuclear functions, and implicated one target as a histone deacetylase complex. Two related cyclophilin A homologs, Cpa1 and Cpa2, are expressed in C. neoformans and mediate cyclosporin A antifungal action and share a function important for growth. These findings provide genetically tractable model systems to explore the in vivo functions of this conserved but enigmatic family of protein folding enzymes. In summary, our studies began with unusual natural product toxins and have led to the identification of conserved targets whose diverse functions in growth and signaling remain to be elucidated.

Much of experimental biology has been based on the premise that studies of model organisms, including bacteria, yeast, insects, and worms, would reveal conserved principles that govern how all organisms function. Our studies support this view and suggest further studies of model organisms will contribute much to our understanding of the molecular basis of life.

Positions:

Chair, Department of Molecular Genetics and Microbiology

Molecular Genetics and Microbiology
School of Medicine

James B. Duke Professor of Medicine

Molecular Genetics and Microbiology
School of Medicine

Professor of Molecular Genetics and Microbiology

Molecular Genetics and Microbiology
School of Medicine

Member of the Duke Cancer Institute

Duke Cancer Institute
School of Medicine

Professor in Medicine

Medicine, Infectious Diseases
School of Medicine

Professor in Pharmacology & Cancer Biology

Pharmacology & Cancer Biology
School of Medicine

Education:

Ph.D. 1989

Ph.D. — Rockefeller University

M.D. 1992

M.D. — Cornell University

News:

Grants:

Genetics of Cryptococcus sexual reproduction

Administered By
Molecular Genetics and Microbiology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
May 01, 1997
End Date
April 30, 2021

Interdisciplinary Research Training Program in AIDS

Administered By
Medicine, Infectious Diseases
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
September 01, 2010
End Date
August 31, 2020

Genetics Training Grant

Administered By
Basic Science Departments
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
September 01, 1979
End Date
June 30, 2020

Organization and Function of Cellular Structure

Administered By
Basic Science Departments
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
July 01, 1975
End Date
June 30, 2020

Structure, function, and evolution of the Cryptococcus MAT locus

Administered By
Molecular Genetics and Microbiology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
June 15, 2002
End Date
November 30, 2019

Molecular Mycology and Pathogenesis Training Program

Administered By
Molecular Genetics and Microbiology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
August 08, 2014
End Date
July 31, 2019

Transplant Infectious Diseases Interdisciplinary Research Training Grant

Administered By
Medicine, Infectious Diseases
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
September 01, 2013
End Date
August 31, 2018

Structural Biological Development of Fungal-Specific Calcineurin Inhibitors

Administered By
Molecular Genetics and Microbiology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
August 04, 2014
End Date
July 31, 2018

Duke Research Training Program for Pediatricians

Administered By
Pediatrics, Infectious Diseases
AwardedBy
National Institutes of Health
Role
Training Faculty
Start Date
July 01, 2002
End Date
April 30, 2018

The Genetic Architecture of Virulence in Cryptococcus neoformans

Administered By
Biology
AwardedBy
National Institutes of Health
Role
Co-Principal Investigator
Start Date
February 14, 2017
End Date
January 31, 2018

Mining the Coprophilous Mycobiome for new Cryptococcus Antiinfectives and Antifungal Synergists

Administered By
Medicine, Infectious Diseases
AwardedBy
University of Iowa
Role
Collaborator
Start Date
January 01, 2017
End Date
November 30, 2017

Center for Molecular & Cellular Studies of Ped Disease

Administered By
Pediatrics
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
April 11, 2003
End Date
November 30, 2017

Training Program in Developmental and Stem Cell Biology

Administered By
Basic Science Departments
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
May 01, 2001
End Date
October 31, 2017

Evolution of Cryptococcus neoformans Strains from Patients with HIV/AIDS

Administered By
Medicine, Infectious Diseases
AwardedBy
National Institutes of Health
Role
Investigator
Start Date
August 05, 2011
End Date
July 31, 2017

Medical Scientist Training Program

Administered By
School of Medicine
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
July 01, 1997
End Date
June 30, 2017

Institutional Training Grant in Pediatric Infectious Disease

Administered By
Pediatrics, Infectious Diseases
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
May 21, 2011
End Date
December 31, 2016

A Computer Modeling Approach to Create an Antifungal to Improve the Treatment of Cryptococcal Infections

Administered By
Molecular Genetics and Microbiology
AwardedBy
Amplyx Pharmaceuticals, Inc.
Role
Principal Investigator
Start Date
August 01, 2014
End Date
October 31, 2016

Improvement of genetic analysis in the pathogenic zygomycete Mucor circinelloides

Administered By
Molecular Genetics and Microbiology
AwardedBy
National Institutes of Health
Role
Co Investigator
Start Date
February 01, 2016
End Date
August 01, 2016

A Combination Therapy Approach to Treating Drug Resistant Fungal Infections

Administered By
Molecular Genetics and Microbiology
AwardedBy
Amplyx Pharmaceuticals, Inc.
Role
Principal Investigator
Start Date
June 01, 2014
End Date
May 31, 2016

Cancer Biology Training Grant

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Cancer Institute
Role
Mentor
Start Date
July 01, 1993
End Date
March 31, 2016

Clinical Oncology Research Career Development Program

Administered By
Surgery, Surgical Sciences
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
September 29, 2009
End Date
July 31, 2015

Instrumentation for Quantitative Phosphoproteomics and Acetylomics

Administered By
Duke Center for Genomic and Computational Biology
AwardedBy
National Institutes of Health
Role
Major User
Start Date
May 15, 2014
End Date
May 14, 2015

Novel Antifungal Therapeutic Approaches

Administered By
Molecular Genetics and Microbiology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
July 01, 2001
End Date
December 31, 2014

A Computer Modeling Approach to Create an Antifungal to Improve the Treatment of Cryptococcal Infections

Administered By
Molecular Genetics and Microbiology
AwardedBy
Amplyx Pharmaceuticals, Inc.
Role
Principal Investigator
Start Date
August 01, 2012
End Date
July 01, 2014

A Structural Approach for Treating Drug Resistant Fungal Pathogens

Administered By
Molecular Genetics and Microbiology
AwardedBy
Amplyx Pharmaceuticals, Inc.
Role
Principal Investigator
Start Date
July 01, 2012
End Date
June 30, 2014

Pulmonary Surfactant and Lung Homeostasis

Administered By
Medicine, Infectious Diseases
AwardedBy
National Institutes of Health
Role
Collaborator
Start Date
July 01, 1993
End Date
March 31, 2014

Sexual reproduction and virulence of zygomycete fungi

Administered By
Molecular Genetics and Microbiology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
May 01, 2010
End Date
April 30, 2013

Role of Calcineurin in Fungal Virulence

Administered By
Molecular Genetics and Microbiology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
May 15, 2004
End Date
April 30, 2010

Genetic analysis of Cryptococcus neoformans virulence

Administered By
Molecular Genetics and Microbiology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
June 01, 2005
End Date
February 28, 2010

GPCR signaling cascades in Cryptococcus neoformans

Administered By
Molecular Genetics and Microbiology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
August 01, 2007
End Date
July 31, 2009

Genetic molecular biology of virulence in C. neoformans

Administered By
Medicine, Infectious Diseases
AwardedBy
National Institutes of Health
Role
Co Investigator
Start Date
January 01, 1991
End Date
March 31, 2008

Role of Calcineurin in Cryptococcus Neoformans Virulence

Administered By
School of Medicine
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
January 01, 1998
End Date
December 31, 2003

Novel Antifungal Drug Targets in Cryptococcus Neoformans

Administered By
School of Medicine
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
September 01, 1997
End Date
June 30, 2001

Novel Antifungal Drug Targets In Cryptococcus Neoformans

Administered By
School of Medicine
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
September 01, 1997
End Date
August 31, 1999

Immunosuppressant Targets In Cryptococcus Neoformans

Administered By
School of Medicine
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
May 01, 1997
End Date
April 30, 1999
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Awards:

Oswald Avery Award. Infectious Diseases Society of America.

Type
National
Awarded By
Infectious Diseases Society of America
Date
January 01, 2003

Investigator/Alumni Investigator. Howard Hughes Medical Institute.

Type
National
Awarded By
Howard Hughes Medical Institute
Date
January 01, 1992

Publications:

A non-canonical RNA degradation pathway suppresses RNAi-dependent epimutations in the human fungal pathogen Mucor circinelloides.

Mucorales are a group of basal fungi that includes the casual agents of the human emerging disease mucormycosis. Recent studies revealed that these pathogens activate an RNAi-based pathway to rapidly generate drug-resistant epimutant strains when exposed to stressful compounds such as the antifungal drug FK506. To elucidate the molecular mechanism of this epimutation pathway, we performed a genetic analysis in Mucor circinelloides that revealed an inhibitory role for the non-canonical RdRP-dependent Dicer-independent silencing pathway, which is an RNAi-based mechanism involved in mRNA degradation that was recently identified. Thus, mutations that specifically block the mRNA degradation pathway, such as those in the genes r3b2 and rdrp3, enhance the production of drug resistant epimutants, similar to the phenotype previously described for mutation of the gene rdrp1. Our genetic analysis also revealed two new specific components of the epimutation pathway related to the quelling induced protein (qip) and a Sad-3-like helicase (rnhA), as mutations in these genes prevented formation of drug-resistant epimutants. Remarkably, drug-resistant epimutant production was notably increased in M. circinelloides f. circinelloides isolates from humans or other animal hosts. The host-pathogen interaction could be a stressful environment in which the phenotypic plasticity provided by the epimutant pathway might provide an advantage for these strains. These results evoke a model whereby balanced regulation of two different RNAi pathways is determined by the activation of the RNAi-dependent epimutant pathway under stress conditions, or its repression when the regular maintenance of the mRNA degradation pathway operates under non-stress conditions.

Authors
Calo, S; Nicolás, FE; Lee, SC; Vila, A; Cervantes, M; Torres-Martinez, S; Ruiz-Vazquez, RM; Cardenas, ME; Heitman, J
MLA Citation
Calo, S, Nicolás, FE, Lee, SC, Vila, A, Cervantes, M, Torres-Martinez, S, Ruiz-Vazquez, RM, Cardenas, ME, and Heitman, J. "A non-canonical RNA degradation pathway suppresses RNAi-dependent epimutations in the human fungal pathogen Mucor circinelloides." PLoS genetics 13.3 (March 24, 2017): e1006686-.
PMID
28339467
Source
epmc
Published In
PLoS genetics
Volume
13
Issue
3
Publish Date
2017
Start Page
e1006686
DOI
10.1371/journal.pgen.1006686

Calcineurin in fungal virulence and drug resistance: Prospects for harnessing targeted inhibition of calcineurin for an antifungal therapeutic approach.

Increases in the incidence and mortality due to the major invasive fungal infections such as aspergillosis, candidiasis and cryptococcosis caused by the species of Aspergillus, Candida and Cryptococcus, are a growing threat to the immunosuppressed patient population. In addition to the limited armamentarium of the current classes of antifungal agents available (pyrimidine analogs, polyenes, azoles, and echinocandins), their toxicity, efficacy and the emergence of resistance are major bottlenecks limiting successful patient outcomes. Although these drugs target distinct fungal pathways, there is an urgent need to develop new antifungals that are more efficacious, fungal-specific, with reduced or no toxicity and simultaneously do not induce resistance. Here we review several lines of evidence which indicate that the calcineurin signaling pathway, a target of the immunosuppressive drugs FK506 and cyclosporine A, orchestrates growth, virulence and drug resistance in a variety of fungal pathogens and can be exploited for novel antifungal drug development.

Authors
Juvvadi, PR; Lee, SC; Heitman, J; Steinbach, WJ
MLA Citation
Juvvadi, PR, Lee, SC, Heitman, J, and Steinbach, WJ. "Calcineurin in fungal virulence and drug resistance: Prospects for harnessing targeted inhibition of calcineurin for an antifungal therapeutic approach." Virulence 8.2 (February 2017): 186-197.
PMID
27325145
Source
epmc
Published In
Virulence
Volume
8
Issue
2
Publish Date
2017
Start Page
186
End Page
197
DOI
10.1080/21505594.2016.1201250

Sexual Reproduction in Dermatophytes.

Sexual reproduction is a rich source of genetic variation and commonly observed among fungi. Basically two different modes of sexual reproduction are observed in fungi, namely heterothallism where two compatible mating types are required to undergo mating and homothallism in which the organism is self-fertile. The genomic region governing the process of sexual reproduction and sex determination is called the mating type (MAT) locus. In filamentous ascomycetes including dermatophytes, the MAT locus harbors two different transcription factor genes in two different mating types. This review focuses on sexual reproduction and the structure of the MAT locus in dermatophytes. The reproductive modes and the observed mating types are summarized for different phylogenetic clades of dermatophytes. In addition, the question of whether or not unisexual reproduction, an interesting form of homothallism, may be the sexual reproduction mode especially in anthropophilic dermatophytes is raised.

Authors
Metin, B; Heitman, J
MLA Citation
Metin, B, and Heitman, J. "Sexual Reproduction in Dermatophytes." Mycopathologia 182.1-2 (February 2017): 45-55.
PMID
27696123
Source
epmc
Published In
Mycopathologia
Volume
182
Issue
1-2
Publish Date
2017
Start Page
45
End Page
55
DOI
10.1007/s11046-016-0072-x

New facets of antifungal therapy.

Invasive fungal infections remain a major cause of morbidity and mortality in immunocompromised patients, and such infections are a substantial burden to healthcare systems around the world. However, the clinically available armamentarium for invasive fungal diseases is limited to 3 main classes (i.e., polyenes, triazoles, and echinocandins), and each has defined limitations related to spectrum of activity, development of resistance, and toxicity. Further, current antifungal therapies are hampered by limited clinical efficacy, high rates of toxicity, and significant variability in pharmacokinetic properties. New antifungal agents, new formulations, and novel combination regimens may improve the care of patients in the future by providing improved strategies to combat challenges associated with currently available antifungal agents. Likewise, therapeutic drug monitoring may be helpful, but its present use remains controversial due to the lack of available data. This article discusses new facets of antifungal therapy with a focus on new antifungal formulations and the synergistic effects between drugs used in combination therapy.

Authors
Chang, Y-L; Yu, S-J; Heitman, J; Wellington, M; Chen, Y-L
MLA Citation
Chang, Y-L, Yu, S-J, Heitman, J, Wellington, M, and Chen, Y-L. "New facets of antifungal therapy." Virulence 8.2 (February 2017): 222-236.
PMID
27820668
Source
epmc
Published In
Virulence
Volume
8
Issue
2
Publish Date
2017
Start Page
222
End Page
236
DOI
10.1080/21505594.2016.1257457

Plants promote mating and dispersal of the human pathogenic fungus Cryptococcus.

Infections due to Cryptococcus are a leading cause of fungal infections worldwide and are acquired as a result of environmental exposure to desiccated yeast or spores. The ability of Cryptococcus to grow, mate, and produce infectious propagules in association with plants is important for the maintenance of the genetic diversity and virulence factors important for infection of animals and humans. In the Western United States and Canada, Cryptococcus has been associated with conifers and tree species other than Eucalyptus; however, to date Cryptococcus has only been studied on live Arabidopsis thaliana, Eucalyptus sp., and Terminalia catappa (almond) seedlings. Previous research has demonstrated the ability of Cryptococcus to colonize live plants, leaves, and vasculature. We investigated the ability of Cryptococcus to grow on live seedlings of the angiosperms, A. thaliana, Eucalyptus camaldulensis, Colophospermum mopane, and the gymnosperms, Pseudotsuga menziesii (Douglas fir), and Tsuga heterophylla (Western hemlock). We observed a broad-range ability of Cryptococcus to colonize both traditional infection models as well as newly tested conifer species. Furthermore, C. neoformans, C. deneoformans, C. gattii (VGI), C. deuterogattii (VGII) and C. bacillisporus (VGIII) were able to colonize live plant leaves and needles but also undergo filamentation and mating on agar seeded with plant materials or in saprobic association with dead plant materials. The ability of Cryptococcus to grow and undergo filamentation and reproduction in saprobic association with both angiosperms and gymnosperms highlights an important role of plant debris in the sexual cycle and exposure to infectious propagules. This study highlights the broad importance of plants (and plant debris) as the ecological niche and reservoirs of infectious propagules of Cryptococcus in the environment.

Authors
Springer, DJ; Mohan, R; Heitman, J
MLA Citation
Springer, DJ, Mohan, R, and Heitman, J. "Plants promote mating and dispersal of the human pathogenic fungus Cryptococcus." PloS one 12.2 (January 2017): e0171695-.
PMID
28212396
Source
epmc
Published In
PloS one
Volume
12
Issue
2
Publish Date
2017
Start Page
e0171695
DOI
10.1371/journal.pone.0171695

Gene Function Analysis in the Ubiquitous Human Commensal and Pathogen Malassezia Genus.

The genus Malassezia includes 14 species that are found on the skin of humans and animals and are associated with a number of diseases. Recent genome sequencing projects have defined the gene content of all 14 species; however, to date, genetic manipulation has not been possible for any species within this genus. Here, we develop and then optimize molecular tools for the transformation of Malassezia furfur and Malassezia sympodialis using Agrobacterium tumefaciens delivery of transfer DNA (T-DNA) molecules. These T-DNAs can insert randomly into the genome. In the case of M. furfur, targeted gene replacements were also achieved via homologous recombination, enabling deletion of the ADE2 gene for purine biosynthesis and of the LAC2 gene predicted to be involved in melanin biosynthesis. Hence, the introduction of exogenous DNA and direct gene manipulation are feasible in Malassezia species.Species in the genus Malassezia are a defining component of the microbiome of the surface of mammals. They are also associated with a wide range of skin disease symptoms. Many species are difficult to culture in vitro, and although genome sequences are available for the species in this genus, it has not been possible to assess gene function to date. In this study, we pursued a series of possible transformation methods and identified one that allows the introduction of DNA into two species of Malassezia, including the ability to make targeted integrations into the genome such that genes can be deleted. This research opens a new direction in terms of now being able to analyze gene functions in this little understood genus. These tools will contribute to define the mechanisms that lead to the commensalism and pathogenicity in this group of obligate fungi that are predominant on the skin of mammals.

Authors
Ianiri, G; Averette, AF; Kingsbury, JM; Heitman, J; Idnurm, A
MLA Citation
Ianiri, G, Averette, AF, Kingsbury, JM, Heitman, J, and Idnurm, A. "Gene Function Analysis in the Ubiquitous Human Commensal and Pathogen Malassezia Genus." mBio 7.6 (November 29, 2016).
PMID
27899504
Source
epmc
Published In
mBio
Volume
7
Issue
6
Publish Date
2016
DOI
10.1128/mbio.01853-16

Metal Chelation as a Powerful Strategy to Probe Cellular Circuitry Governing Fungal Drug Resistance and Morphogenesis.

Fungal pathogens have evolved diverse strategies to sense host-relevant cues and coordinate cellular responses, which enable virulence and drug resistance. Defining circuitry controlling these traits opens new opportunities for chemical diversity in therapeutics, as the cognate inhibitors are rarely explored by conventional screening approaches. This has great potential to address the pressing need for new therapeutic strategies for invasive fungal infections, which have a staggering impact on human health. To explore this approach, we focused on a leading human fungal pathogen, Candida albicans, and screened 1,280 pharmacologically active compounds to identify those that potentiate the activity of echinocandins, which are front-line therapeutics that target fungal cell wall synthesis. We identified 19 compounds that enhance activity of the echinocandin caspofungin against an echinocandin-resistant clinical isolate, with the broad-spectrum chelator DTPA demonstrating the greatest synergistic activity. We found that DTPA increases susceptibility to echinocandins via chelation of magnesium. Whole genome sequencing of mutants resistant to the combination of DTPA and caspofungin identified mutations in the histidine kinase gene NIK1 that confer resistance to the combination. Functional analyses demonstrated that DTPA activates the mitogen-activated protein kinase Hog1, and that NIK1 mutations block Hog1 activation in response to both caspofungin and DTPA. The combination has therapeutic relevance as DTPA enhanced the efficacy of caspofungin in a mouse model of echinocandin-resistant candidiasis. We found that DTPA not only reduces drug resistance but also modulates morphogenesis, a key virulence trait that is normally regulated by environmental cues. DTPA induced filamentation via depletion of zinc, in a manner that is contingent upon Ras1-PKA signaling, as well as the transcription factors Brg1 and Rob1. Thus, we establish a new mechanism by which metal chelation modulates morphogenetic circuitry and echinocandin resistance, and illuminate a novel facet to metal homeostasis at the host-pathogen interface, with broad therapeutic potential.

Authors
Polvi, EJ; Averette, AF; Lee, SC; Kim, T; Bahn, Y-S; Veri, AO; Robbins, N; Heitman, J; Cowen, LE
MLA Citation
Polvi, EJ, Averette, AF, Lee, SC, Kim, T, Bahn, Y-S, Veri, AO, Robbins, N, Heitman, J, and Cowen, LE. "Metal Chelation as a Powerful Strategy to Probe Cellular Circuitry Governing Fungal Drug Resistance and Morphogenesis." PLoS genetics 12.10 (October 3, 2016): e1006350-.
Website
http://hdl.handle.net/10161/13050
PMID
27695031
Source
epmc
Published In
PLoS genetics
Volume
12
Issue
10
Publish Date
2016
Start Page
e1006350
DOI
10.1371/journal.pgen.1006350

Dual action antifungal small molecule modulates multidrug efflux and TOR signaling.

There is an urgent need for new strategies to treat invasive fungal infections, which are a leading cause of human mortality. Here, we establish two activities of the natural product beauvericin, which potentiates the activity of the most widely deployed class of antifungal against the leading human fungal pathogens, blocks the emergence of drug resistance, and renders antifungal-resistant pathogens responsive to treatment in mammalian infection models. Harnessing genome sequencing of beauvericin-resistant mutants, affinity purification of a biotinylated beauvericin analog, and biochemical and genetic assays reveals that beauvericin blocks multidrug efflux and inhibits the global regulator TORC1 kinase, thereby activating the protein kinase CK2 and inhibiting the molecular chaperone Hsp90. Substitutions in the multidrug transporter Pdr5 that enable beauvericin efflux impair antifungal efflux, thereby impeding resistance to the drug combination. Thus, dual targeting of multidrug efflux and TOR signaling provides a powerful, broadly effective therapeutic strategy for treating fungal infectious disease that evades resistance.

Authors
Shekhar-Guturja, T; Gunaherath, GMKB; Wijeratne, EMK; Lambert, J-P; Averette, AF; Lee, SC; Kim, T; Bahn, Y-S; Tripodi, F; Ammar, R; Döhl, K; Niewola-Staszkowska, K; Schmitt, L; Loewith, RJ; Roth, FP; Sanglard, D; Andes, D; Nislow, C; Coccetti, P; Gingras, A-C; Heitman, J; Gunatilaka, AAL; Cowen, LE
MLA Citation
Shekhar-Guturja, T, Gunaherath, GMKB, Wijeratne, EMK, Lambert, J-P, Averette, AF, Lee, SC, Kim, T, Bahn, Y-S, Tripodi, F, Ammar, R, Döhl, K, Niewola-Staszkowska, K, Schmitt, L, Loewith, RJ, Roth, FP, Sanglard, D, Andes, D, Nislow, C, Coccetti, P, Gingras, A-C, Heitman, J, Gunatilaka, AAL, and Cowen, LE. "Dual action antifungal small molecule modulates multidrug efflux and TOR signaling." Nature chemical biology 12.10 (October 2016): 867-875.
PMID
27571477
Source
epmc
Published In
Nature Chemical Biology
Volume
12
Issue
10
Publish Date
2016
Start Page
867
End Page
875
DOI
10.1038/nchembio.2165

Systematic functional analysis of kinases in the fungal pathogen Cryptococcus neoformans.

Cryptococcus neoformans is the leading cause of death by fungal meningoencephalitis; however, treatment options remain limited. Here we report the construction of 264 signature-tagged gene-deletion strains for 129 putative kinases, and examine their phenotypic traits under 30 distinct in vitro growth conditions and in two different hosts (insect larvae and mice). Clustering analysis of in vitro phenotypic traits indicates that several of these kinases have roles in known signalling pathways, and identifies hitherto uncharacterized signalling cascades. Virulence assays in the insect and mouse models provide evidence of pathogenicity-related roles for 63 kinases involved in the following biological categories: growth and cell cycle, nutrient metabolism, stress response and adaptation, cell signalling, cell polarity and morphology, vacuole trafficking, transfer RNA (tRNA) modification and other functions. Our study provides insights into the pathobiological signalling circuitry of C. neoformans and identifies potential anticryptococcal or antifungal drug targets.

Authors
Lee, K-T; So, Y-S; Yang, D-H; Jung, K-W; Choi, J; Lee, D-G; Kwon, H; Jang, J; Wang, LL; Cha, S; Meyers, GL; Jeong, E; Jin, J-H; Lee, Y; Hong, J; Bang, S; Ji, J-H; Park, G; Byun, H-J; Park, SW; Park, Y-M; Adedoyin, G; Kim, T; Averette, AF; Choi, J-S; Heitman, J; Cheong, E; Lee, Y-H; Bahn, Y-S
MLA Citation
Lee, K-T, So, Y-S, Yang, D-H, Jung, K-W, Choi, J, Lee, D-G, Kwon, H, Jang, J, Wang, LL, Cha, S, Meyers, GL, Jeong, E, Jin, J-H, Lee, Y, Hong, J, Bang, S, Ji, J-H, Park, G, Byun, H-J, Park, SW, Park, Y-M, Adedoyin, G, Kim, T, Averette, AF, Choi, J-S, Heitman, J, Cheong, E, Lee, Y-H, and Bahn, Y-S. "Systematic functional analysis of kinases in the fungal pathogen Cryptococcus neoformans." Nature communications 7 (September 28, 2016): 12766-.
Website
http://hdl.handle.net/10161/13057
PMID
27677328
Source
epmc
Published In
Nature Communications
Volume
7
Publish Date
2016
Start Page
12766
DOI
10.1038/ncomms12766

Calcineurin Targets Involved in Stress Survival and Fungal Virulence.

Calcineurin governs stress survival, sexual differentiation, and virulence of the human fungal pathogen Cryptococcus neoformans. Calcineurin is activated by increased Ca2+ levels caused by stress, and transduces signals by dephosphorylating protein substrates. Herein, we identified and characterized calcineurin substrates in C. neoformans by employing phosphoproteomic TiO2 enrichment and quantitative mass spectrometry. The identified targets include the transactivator Crz1 as well as novel substrates whose functions are linked to P-bodies/stress granules (PBs/SGs) and mRNA translation and decay, such as Pbp1 and Puf4. We show that Crz1 is a bona fide calcineurin substrate, and Crz1 localization and transcriptional activity are controlled by calcineurin. We previously demonstrated that thermal and other stresses trigger calcineurin localization to PBs/SGs. Several calcineurin targets localized to PBs/SGs, including Puf4 and Pbp1, contribute to stress resistance and virulence individually or in conjunction with Crz1. Moreover, Pbp1 is also required for sexual development. Genetic epistasis analysis revealed that Crz1 and the novel targets Lhp1, Puf4, and Pbp1 function in a branched calcineurin pathway that orchestrates stress survival and virulence. These findings support a model whereby calcineurin controls stress and virulence, at the transcriptional level via Crz1, and post-transcriptionally by localizing to PBs/SGs and acting on targets involved in mRNA metabolism. The calcineurin targets identified in this study share little overlap with known calcineurin substrates, with the exception of Crz1. In particular, the mRNA binding proteins and PBs/SGs residents comprise a cohort of novel calcineurin targets that have not been previously linked to calcineurin in mammals or in Saccharomyces cerevisiae. This study suggests either extensive evolutionary rewiring of the calcineurin pathway, or alternatively that these novel calcineurin targets have yet to be characterized as calcineurin targets in other organisms. These findings further highlight C. neoformans as an outstanding model to define calcineurin-responsive virulence networks as targets for antifungal therapy.

Authors
Park, H-S; Chow, EWL; Fu, C; Soderblom, EJ; Moseley, MA; Heitman, J; Cardenas, ME
MLA Citation
Park, H-S, Chow, EWL, Fu, C, Soderblom, EJ, Moseley, MA, Heitman, J, and Cardenas, ME. "Calcineurin Targets Involved in Stress Survival and Fungal Virulence." PLoS pathogens 12.9 (September 9, 2016): e1005873-.
Website
http://hdl.handle.net/10161/13053
PMID
27611567
Source
epmc
Published In
PLoS pathogens
Volume
12
Issue
9
Publish Date
2016
Start Page
e1005873
DOI
10.1371/journal.ppat.1005873

Cancer-associated isocitrate dehydrogenase mutations induce mitochondrial DNA instability.

A major advance in understanding the progression and prognostic outcome of certain cancers, such as low-grade gliomas, acute myeloid leukaemia, and chondrosarcomas, has been the identification of early-occurring mutations in the NADP+-dependent isocitrate dehydrogenase genes IDH1 and IDH2 These mutations result in the production of the onco-metabolite D-2-hydroxyglutarate (2HG), thought to contribute to disease progression. To better understand the mechanisms of 2HG pathophysiology, we introduced the analogous glioma-associated mutations into the NADP+ isocitrate dehydrogenase genes (IDP1, IDP2, IDP3) in Saccharomyces cerevisiae Intriguingly, expression of the mitochondrial IDP1R148H mutant allele results in high levels of 2HG production as well as extensive mtDNA loss and respiration defects. We find no evidence for a reactive oxygen-mediated mechanism mediating this mtDNA loss. Instead, we show that 2HG production perturbs the iron sensing mechanisms as indicated by upregulation of the Aft1-controlled iron regulon and a concomitant increase in iron levels. Accordingly, iron chelation, or overexpression of a truncated AFT1 allele that dampens transcription of the iron regulon, suppresses the loss of respirative capacity. Additional suppressing factors include overexpression of the mitochondrial aldehyde dehydrogenase gene ALD5 or disruption of the retrograde response transcription factor RTG1 Furthermore, elevated α-ketoglutarate levels also suppress 2HG-mediated respiration loss; consistent with a mechanism by which 2HG contributes to mtDNA loss by acting as a toxic α-ketoglutarate analog. Our findings provide insight into the mechanisms that may contribute to 2HG oncogenicity in glioma and acute myeloid leukaemia progression, with the promise for innovative diagnostic and prognostic strategies and novel therapeutic modalities.

Authors
Kingsbury, JM; Shamaprasad, N; Billmyre, RB; Heitman, J; Cardenas, ME
MLA Citation
Kingsbury, JM, Shamaprasad, N, Billmyre, RB, Heitman, J, and Cardenas, ME. "Cancer-associated isocitrate dehydrogenase mutations induce mitochondrial DNA instability." Human molecular genetics 25.16 (August 2016): 3524-3538.
PMID
27427385
Source
epmc
Published In
Human Molecular Genetics
Volume
25
Issue
16
Publish Date
2016
Start Page
3524
End Page
3538
DOI
10.1093/hmg/ddw195

Expansion of Signal Transduction Pathways in Fungi by Extensive Genome Duplication.

Plants and fungi use light and other signals to regulate development, growth, and metabolism. The fruiting bodies of the fungus Phycomyces blakesleeanus are single cells that react to environmental cues, including light, but the mechanisms are largely unknown [1]. The related fungus Mucor circinelloides is an opportunistic human pathogen that changes its mode of growth upon receipt of signals from the environment to facilitate pathogenesis [2]. Understanding how these organisms respond to environmental cues should provide insights into the mechanisms of sensory perception and signal transduction by a single eukaryotic cell, and their role in pathogenesis. We sequenced the genomes of P. blakesleeanus and M. circinelloides and show that they have been shaped by an extensive genome duplication or, most likely, a whole-genome duplication (WGD), which is rarely observed in fungi [3-6]. We show that the genome duplication has expanded gene families, including those involved in signal transduction, and that duplicated genes have specialized, as evidenced by differences in their regulation by light. The transcriptional response to light varies with the developmental stage and is still observed in a photoreceptor mutant of P. blakesleeanus. A phototropic mutant of P. blakesleeanus with a heterozygous mutation in the photoreceptor gene madA demonstrates that photosensor dosage is important for the magnitude of signal transduction. We conclude that the genome duplication provided the means to improve signal transduction for enhanced perception of environmental signals. Our results will help to understand the role of genome dynamics in the evolution of sensory perception in eukaryotes.

Authors
Corrochano, LM; Kuo, A; Marcet-Houben, M; Polaino, S; Salamov, A; Villalobos-Escobedo, JM; Grimwood, J; Álvarez, MI; Avalos, J; Bauer, D; Benito, EP; Benoit, I; Burger, G; Camino, LP; Cánovas, D; Cerdá-Olmedo, E; Cheng, J-F; Domínguez, A; Eliáš, M; Eslava, AP; Glaser, F; Gutiérrez, G; Heitman, J; Henrissat, B; Iturriaga, EA; Lang, BF; Lavín, JL; Lee, SC; Li, W; Lindquist, E; López-García, S; Luque, EM; Marcos, AT; Martin, J; McCluskey, K; Medina, HR; Miralles-Durán, A; Miyazaki, A et al.
MLA Citation
Corrochano, LM, Kuo, A, Marcet-Houben, M, Polaino, S, Salamov, A, Villalobos-Escobedo, JM, Grimwood, J, Álvarez, MI, Avalos, J, Bauer, D, Benito, EP, Benoit, I, Burger, G, Camino, LP, Cánovas, D, Cerdá-Olmedo, E, Cheng, J-F, Domínguez, A, Eliáš, M, Eslava, AP, Glaser, F, Gutiérrez, G, Heitman, J, Henrissat, B, Iturriaga, EA, Lang, BF, Lavín, JL, Lee, SC, Li, W, Lindquist, E, López-García, S, Luque, EM, Marcos, AT, Martin, J, McCluskey, K, Medina, HR, Miralles-Durán, A, and Miyazaki, A et al. "Expansion of Signal Transduction Pathways in Fungi by Extensive Genome Duplication." Current biology : CB 26.12 (June 2016): 1577-1584.
PMID
27238284
Source
epmc
Published In
Current Biology
Volume
26
Issue
12
Publish Date
2016
Start Page
1577
End Page
1584
DOI
10.1016/j.cub.2016.04.038

Structures of Pathogenic Fungal FKBP12s Reveal Possible Self-Catalysis Function.

Invasive fungal infections remain difficult to treat and require novel targeting strategies. The 12-kDa FK506-binding protein (FKBP12) is a ubiquitously expressed peptidyl-prolyl isomerase with considerable homology between fungal pathogens and is thus a prime candidate for future targeting efforts to generate a panfungal strategy. Despite decades of research on FKBPs, their substrates and mechanisms of action remain unclear. Here we describe structural, biochemical, and in vivo analyses of FKBP12s from the pathogenic fungi Candida albicans, Candida glabrata, and Aspergillus fumigatus Strikingly, multiple apo A. fumigatus and C. albicans FKBP12 crystal structures revealed a symmetric, intermolecular interaction involving the deep insertion of an active-site loop proline into the active-site pocket of an adjacent subunit. Such interactions have not been observed in previous FKBP structures. This finding indicates the possibility that this is a self-substrate interaction unique to the A. fumigatus and C. albicans fungal proteins that contain this central proline. Structures obtained with the proline in the cis and trans states provide more data in support of self-catalysis. Moreover, cysteine cross-linking experiments captured the interacting dimer, supporting the idea that it forms in solution. Finally, genetic studies exploring the impact of mutations altering the central proline and an adjacent residue provide evidence that any dimeric state formed in vivo, where FKBP12 concentrations are low, is transient. Taken together, these findings suggest a unique mechanism of self-substrate regulation by fungal FKBP12s, lending further novel understanding of this protein for future drug-targeting efforts.FKBP12 is a cis-trans peptidyl-prolyl isomerase that plays key roles in cellular protein homeostasis. FKBP12s also bind the immunosuppressive drug FK506 to inhibit the phosphatase calcineurin (CaN). CaN is required for virulence of A. fumigatus, C. albicans, C. glabrata, and other deadly fungal pathogens, marking FKBP12 and CaN as potential broad-spectrum drug targets. Here we describe structures of fungal FKBP12s. Multiple apo A. fumigatus and C. albicans FKBP12 structures reveal the insertion of a proline, conspicuously conserved in these proteins, into the active sites of adjacent molecules. This suggests that these proteins might serve as their own substrates. Cysteine disulfide trapping experiments provide support for this self-interaction and hence possible intermolecular catalysis by these enzymes.

Authors
Tonthat, NK; Juvvadi, PR; Zhang, H; Lee, SC; Venters, R; Spicer, L; Steinbach, WJ; Heitman, J; Schumacher, MA
MLA Citation
Tonthat, NK, Juvvadi, PR, Zhang, H, Lee, SC, Venters, R, Spicer, L, Steinbach, WJ, Heitman, J, and Schumacher, MA. "Structures of Pathogenic Fungal FKBP12s Reveal Possible Self-Catalysis Function." mBio 7.2 (April 26, 2016): e00492-e00416.
PMID
27118592
Source
epmc
Published In
mBio
Volume
7
Issue
2
Publish Date
2016
Start Page
e00492
End Page
e00416
DOI
10.1128/mbio.00492-16

Gene Network Polymorphism Illuminates Loss and Retention of Novel RNAi Silencing Components in the Cryptococcus Pathogenic Species Complex.

RNAi is a ubiquitous pathway that serves central functions throughout eukaryotes, including maintenance of genome stability and repression of transposon expression and movement. However, a number of organisms have lost their RNAi pathways, including the model yeast Saccharomyces cerevisiae, the maize pathogen Ustilago maydis, the human pathogen Cryptococcus deuterogattii, and some human parasite pathogens, suggesting there may be adaptive benefits associated with both retention and loss of RNAi. By comparing the RNAi-deficient genome of the Pacific Northwest Outbreak C. deuterogattii strain R265 with the RNAi-proficient genomes of the Cryptococcus pathogenic species complex, we identified a set of conserved genes that were lost in R265 and all other C. deuterogattii isolates examined. Genetic and molecular analyses reveal several of these lost genes play roles in RNAi pathways. Four novel components were examined further. Znf3 (a zinc finger protein) and Qip1 (a homolog of N. crassa Qip) were found to be essential for RNAi, while Cpr2 (a constitutive pheromone receptor) and Fzc28 (a transcription factor) are involved in sex-induced but not mitosis-induced silencing. Our results demonstrate that the mitotic and sex-induced RNAi pathways rely on the same core components, but sex-induced silencing may be a more specific, highly induced variant that involves additional specialized or regulatory components. Our studies further illustrate how gene network polymorphisms involving known components of key cellular pathways can inform identification of novel elements and suggest that RNAi loss may have been a core event in the speciation of C. deuterogattii and possibly contributed to its pathogenic trajectory.

Authors
Feretzaki, M; Billmyre, RB; Clancey, SA; Wang, X; Heitman, J
MLA Citation
Feretzaki, M, Billmyre, RB, Clancey, SA, Wang, X, and Heitman, J. "Gene Network Polymorphism Illuminates Loss and Retention of Novel RNAi Silencing Components in the Cryptococcus Pathogenic Species Complex." PLoS genetics 12.3 (March 4, 2016): e1005868-.
Website
http://hdl.handle.net/10161/11693
PMID
26943821
Source
epmc
Published In
PLoS genetics
Volume
12
Issue
3
Publish Date
2016
Start Page
e1005868
DOI
10.1371/journal.pgen.1005868

Sex in the Mucoralean Fungi

© 2014 Blackwell Verlag GmbH.Sexual development is extant in virtually all eukaryotic species, including throughout the kingdom Fungi. Positioned within the opisthokonts along with metazoans, fungi serve as model systems to elucidate the genetics and impact of sexual development. Basal fungal lineages such as the Mucoralean fungi provide a unique basis to study sexual reproduction, in which common ancestral traits found in both animal and fungal lineages may be conserved. This review discusses the sexual development, sex loci, and evolution of the sex locus in the Mucoralean fungi, which sheds light on our understanding of the evolution and functions of sex.

Authors
Lee, SC; Heitman, J
MLA Citation
Lee, SC, and Heitman, J. "Sex in the Mucoralean Fungi." Mycoses 57.s3 (January 1, 2016): 18-24.
Source
scopus
Published In
Mycoses
Volume
57
Issue
s3
Publish Date
2016
Start Page
18
End Page
24
DOI
10.1111/myc.12244

Chromosomal Translocations via Intercentromeric Recombination Underlie Mating Transitions during Fungal Evolution

Authors
Sun, S; Yadav, V; Billmyre, B; Sanyal, K; Heitman, J
MLA Citation
Sun, S, Yadav, V, Billmyre, B, Sanyal, K, and Heitman, J. "Chromosomal Translocations via Intercentromeric Recombination Underlie Mating Transitions during Fungal Evolution." 2016.
Source
wos-lite
Published In
Cytogenetic and genome research
Volume
148
Issue
2-3
Publish Date
2016
Start Page
86
End Page
87

From Two to One: Unipolar Sexual Reproduction.

While sexual reproduction is universal in eukaryotes, and shares conserved core features, the specific aspects of sexual reproduction can differ dramatically from species to species. This is also true in Fungi. Among fungal species, mating determination can vary from tetrapolar with more than a thousand different mating types, to bipolar with only two opposite mating types, and finally to unipolar without the need of a compatible mating partner for sexual reproduction. Cryptococcus neoformans is a human pathogenic fungus that belongs to the Basidiomycota. While C. neoformans has a well-defined bipolar mating system with two opposite mating types, MATa and MATα, it can also undergo homothallic unisexual reproduction from one single cell or between two cells of the same mating type. Recently, it was shown that, as in a-α bisexual reproduction, meiosis is also involved in α-α unisexual reproduction in C. neoformans. Briefly, recombination frequencies, the number of crossovers along chromosomes, as well as frequencies at which aneuploid and diploid progeny are produced, are all comparable between a-α bisexual and α-α unisexual reproduction. The plasticity observed in C. neoformans sexual reproduction highlights the extensive diversity in mating type determination, mating recognition, as well as modes of sexual reproduction across fungal species.

Authors
Sun, S; Heitman, J
MLA Citation
Sun, S, and Heitman, J. "From Two to One: Unipolar Sexual Reproduction." Fungal biology reviews 29.3-4 (December 2015): 118-125.
PMID
26744600
Source
epmc
Published In
Fungal Biology Reviews
Volume
29
Issue
3-4
Publish Date
2015
Start Page
118
End Page
125
DOI
10.1016/j.fbr.2015.06.002

Evolution of sexual reproduction: a view from the Fungal Kingdom supports an evolutionary epoch with sex before sexes.

Sexual reproduction is conserved throughout each supergroup within the eukaryotic tree of life, and therefore thought to have evolved once and to have been present in the last eukaryotic common ancestor (LECA). Given the antiquity of sex, there are features of sexual reproduction that are ancient and ancestral, and thus shared in diverse extant organisms. On the other hand, the vast evolutionary distance that separates any given extant species from the LECA necessarily implies that other features of sex will be derived. While most types of sex we are familiar with involve two opposite sexes or mating types, recent studies in the fungal kingdom have revealed novel and unusual patterns of sexual reproduction, including unisexual reproduction. In this mode of reproduction a single mating type can on its own undergo self-fertile/homothallic reproduction, either with itself or with other members of the population of the same mating type. Unisexual reproduction has arisen independently as a derived feature in several different lineages. That a myriad of different types of sex determination and sex determinants abound in animals, plants, protists, and fungi suggests that sex specification itself may not be ancestral and instead may be a derived trait. If so, then the original form of sexual reproduction may have been unisexual, onto which sexes were superimposed as a later feature. In this model, unisexual reproduction is both an ancestral and a derived trait. In this review, we consider what is new and what is old about sexual reproduction from the unique vantage point of the fungal kingdom.

Authors
Heitman, J
MLA Citation
Heitman, J. "Evolution of sexual reproduction: a view from the Fungal Kingdom supports an evolutionary epoch with sex before sexes." Fungal biology reviews 29.3-4 (December 2015): 108-117.
PMID
26834823
Source
epmc
Published In
Fungal Biology Reviews
Volume
29
Issue
3-4
Publish Date
2015
Start Page
108
End Page
117

Genus-Wide Comparative Genomics of Malassezia Delineates Its Phylogeny, Physiology, and Niche Adaptation on Human Skin.

Malassezia is a unique lipophilic genus in class Malasseziomycetes in Ustilaginomycotina, (Basidiomycota, fungi) that otherwise consists almost exclusively of plant pathogens. Malassezia are typically isolated from warm-blooded animals, are dominant members of the human skin mycobiome and are associated with common skin disorders. To characterize the genetic basis of the unique phenotypes of Malassezia spp., we sequenced the genomes of all 14 accepted species and used comparative genomics against a broad panel of fungal genomes to comprehensively identify distinct features that define the Malassezia gene repertoire: gene gain and loss; selection signatures; and lineage-specific gene family expansions. Our analysis revealed key gene gain events (64) with a single gene conserved across all Malassezia but absent in all other sequenced Basidiomycota. These likely horizontally transferred genes provide intriguing gain-of-function events and prime candidates to explain the emergence of Malassezia. A larger set of genes (741) were lost, with enrichment for glycosyl hydrolases and carbohydrate metabolism, concordant with adaptation to skin's carbohydrate-deficient environment. Gene family analysis revealed extensive turnover and underlined the importance of secretory lipases, phospholipases, aspartyl proteases, and other peptidases. Combining genomic analysis with a re-evaluation of culture characteristics, we establish the likely lipid-dependence of all Malassezia. Our phylogenetic analysis sheds new light on the relationship between Malassezia and other members of Ustilaginomycotina, as well as phylogenetic lineages within the genus. Overall, our study provides a unique genomic resource for understanding Malassezia niche-specificity and potential virulence, as well as their abundance and distribution in the environment and on human skin.

Authors
Wu, G; Zhao, H; Li, C; Rajapakse, MP; Wong, WC; Xu, J; Saunders, CW; Reeder, NL; Reilman, RA; Scheynius, A; Sun, S; Billmyre, BR; Li, W; Averette, AF; Mieczkowski, P; Heitman, J; Theelen, B; Schröder, MS; De Sessions, PF; Butler, G; Maurer-Stroh, S; Boekhout, T; Nagarajan, N; Dawson, TL
MLA Citation
Wu, G, Zhao, H, Li, C, Rajapakse, MP, Wong, WC, Xu, J, Saunders, CW, Reeder, NL, Reilman, RA, Scheynius, A, Sun, S, Billmyre, BR, Li, W, Averette, AF, Mieczkowski, P, Heitman, J, Theelen, B, Schröder, MS, De Sessions, PF, Butler, G, Maurer-Stroh, S, Boekhout, T, Nagarajan, N, and Dawson, TL. "Genus-Wide Comparative Genomics of Malassezia Delineates Its Phylogeny, Physiology, and Niche Adaptation on Human Skin." PLoS genetics 11.11 (November 5, 2015): e1005614-.
PMID
26539826
Source
epmc
Published In
PLoS genetics
Volume
11
Issue
11
Publish Date
2015
Start Page
e1005614
DOI
10.1371/journal.pgen.1005614

An Antifungal Combination Matrix Identifies a Rich Pool of Adjuvant Molecules that Enhance Drug Activity against Diverse Fungal Pathogens.

There is an urgent need to identify new treatments for fungal infections. By combining sub-lethal concentrations of the known antifungals fluconazole, caspofungin, amphotericin B, terbinafine, benomyl, and cyprodinil with ∼3,600 compounds in diverse fungal species, we generated a deep reservoir of chemical-chemical interactions termed the Antifungal Combinations Matrix (ACM). Follow-up susceptibility testing against a fluconazole-resistant isolate of C. albicans unveiled ACM combinations capable of potentiating fluconazole in this clinical strain. We used chemical genetics to elucidate the mode of action of the antimycobacterial drug clofazimine, a compound with unreported antifungal activity that synergized with several antifungals. Clofazimine induces a cell membrane stress for which the Pkc1 signaling pathway is required for tolerance. Additional tests against additional fungal pathogens, including Aspergillus fumigatus, highlighted that clofazimine exhibits efficacy as a combination agent against multiple fungi. Thus, the ACM is a rich reservoir of chemical combinations with therapeutic potential against diverse fungal pathogens.

Authors
Robbins, N; Spitzer, M; Yu, T; Cerone, RP; Averette, AK; Bahn, Y-S; Heitman, J; Sheppard, DC; Tyers, M; Wright, GD
MLA Citation
Robbins, N, Spitzer, M, Yu, T, Cerone, RP, Averette, AK, Bahn, Y-S, Heitman, J, Sheppard, DC, Tyers, M, and Wright, GD. "An Antifungal Combination Matrix Identifies a Rich Pool of Adjuvant Molecules that Enhance Drug Activity against Diverse Fungal Pathogens." Cell reports 13.7 (November 4, 2015): 1481-1492.
PMID
26549450
Source
epmc
Published In
Cell Reports
Volume
13
Issue
7
Publish Date
2015
Start Page
1481
End Page
1492
DOI
10.1016/j.celrep.2015.10.018

Development of Fungal-Specific Calcineurin Inhibitors Based on Molecular Structure and Dynamics

Authors
Venters, R; Spicer, L; Heitman, J; Steinbach, W; Juvvadi, P; Schumacher, M
MLA Citation
Venters, R, Spicer, L, Heitman, J, Steinbach, W, Juvvadi, P, and Schumacher, M. "Development of Fungal-Specific Calcineurin Inhibitors Based on Molecular Structure and Dynamics." October 2015.
Source
wos-lite
Published In
Protein Science
Volume
24
Publish Date
2015
Start Page
113
End Page
114

Calcineurin orchestrates dimorphic transitions, antifungal drug responses and host-pathogen interactions of the pathogenic mucoralean fungus Mucor circinelloides.

Calcineurin plays essential roles in virulence and growth of pathogenic fungi and is a target of the natural products FK506 and Cyclosporine A. In the pathogenic mucoralean fungus Mucor circinelloides, calcineurin mutation or inhibition confers a yeast-locked phenotype indicating that calcineurin governs the dimorphic transition. Genetic analysis in this study reveals that two calcineurin A catalytic subunits (out of three) are functionally diverged. Homology modeling illustrates modes of resistance resulting from amino substitutions in the interface between each calcineurin subunit and the inhibitory drugs. In addition, we show how the dimorphic transition orchestrated by calcineurin programs different outcomes during host-pathogen interactions. For example, when macrophages phagocytose Mucor yeast, subsequent phagosomal maturation occurs, indicating host cells respond appropriately to control the pathogen. On the other hand, upon phagocytosis of spores, macrophages fail to form mature phagosomes. Cytokine production from immune cells differs following exposure to yeast versus spores (which germinate into hyphae). Thus, the morphogenic transition can be targeted as an efficient treatment option against Mucor infection. In addition, genetic analysis (including gene disruption and mutational studies) further strengthens the understanding of calcineurin and provides a foundation to develop antifungal agents targeting calcineurin to deploy against Mucor and other pathogenic fungi.

Authors
Lee, SC; Li, A; Calo, S; Inoue, M; Tonthat, NK; Bain, JM; Louw, J; Shinohara, ML; Erwig, LP; Schumacher, MA; Ko, DC; Heitman, J
MLA Citation
Lee, SC, Li, A, Calo, S, Inoue, M, Tonthat, NK, Bain, JM, Louw, J, Shinohara, ML, Erwig, LP, Schumacher, MA, Ko, DC, and Heitman, J. "Calcineurin orchestrates dimorphic transitions, antifungal drug responses and host-pathogen interactions of the pathogenic mucoralean fungus Mucor circinelloides." Molecular microbiology 97.5 (September 2015): 844-865.
PMID
26010100
Source
epmc
Published In
Molecular Microbiology
Volume
97
Issue
5
Publish Date
2015
Start Page
844
End Page
865
DOI
10.1111/mmi.13071

Genome Evolution and Innovation across the Four Major Lineages of Cryptococcus gattii.

Cryptococcus gattii is a fungal pathogen of humans, causing pulmonary infections in otherwise healthy hosts. To characterize genomic variation among the four major lineages of C. gattii (VGI, -II, -III, and -IV), we generated, annotated, and compared 16 de novo genome assemblies, including the first for the rarely isolated lineages VGIII and VGIV. By identifying syntenic regions across assemblies, we found 15 structural rearrangements, which were almost exclusive to the VGI-III-IV lineages. Using synteny to inform orthology prediction, we identified a core set of 87% of C. gattii genes present as single copies in all four lineages. Remarkably, 737 genes are variably inherited across lineages and are overrepresented for response to oxidative stress, mitochondrial import, and metal binding and transport. Specifically, VGI has an expanded set of iron-binding genes thought to be important to the virulence of Cryptococcus, while VGII has expansions in the stress-related heat shock proteins relative to the other lineages. We also characterized genes uniquely absent in each lineage, including a copper transporter absent from VGIV, which influences Cryptococcus survival during pulmonary infection and the onset of meningoencephalitis. Through inclusion of population-level data for an additional 37 isolates, we identified a new transcontinental clonal group that we name VGIIx, mitochondrial recombination between VGII and VGIII, and positive selection of multidrug transporters and the iron-sulfur protein aconitase along multiple branches of the phylogenetic tree. Our results suggest that gene expansion or contraction and positive selection have introduced substantial variation with links to mechanisms of pathogenicity across this species complex.The genetic differences between phenotypically different pathogens provide clues to the underlying mechanisms of those traits and can lead to new drug targets and improved treatments for those diseases. In this paper, we compare 16 genomes belonging to four highly differentiated lineages of Cryptococcus gattii, which cause pulmonary infections in otherwise healthy humans and other animals. Half of these lineages have not had their genomes previously assembled and annotated. We identified 15 ancestral rearrangements in the genome and over 700 genes that are unique to one or more lineages, many of which are associated with virulence. In addition, we found evidence for recent transcontinental spread, mitochondrial genetic exchange, and positive selection in multidrug transporters. Our results suggest that gene expansion/contraction and positive selection are diversifying the mechanisms of pathogenicity across this species complex.

Authors
Farrer, RA; Desjardins, CA; Sakthikumar, S; Gujja, S; Saif, S; Zeng, Q; Chen, Y; Voelz, K; Heitman, J; May, RC; Fisher, MC; Cuomo, CA
MLA Citation
Farrer, RA, Desjardins, CA, Sakthikumar, S, Gujja, S, Saif, S, Zeng, Q, Chen, Y, Voelz, K, Heitman, J, May, RC, Fisher, MC, and Cuomo, CA. "Genome Evolution and Innovation across the Four Major Lineages of Cryptococcus gattii." mBio 6.5 (September 2015): e00868-e00815.
PMID
26330512
Source
epmc
Published In
mBio
Volume
6
Issue
5
Publish Date
2015
Start Page
e00868
End Page
e00815
DOI
10.1128/mbio.00868-15

Genomics and Transcriptomics Analyses of the Oil-Accumulating Basidiomycete Yeast Trichosporon oleaginosus: Insights into Substrate Utilization and Alternative Evolutionary Trajectories of Fungal Mating Systems.

Microbial fermentation of agro-industrial waste holds great potential for reducing the environmental impact associated with the production of lipids for industrial purposes from plant biomass. However, the chemical complexity of many residues currently prevents efficient conversion into lipids, creating a high demand for strains with the ability to utilize all energy-rich components of agricultural residues. Here, we present results of genome and transcriptome analyses of Trichosporon oleaginosus. This oil-accumulating yeast is able to grow on a wide variety of substrates, including pentoses and N-acetylglucosamine, making it an interesting candidate for biotechnological applications. Transcriptomics shows specific changes in gene expression patterns under lipid-accumulating conditions. Furthermore, gene content and expression analyses indicate that T. oleaginosus is well-adapted for the utilization of chitin-rich biomass. We also focused on the T. oleaginosus mating type, because this species is a member of the Tremellomycetes, a group that has been intensively analyzed as a model for the evolution of sexual development, the best-studied member being Cryptococcus neoformans. The structure of the T. oleaginosus mating-type regions differs significantly from that of other Tremellomycetes and reveals a new evolutionary trajectory paradigm. Comparative analysis shows that recruitment of developmental genes to the ancestral tetrapolar mating-type loci occurred independently in the Trichosporon and Cryptococcus lineages, supporting the hypothesis of a trend toward larger mating-type regions in fungi.Finite fossil fuel resources pose sustainability challenges to society and industry. Microbial oils are a sustainable feedstock for biofuel and chemical production that does not compete with food production. We describe genome and transcriptome analyses of the oleaginous yeast Trichosporon oleaginosus, which can accumulate up to 70% of its dry weight as lipids. In contrast to conventional yeasts, this organism not only shows an absence of diauxic effect while fermenting hexoses and pentoses but also effectively utilizes xylose and N-acetylglucosamine, which are building blocks of lignocellulose and chitin, respectively. Transcriptome analysis revealed metabolic networks that govern conversion of xylose or N-acetylglucosamine as well as lipid accumulation. These data form the basis for a targeted strain optimization strategy. Furthermore, analysis of the mating type of T. oleaginosus supports the hypothesis of a trend toward larger mating-type regions in fungi, similar to the evolution of sex chromosomes in animals and plants.

Authors
Kourist, R; Bracharz, F; Lorenzen, J; Kracht, ON; Chovatia, M; Daum, C; Deshpande, S; Lipzen, A; Nolan, M; Ohm, RA; Grigoriev, IV; Sun, S; Heitman, J; Brück, T; Nowrousian, M
MLA Citation
Kourist, R, Bracharz, F, Lorenzen, J, Kracht, ON, Chovatia, M, Daum, C, Deshpande, S, Lipzen, A, Nolan, M, Ohm, RA, Grigoriev, IV, Sun, S, Heitman, J, Brück, T, and Nowrousian, M. "Genomics and Transcriptomics Analyses of the Oil-Accumulating Basidiomycete Yeast Trichosporon oleaginosus: Insights into Substrate Utilization and Alternative Evolutionary Trajectories of Fungal Mating Systems." mBio 6.4 (July 21, 2015): e00918-.
PMID
26199329
Source
epmc
Published In
mBio
Volume
6
Issue
4
Publish Date
2015
Start Page
e00918
DOI
10.1128/mbio.00918-15

Cryptococcosis Serotypes Impact Outcome and Provide Evidence of Cryptococcus neoformans Speciation.

Cryptococcus neoformans is a human opportunistic fungal pathogen causing severe disseminated meningoencephalitis, mostly in patients with cellular immune defects. This species is divided into three serotypes: A, D, and the AD hybrid. Our objectives were to compare population structures of serotype A and D clinical isolates and to assess whether infections with AD hybrids differ from infections with the other serotypes. For this purpose, we analyzed 483 isolates and the corresponding clinical data from 234 patients enrolled during the CryptoA/D study or the nationwide survey on cryptococcosis in France. Isolates were characterized in terms of ploidy, serotype, mating type, and genotype, utilizing flow cytometry, serotype- and mating type-specific PCR amplifications, and multilocus sequence typing (MLST) methods. Our results suggest that C. neoformans serotypes A and D have different routes of multiplication (primarily clonal expansion versus recombination events for serotype A and serotype D, respectively) and important genomic differences. Cryptococcosis includes a high proportion of proven or probable infections (21.5%) due to a mixture of genotypes, serotypes, and/or ploidies. Multivariate analysis showed that parameters independently associated with failure to achieve cerebrospinal fluid (CSF) sterilization by week 2 were a high serum antigen titer, the lack of flucytosine during induction therapy, and the occurrence of mixed infection, while infections caused by AD hybrids were more likely to be associated with CSF sterilization. Our study provides additional evidence for the possible speciation of C. neoformans var. neoformans and grubii and highlights the importance of careful characterization of causative isolates.Cryptococcus neoformans is an environmental fungus causing severe disease, estimated to be responsible for 600,000 deaths per year worldwide. This species is divided into serotypes A and D and an AD hybrid, and these could be considered two different species and an interspecies hybrid. The objectives of our study were to compare population structures of serotype A and serotype D and to assess whether infections with AD hybrids differ from infections with serotype A or D isolates in terms of clinical presentation and outcome. For this purpose, we used clinical data and strains from patients diagnosed with cryptococcosis in France. Our results suggest that, according to the serotype, isolates have different routes of multiplication and high genomic differences, confirming the possible speciation of serotypes A and D. Furthermore, we observed a better prognosis for infections caused by AD hybrid than those caused by serotype A or D, at least for those diagnosed in France.

Authors
Desnos-Ollivier, M; Patel, S; Raoux-Barbot, D; Heitman, J; Dromer, F
MLA Citation
Desnos-Ollivier, M, Patel, S, Raoux-Barbot, D, Heitman, J, and Dromer, F. "Cryptococcosis Serotypes Impact Outcome and Provide Evidence of Cryptococcus neoformans Speciation." mBio 6.3 (June 9, 2015): e00311-.
PMID
26060271
Source
epmc
Published In
mBio
Volume
6
Issue
3
Publish Date
2015
Start Page
e00311
DOI
10.1128/mbio.00311-15

Cryptococcal osteomyelitis in an adolescent survivor of T-cell acute lymphoblastic leukemia.

Cryptococcosis is infrequent in children, and isolated cryptococcal osteomyelitis is rarely encountered. Here, we describe a 14-year-old patient in remission from T-cell acute lymphoblastic leukemia with osteomyelitis because of Cryptococcus neoformans var. grubii. The patient was effectively treated with antifungal therapy.

Authors
Oh, D-Y; Madhusoodhan, PP; Springer, DJ; Inglima, K; Chaudhri, AA; Heitman, J; Raetz, EA; Khaitan, A; Rigaud, M
MLA Citation
Oh, D-Y, Madhusoodhan, PP, Springer, DJ, Inglima, K, Chaudhri, AA, Heitman, J, Raetz, EA, Khaitan, A, and Rigaud, M. "Cryptococcal osteomyelitis in an adolescent survivor of T-cell acute lymphoblastic leukemia." The Pediatric infectious disease journal 34.6 (June 2015): 662-666.
PMID
25806844
Source
epmc
Published In
Pediatric Infectious Disease Journal
Volume
34
Issue
6
Publish Date
2015
Start Page
662
End Page
666
DOI
10.1097/inf.0000000000000687

Unisexual versus bisexual mating in Cryptococcus neoformans: Consequences and biological impacts.

Cryptococcus neoformans is an opportunistic human fungal pathogen and can undergo both bisexual and unisexual mating. Despite the fact that one mating type is dispensable for unisexual mating, the two sexual cycles share surprisingly similar features. Both mating cycles are affected by similar environmental factors and regulated by the same pheromone response pathway. Recombination takes place during unisexual reproduction in a fashion similar to bisexual reproduction and can both admix pre-existing genetic diversity and also generate diversity de novo just like bisexual reproduction. These common features may allow the unisexual life cycle to provide phenotypic and genotypic plasticity for the natural Cryptococcus population, which is predominantly α mating type, and to avoid Muller's ratchet. The morphological transition from yeast to hyphal growth during both bisexual and unisexual mating may provide increased opportunities for outcrossing and the ability to forage for nutrients at a distance. The unisexual life cycle is a key evolutionary factor for Cryptococcus as a highly successful global fungal pathogen.

Authors
Fu, C; Sun, S; Billmyre, RB; Roach, KC; Heitman, J
MLA Citation
Fu, C, Sun, S, Billmyre, RB, Roach, KC, and Heitman, J. "Unisexual versus bisexual mating in Cryptococcus neoformans: Consequences and biological impacts." Fungal genetics and biology : FG & B 78 (May 2015): 65-75. (Review)
PMID
25173822
Source
epmc
Published In
Fungal Genetics and Biology
Volume
78
Publish Date
2015
Start Page
65
End Page
75
DOI
10.1016/j.fgb.2014.08.008

Systematic functional profiling of transcription factor networks in Cryptococcus neoformans.

Cryptococcus neoformans causes life-threatening meningoencephalitis in humans, but its overall biological and pathogenic regulatory circuits remain elusive, particularly due to the presence of an evolutionarily divergent set of transcription factors (TFs). Here, we report the construction of a high-quality library of 322 signature-tagged gene-deletion strains for 155 putative TF genes previously predicted using the DNA-binding domain TF database, and examine their in vitro and in vivo phenotypic traits under 32 distinct growth conditions. At least one phenotypic trait is exhibited by 145 out of 155 TF mutants (93%) and ∼85% of them (132/155) are functionally characterized for the first time in this study. The genotypic and phenotypic data for each TF are available in the C. neoformans TF phenome database (http://tf.cryptococcus.org). In conclusion, our phenome-based functional analysis of the C. neoformans TF mutant library provides key insights into transcriptional networks of basidiomycetous fungi and human fungal pathogens.

Authors
Jung, K-W; Yang, D-H; Maeng, S; Lee, K-T; So, Y-S; Hong, J; Choi, J; Byun, H-J; Kim, H; Bang, S; Song, M-H; Lee, J-W; Kim, MS; Kim, S-Y; Ji, J-H; Park, G; Kwon, H; Cha, S; Meyers, GL; Wang, LL; Jang, J; Janbon, G; Adedoyin, G; Kim, T; Averette, AK; Heitman, J; Cheong, E; Lee, Y-H; Lee, Y-W; Bahn, Y-S
MLA Citation
Jung, K-W, Yang, D-H, Maeng, S, Lee, K-T, So, Y-S, Hong, J, Choi, J, Byun, H-J, Kim, H, Bang, S, Song, M-H, Lee, J-W, Kim, MS, Kim, S-Y, Ji, J-H, Park, G, Kwon, H, Cha, S, Meyers, GL, Wang, LL, Jang, J, Janbon, G, Adedoyin, G, Kim, T, Averette, AK, Heitman, J, Cheong, E, Lee, Y-H, Lee, Y-W, and Bahn, Y-S. "Systematic functional profiling of transcription factor networks in Cryptococcus neoformans." Nature communications 6 (April 7, 2015): 6757-.
PMID
25849373
Source
epmc
Published In
Nature Communications
Volume
6
Publish Date
2015
Start Page
6757
DOI
10.1038/ncomms7757

A case of cryptococcus gattii in Western Florida: An aggressive pathogen in an immunocompetent host

Copyright © 2014 Wolters Kluwer Health, Inc. All rights reserved.Background Cryptococcus gattii is an environmental pathogenic yeast that produces disease in the skin, the respiratory tract, and the central nervous system of both immunocompromised and immunocompetent hosts. The incidence of C. gattii disease is increasing in frequency and, importantly, appears to be spreading its geographic clinical appearance since the first identified case series, which had shown defined boundaries. Case A 56-year-old previously healthy woman presented for medical care with 3 weeks of progressive weakness, headache, neck pain, and back pain. Initial computed tomographic scan results of the head and the spine were normal. However, chest x-ray showed a large right upper lobe mass. Bronchoscopic examination revealed a partially obstructing endobronchial polyp, on which a biopsy was performed, and culture of the tissue grew encapsulated yeast cells. Magnetic resonance imaging of the brain revealed multiple rim-enhancing lesions, and cerebrospinal fluid grew encapsulated yeasts that were identified as C. gattii, molecular-type VGI. Conclusions This is a virulent VGI strain of C. gattii, causing infection in an immunocompetent host in Western Florida with no recent travel history. Here, we discuss clinical, laboratory, diagnostic, and pathologic findings of the clinical presentation and its delayed diagnostic appearance in a nonendemic area.

Authors
Tiro, AV; Springer, DJ; Irby, R; Averill, F; Ottaviani, A; Heitman, J; Perfect, JR
MLA Citation
Tiro, AV, Springer, DJ, Irby, R, Averill, F, Ottaviani, A, Heitman, J, and Perfect, JR. "A case of cryptococcus gattii in Western Florida: An aggressive pathogen in an immunocompetent host." Infectious Diseases in Clinical Practice 23.2 (March 12, 2015): 105-108.
Source
scopus
Published In
Infectious Diseases in Clinical Practice
Volume
23
Issue
2
Publish Date
2015
Start Page
105
End Page
108
DOI
10.1097/IPC.0000000000000211

Network-assisted genetic dissection of pathogenicity and drug resistance in the opportunistic human pathogenic fungus Cryptococcus neoformans.

Cryptococcus neoformans is an opportunistic human pathogenic fungus that causes meningoencephalitis. Due to the increasing global risk of cryptococcosis and the emergence of drug-resistant strains, the development of predictive genetics platforms for the rapid identification of novel genes governing pathogenicity and drug resistance of C. neoformans is imperative. The analysis of functional genomics data and genome-scale mutant libraries may facilitate the genetic dissection of such complex phenotypes but with limited efficiency. Here, we present a genome-scale co-functional network for C. neoformans, CryptoNet, which covers ~81% of the coding genome and provides an efficient intermediary between functional genomics data and reverse-genetics resources for the genetic dissection of C. neoformans phenotypes. CryptoNet is the first genome-scale co-functional network for any fungal pathogen. CryptoNet effectively identified novel genes for pathogenicity and drug resistance using guilt-by-association and context-associated hub algorithms. CryptoNet is also the first genome-scale co-functional network for fungi in the basidiomycota phylum, as Saccharomyces cerevisiae belongs to the ascomycota phylum. CryptoNet may therefore provide insights into pathway evolution between two distinct phyla of the fungal kingdom. The CryptoNet web server (www.inetbio.org/cryptonet) is a public resource that provides an interactive environment of network-assisted predictive genetics for C. neoformans.

Authors
Kim, H; Jung, K-W; Maeng, S; Chen, Y-L; Shin, J; Shim, JE; Hwang, S; Janbon, G; Kim, T; Heitman, J; Bahn, Y-S; Lee, I
MLA Citation
Kim, H, Jung, K-W, Maeng, S, Chen, Y-L, Shin, J, Shim, JE, Hwang, S, Janbon, G, Kim, T, Heitman, J, Bahn, Y-S, and Lee, I. "Network-assisted genetic dissection of pathogenicity and drug resistance in the opportunistic human pathogenic fungus Cryptococcus neoformans." Scientific reports 5 (March 5, 2015): 8767-.
PMID
25739925
Source
epmc
Published In
Scientific Reports
Volume
5
Publish Date
2015
Start Page
8767
DOI
10.1038/srep08767

A Case of Cryptococcus gattii in Western Florida

Authors
Tiro, AV; Springer, DJ; Irby, R; Averill, F; Ottaviani, A; Heitman, J; Perfect, JR
MLA Citation
Tiro, AV, Springer, DJ, Irby, R, Averill, F, Ottaviani, A, Heitman, J, and Perfect, JR. "A Case of Cryptococcus gattii in Western Florida." Infectious Diseases in Clinical Practice 23.2 (March 2015): 105-108.
Source
crossref
Published In
Infectious Diseases in Clinical Practice
Volume
23
Issue
2
Publish Date
2015
Start Page
105
End Page
108
DOI
10.1097/IPC.0000000000000211

On the Discovery of TOR As the Target of Rapamycin.

Authors
Heitman, J
MLA Citation
Heitman, J. "On the Discovery of TOR As the Target of Rapamycin." PLoS pathogens 11.11 (January 2015): e1005245-. (Review)
PMID
26540102
Source
epmc
Published In
PLoS pathogens
Volume
11
Issue
11
Publish Date
2015
Start Page
e1005245
DOI
10.1371/journal.ppat.1005245

Unisexual reproduction drives meiotic recombination and phenotypic and karyotypic plasticity in Cryptococcus neoformans.

In fungi, unisexual reproduction, where sexual development is initiated without the presence of two compatible mating type alleles, has been observed in several species that can also undergo traditional bisexual reproduction, including the important human fungal pathogens Cryptococcus neoformans and Candida albicans. While unisexual reproduction has been well characterized qualitatively, detailed quantifications are still lacking for aspects of this process, such as the frequency of recombination during unisexual reproduction, and how this compares with bisexual reproduction. Here, we analyzed meiotic recombination during α-α unisexual and a-α bisexual reproduction of C. neoformans. We found that meiotic recombination operates in a similar fashion during both modes of sexual reproduction. Specifically, we observed that in α-α unisexual reproduction, the numbers of crossovers along the chromosomes during meiosis, recombination frequencies at specific chromosomal regions, as well as meiotic recombination hot and cold spots, are all similar to those observed during a-α bisexual reproduction. The similarity in meiosis is also reflected by the fact that phenotypic segregation among progeny collected from the two modes of sexual reproduction is also similar, with transgressive segregation being observed in both. Additionally, we found diploid meiotic progeny were also produced at similar frequencies in the two modes of sexual reproduction, and transient chromosomal loss and duplication likely occurs frequently and results in aneuploidy and loss of heterozygosity that can span entire chromosomes. Furthermore, in both α-α unisexual and a-α bisexual reproduction, we observed biased allele inheritance in regions on chromosome 4, suggesting the presence of fragile chromosomal regions that might be vulnerable to mitotic recombination. Interestingly, we also observed a crossover event that occurred within the MAT locus during α-α unisexual reproduction. Our results provide definitive evidence that α-α unisexual reproduction is a meiotic process similar to a-α bisexual reproduction.

Authors
Sun, S; Billmyre, RB; Mieczkowski, PA; Heitman, J
MLA Citation
Sun, S, Billmyre, RB, Mieczkowski, PA, and Heitman, J. "Unisexual reproduction drives meiotic recombination and phenotypic and karyotypic plasticity in Cryptococcus neoformans." PLoS genetics 10.12 (December 11, 2014): e1004849-.
PMID
25503976
Source
epmc
Published In
PLoS genetics
Volume
10
Issue
12
Publish Date
2014
Start Page
e1004849
DOI
10.1371/journal.pgen.1004849

Sex in the Mucoralean fungi.

Sexual development is extant in virtually all eukaryotic species, including throughout the kingdom Fungi. Positioned within the opisthokonts along with metazoans, fungi serve as model systems to elucidate the genetics and impact of sexual development. Basal fungal lineages such as the Mucoralean fungi provide a unique basis to study sexual reproduction, in which common ancestral traits found in both animal and fungal lineages may be conserved. This review discusses the sexual development, sex loci, and evolution of the sex locus in the Mucoralean fungi, which sheds light on our understanding of the evolution and functions of sex.

Authors
Lee, SC; Heitman, J
MLA Citation
Lee, SC, and Heitman, J. "Sex in the Mucoralean fungi." Mycoses 57 Suppl 3 (December 2014): 18-24. (Review)
PMID
25175551
Source
epmc
Published In
Mycoses
Volume
57 Suppl 3
Publish Date
2014
Start Page
18
End Page
24
DOI
10.1111/myc.12244

Distinct and redundant roles of exonucleases in Cryptococcus neoformans: implications for virulence and mating.

Opportunistic pathogens like Cryptococcus neoformans are constantly exposed to changing environments, in their natural habitat as well as when encountering a human host. This requires a coordinated program to regulate gene expression that can act at the levels of mRNA synthesis and also mRNA degradation. Here, we find that deletion of the gene encoding the major cytoplasmic 5'→3' exonuclease Xrn1p in C. neoformans has important consequences for virulence associated phenotypes such as growth at 37 °C, capsule and melanin. In an invertebrate model of cryptococcosis the alteration of these virulence properties corresponds to avirulence of the xrn1Δ mutant strains. Additionally, deletion of XRN1 impairs uni- and bisexual mating. On a molecular level, the absence of XRN1 is associated with the upregulation of other major exonuclease encoding genes (i.e. XRN2 and RRP44). Using inducible alleles of RRP44 and XRN2, we show that artificial overexpression of these genes alters LAC1 gene expression and mating. Our data thus suggest the existence of a complex interdependent regulation of exonuclease encoding genes that impact upon virulence and mating in C. neoformans.

Authors
Wollschlaeger, C; Trevijano-Contador, N; Wang, X; Legrand, M; Zaragoza, O; Heitman, J; Janbon, G
MLA Citation
Wollschlaeger, C, Trevijano-Contador, N, Wang, X, Legrand, M, Zaragoza, O, Heitman, J, and Janbon, G. "Distinct and redundant roles of exonucleases in Cryptococcus neoformans: implications for virulence and mating." Fungal genetics and biology : FG & B 73 (December 2014): 20-28.
PMID
25267175
Source
epmc
Published In
Fungal Genetics and Biology
Volume
73
Publish Date
2014
Start Page
20
End Page
28
DOI
10.1016/j.fgb.2014.09.007

Unisexual reproduction reverses Muller's ratchet.

Cryptococcus neoformans is a pathogenic basidiomycetous fungus that engages in outcrossing, inbreeding, and selfing forms of unisexual reproduction as well as canonical sexual reproduction between opposite mating types. Long thought to be clonal, >99% of sampled environmental and clinical isolates of C. neoformans are MATα, limiting the frequency of opposite mating-type sexual reproduction. Sexual reproduction allows eukaryotic organisms to exchange genetic information and shuffle their genomes to avoid the irreversible accumulation of deleterious changes that occur in asexual populations, known as Muller's ratchet. We tested whether unisexual reproduction, which dispenses with the requirement for an opposite mating-type partner, is able to purge the genome of deleterious mutations. We report that the unisexual cycle can restore mutant strains of C. neoformans to wild-type genotype and phenotype, including prototrophy and growth rate. Furthermore, the unisexual cycle allows attenuated strains to purge deleterious mutations and produce progeny that are returned to wild-type virulence. Our results show that unisexual populations of C. neoformans are able to avoid Muller's ratchet and loss of fitness through a unisexual reproduction cycle involving α-α cell fusion, nuclear fusion, and meiosis. Similar types of unisexual reproduction may operate in other pathogenic and saprobic eukaryotic taxa.

Authors
Roach, KC; Heitman, J
MLA Citation
Roach, KC, and Heitman, J. "Unisexual reproduction reverses Muller's ratchet." Genetics 198.3 (November 2014): 1059-1069.
PMID
25217049
Source
epmc
Published In
Genetics
Volume
198
Issue
3
Publish Date
2014
Start Page
1059
End Page
1069
DOI
10.1534/genetics.114.170472

Sex and the Microsporidia

© 2014 John Wiley & Sons, Inc.In this chapter, the author brings the various types of evidence for sexuality and asexuality in the microsporidia, ranging from observations of life cycles under the light and electron microscope to population genetic analyses of recombination and surveys of whole genomes for sex-related genes. Sexual reproduction in fungi includes the three crucial steps: plasmogamy, karyogamy and meiosis that are common to most sexual multicellular organisms. Microsporidia are descended from fungal or fungal-like ancestors. Given that most major groups of fungi include sexual species and that asexual species tend to have reduced evolutionary potential, the most parsimonious hypothesis is that microsporidia are descended from an ancestor that reproduced sexually in a similar manner to existing sexual fungi. Various types of evidence suggest that some or all microsporidia reproduce sexually in a similar manner to fungi. Observations of microsporidian life cycles have identified putative meiotic structures and gametes in certain microsporidian species.

Authors
Lee, SC; Heitman, J; Ironside, JE
MLA Citation
Lee, SC, Heitman, J, and Ironside, JE. "Sex and the Microsporidia." Microsporidia: Pathogens of Opportunity: First Edition. October 20, 2014. 231-243.
Source
scopus
Publish Date
2014
Start Page
231
End Page
243
DOI
10.1002/9781118395264.ch8

Antifungal drug resistance evoked via RNAi-dependent epimutations.

Microorganisms evolve via a range of mechanisms that may include or involve sexual/parasexual reproduction, mutators, aneuploidy, Hsp90 and even prions. Mechanisms that may seem detrimental can be repurposed to generate diversity. Here we show that the human fungal pathogen Mucor circinelloides develops spontaneous resistance to the antifungal drug FK506 (tacrolimus) via two distinct mechanisms. One involves Mendelian mutations that confer stable drug resistance; the other occurs via an epigenetic RNA interference (RNAi)-mediated pathway resulting in unstable drug resistance. The peptidylprolyl isomerase FKBP12 interacts with FK506 forming a complex that inhibits the protein phosphatase calcineurin. Calcineurin inhibition by FK506 blocks M. circinelloides transition to hyphae and enforces yeast growth. Mutations in the fkbA gene encoding FKBP12 or the calcineurin cnbR or cnaA genes confer FK506 resistance and restore hyphal growth. In parallel, RNAi is spontaneously triggered to silence the fkbA gene, giving rise to drug-resistant epimutants. FK506-resistant epimutants readily reverted to the drug-sensitive wild-type phenotype when grown without exposure to the drug. The establishment of these epimutants is accompanied by generation of abundant fkbA small RNAs and requires the RNAi pathway as well as other factors that constrain or reverse the epimutant state. Silencing involves the generation of a double-stranded RNA trigger intermediate using the fkbA mature mRNA as a template to produce antisense fkbA RNA. This study uncovers a novel epigenetic RNAi-based epimutation mechanism controlling phenotypic plasticity, with possible implications for antimicrobial drug resistance and RNAi-regulatory mechanisms in fungi and other eukaryotes.

Authors
Calo, S; Shertz-Wall, C; Lee, SC; Bastidas, RJ; Nicolás, FE; Granek, JA; Mieczkowski, P; Torres-Martínez, S; Ruiz-Vázquez, RM; Cardenas, ME; Heitman, J
MLA Citation
Calo, S, Shertz-Wall, C, Lee, SC, Bastidas, RJ, Nicolás, FE, Granek, JA, Mieczkowski, P, Torres-Martínez, S, Ruiz-Vázquez, RM, Cardenas, ME, and Heitman, J. "Antifungal drug resistance evoked via RNAi-dependent epimutations." Nature 513.7519 (September 2014): 555-558.
PMID
25079329
Source
epmc
Published In
Nature
Volume
513
Issue
7519
Publish Date
2014
Start Page
555
End Page
558
DOI
10.1038/nature13575

Cryptococcus gattii VGIII Isolates Causing Infections in HIV/AIDS Patients in Southern California: Identification of the Local Environmental Source as Arboreal

© 2014 Springer et al.Ongoing Cryptococcus gattii outbreaks in the Western United States and Canada illustrate the impact of environmental reservoirs and both clonal and recombining propagation in driving emergence and expansion of microbial pathogens. C. gattii comprises four distinct molecular types: VGI, VGII, VGIII, and VGIV, with no evidence of nuclear genetic exchange, indicating these represent distinct species. C. gattii VGII isolates are causing the Pacific Northwest outbreak, whereas VGIII isolates frequently infect HIV/AIDS patients in Southern California. VGI, VGII, and VGIII have been isolated from patients and animals in the Western US, suggesting these molecular types occur in the environment. However, only two environmental isolates of C. gattii have ever been reported from California: CBS7750 (VGII) and WM161 (VGIII). The incongruence of frequent clinical presence and uncommon environmental isolation suggests an unknown C. gattii reservoir in California. Here we report frequent isolation of C. gattii VGIII MATα and MATa isolates and infrequent isolation of VGI MATα from environmental sources in Southern California. VGIII isolates were obtained from soil debris associated with tree species not previously reported as hosts from sites near residences of infected patients. These isolates are fertile under laboratory conditions, produce abundant spores, and are part of both locally and more distantly recombining populations. MLST and whole genome sequence analysis provide compelling evidence that these environmental isolates are the source of human infections. Isolates displayed wide-ranging virulence in macrophage and animal models. When clinical and environmental isolates with indistinguishable MLST profiles were compared, environmental isolates were less virulent. Taken together, our studies reveal an environmental source and risk of C. gattii to HIV/AIDS patients with implications for the >1,000,000 cryptococcal infections occurring annually for which the causative isolate is rarely assigned species status. Thus, the C. gattii global health burden could be more substantial than currently appreciated.

Authors
Springer, DJ; Billmyre, RB; Filler, EE; Voelz, K; Pursall, R; Mieczkowski, PA; Larsen, RA; Dietrich, FS; May, RC; Filler, SG; Heitman, J
MLA Citation
Springer, DJ, Billmyre, RB, Filler, EE, Voelz, K, Pursall, R, Mieczkowski, PA, Larsen, RA, Dietrich, FS, May, RC, Filler, SG, and Heitman, J. "Cryptococcus gattii VGIII Isolates Causing Infections in HIV/AIDS Patients in Southern California: Identification of the Local Environmental Source as Arboreal." PLoS Pathogens 10.8 (August 21, 2014).
Source
scopus
Published In
PLoS pathogens
Volume
10
Issue
8
Publish Date
2014
DOI
10.1371/journal.ppat.1004285

Cryptococcus gattii VGIII isolates causing infections in HIV/AIDS patients in Southern California: identification of the local environmental source as arboreal.

Ongoing Cryptococcus gattii outbreaks in the Western United States and Canada illustrate the impact of environmental reservoirs and both clonal and recombining propagation in driving emergence and expansion of microbial pathogens. C. gattii comprises four distinct molecular types: VGI, VGII, VGIII, and VGIV, with no evidence of nuclear genetic exchange, indicating these represent distinct species. C. gattii VGII isolates are causing the Pacific Northwest outbreak, whereas VGIII isolates frequently infect HIV/AIDS patients in Southern California. VGI, VGII, and VGIII have been isolated from patients and animals in the Western US, suggesting these molecular types occur in the environment. However, only two environmental isolates of C. gattii have ever been reported from California: CBS7750 (VGII) and WM161 (VGIII). The incongruence of frequent clinical presence and uncommon environmental isolation suggests an unknown C. gattii reservoir in California. Here we report frequent isolation of C. gattii VGIII MATα and MATa isolates and infrequent isolation of VGI MATα from environmental sources in Southern California. VGIII isolates were obtained from soil debris associated with tree species not previously reported as hosts from sites near residences of infected patients. These isolates are fertile under laboratory conditions, produce abundant spores, and are part of both locally and more distantly recombining populations. MLST and whole genome sequence analysis provide compelling evidence that these environmental isolates are the source of human infections. Isolates displayed wide-ranging virulence in macrophage and animal models. When clinical and environmental isolates with indistinguishable MLST profiles were compared, environmental isolates were less virulent. Taken together, our studies reveal an environmental source and risk of C. gattii to HIV/AIDS patients with implications for the >1,000,000 cryptococcal infections occurring annually for which the causative isolate is rarely assigned species status. Thus, the C. gattii global health burden could be more substantial than currently appreciated.

Authors
Springer, DJ; Billmyre, RB; Filler, EE; Voelz, K; Pursall, R; Mieczkowski, PA; Larsen, RA; Dietrich, FS; May, RC; Filler, SG; Heitman, J
MLA Citation
Springer, DJ, Billmyre, RB, Filler, EE, Voelz, K, Pursall, R, Mieczkowski, PA, Larsen, RA, Dietrich, FS, May, RC, Filler, SG, and Heitman, J. "Cryptococcus gattii VGIII isolates causing infections in HIV/AIDS patients in Southern California: identification of the local environmental source as arboreal." PLoS pathogens 10.8 (August 21, 2014): e1004285-.
Website
http://hdl.handle.net/10161/9025
PMID
25144534
Source
epmc
Published In
PLoS pathogens
Volume
10
Issue
8
Publish Date
2014
Start Page
e1004285
DOI
10.1371/journal.ppat.1004285

Sexual reproduction of human fungal pathogens.

We review here recent advances in our understanding of sexual reproduction in fungal pathogens that commonly infect humans, including Candida albicans, Cryptococcus neoformans/gattii, and Aspergillus fumigatus. Where appropriate or relevant, we introduce findings on other species associated with human infections. In particular, we focus on rapid advances involving genetic, genomic, and population genetic approaches that have reshaped our view of how fungal pathogens evolve. Rather than being asexual, mitotic, and largely clonal, as was thought to be prevalent as recently as a decade ago, we now appreciate that the vast majority of pathogenic fungi have retained extant sexual, or parasexual, cycles. In some examples, sexual and parasexual unions of pathogenic fungi involve closely related individuals, generating diversity in the population but with more restricted recombination than expected from fertile, sexual, outcrossing and recombining populations. In other cases, species and isolates participate in global outcrossing populations with the capacity for considerable levels of gene flow. These findings illustrate general principles of eukaryotic pathogen emergence with relevance for other fungi, parasitic eukaryotic pathogens, and both unicellular and multicellular eukaryotic organisms.

Authors
Heitman, J; Carter, DA; Dyer, PS; Soll, DR
MLA Citation
Heitman, J, Carter, DA, Dyer, PS, and Soll, DR. "Sexual reproduction of human fungal pathogens." Cold Spring Harbor perspectives in medicine 4.8 (August 2014). (Review)
PMID
25085958
Source
epmc
Published In
Cold Spring Harbor perspectives in medicine
Volume
4
Issue
8
Publish Date
2014
DOI
10.1101/cshperspect.a019281

Highly recombinant VGII Cryptococcus gattii population develops clonal outbreak clusters through both sexual macroevolution and asexual microevolution.

An outbreak of the fungal pathogen Cryptococcus gattii began in the Pacific Northwest (PNW) in the late 1990s. This outbreak consists of three clonal subpopulations: VGIIa/major, VGIIb/minor, and VGIIc/novel. Both VGIIa and VGIIc are unique to the PNW and exhibit increased virulence. In this study, we sequenced the genomes of isolates from these three groups, as well as global isolates, and analyzed a total of 53 isolates. We found that VGIIa/b/c populations show evidence of clonal expansion in the PNW. Whole-genome sequencing provided evidence that VGIIb originated in Australia, while VGIIa may have originated in South America, and these were likely independently introduced. Additionally, the VGIIa outbreak lineage may have arisen from a less virulent clade that contained a mutation in the MSH2 ortholog, but this appears to have reverted in the VGIIa outbreak strains, suggesting that a transient mutator phenotype may have contributed to adaptation and evolution of virulence in the PNW outbreak. PNW outbreak isolates share genomic islands, both between the clonal lineages and with global isolates, indicative of sexual recombination. This suggests that VGII C. gattii has undergone sexual reproduction, either bisexual or unisexual, in multiple locales contributing to the production of novel, virulent subtypes. We also found that the genomes of two basal VGII isolates from HIV(+) patients contain an introgression tract spanning three genes. Introgression substantially contributed to intra-VGII polymorphism and likely occurred through sexual reproduction with VGI. More broadly, these findings illustrate how both microevolution and sexual reproduction play central roles in the development of infectious outbreaks from avirulent or less virulent progenitors. Importance: Cryptococcus gattii is the causative agent responsible for ongoing infections in the Pacific Northwest of the United States and western Canada. The incidence of these infections increased dramatically in the 1990s and remains elevated. These infections are attributable to three clonal lineages of C. gattii, VGIIa, VGIIb, and VGIIc, with only VGIIa identified once previously in the Pacific Northwest prior to the start of the outbreak, albeit in a less virulent form. This study addresses the origin and emergence of this outbreak, using whole-genome sequencing and comparison of both outbreak and global isolates. We show that VGIIa arose mitotically from a less virulent clonal group, possibly via the action of a mutator phenotype, while VGIIb was likely introduced from Australia, and VGIIc appears to have emerged in the United States or in an undersampled locale via sexual reproduction. This work shows that multiple processes can contribute to the emergence of an outbreak.

Authors
Billmyre, RB; Croll, D; Li, W; Mieczkowski, P; Carter, DA; Cuomo, CA; Kronstad, JW; Heitman, J
MLA Citation
Billmyre, RB, Croll, D, Li, W, Mieczkowski, P, Carter, DA, Cuomo, CA, Kronstad, JW, and Heitman, J. "Highly recombinant VGII Cryptococcus gattii population develops clonal outbreak clusters through both sexual macroevolution and asexual microevolution." mBio 5.4 (July 29, 2014): e01494-e01414.
PMID
25073643
Source
epmc
Published In
mBio
Volume
5
Issue
4
Publish Date
2014
Start Page
e01494
End Page
e01414
DOI
10.1128/mbio.01494-14

Analysis of a food-borne fungal pathogen outbreak: virulence and genome of a Mucor circinelloides isolate from yogurt.

Food-borne pathogens are ongoing problems, and new pathogens are emerging. The impact of fungi, however, is largely underestimated. Recently, commercial yogurts contaminated with Mucor circinelloides were sold, and >200 consumers became ill with nausea, vomiting, and diarrhea. Mucoralean fungi cause the fatal fungal infection mucormycosis, whose incidence has been continuously increasing. In this study, we isolated an M. circinelloides strain from a yogurt container, and multilocus sequence typing identified the strain as Mucor circinelloides f. circinelloides. M. circinelloides f. circinelloides is the most virulent M. circinelloides subspecies and is commonly associated with human infections, whereas M. circinelloides f. lusitanicus and M. circinelloides f. griseocyanus are less common causes of infection. Whole-genome analysis of the yogurt isolate confirmed it as being close to the M. circinelloides f. circinelloides subgroup, with a higher percentage of divergence with the M. circinelloides f. lusitanicus subgroup. In mating assays, the yogurt isolate formed sexual zygospores with the (-) M. circinelloides f. circinelloides tester strain, which is congruent with its sex locus encoding SexP, the (+) mating type sex determinant. The yogurt isolate was virulent in murine and wax moth larva host systems. In a murine gastromucormycosis model, Mucor was recovered from fecal samples of infected mice for up to 10 days, indicating that Mucor can survive transit through the GI tract. In interactions with human immune cells, M. circinelloides f. lusitanicus induced proinflammatory cytokines but M. circinelloides f. circinelloides did not, which may explain the different levels of virulence in mammalian hosts. This study demonstrates that M. circinelloides can spoil food products and cause gastrointestinal illness in consumers and may pose a particular risk to immunocompromised patients. Importance: The U.S. FDA reported that yogurt products were contaminated with M. circinelloides, a mucoralean fungal pathogen, and >200 consumers complained of symptoms, including vomiting, nausea, and diarrhea. The manufacturer voluntarily withdrew the affected yogurt products from the market. Compared to other food-borne pathogens, including bacteria, viruses, and parasites, less focus has been placed on the risk of fungal pathogens. This study evaluates the potential risk from the food-borne fungal pathogen M. circinelloides that was isolated from the contaminated commercial yogurt. We successfully cultured an M. circinelloides isolate and found that the isolate belongs to the species M. circinelloides f. circinelloides, which is often associated with human infections. In murine and insect host models, the isolate was virulent. While information disseminated in the popular press would suggest this fungal contaminant poses little or no risk to consumers, our results show instead that it is capable of causing significant infections in animals.

Authors
Lee, SC; Billmyre, RB; Li, A; Carson, S; Sykes, SM; Huh, EY; Mieczkowski, P; Ko, DC; Cuomo, CA; Heitman, J
MLA Citation
Lee, SC, Billmyre, RB, Li, A, Carson, S, Sykes, SM, Huh, EY, Mieczkowski, P, Ko, DC, Cuomo, CA, and Heitman, J. "Analysis of a food-borne fungal pathogen outbreak: virulence and genome of a Mucor circinelloides isolate from yogurt." mBio 5.4 (July 8, 2014): e01390-e01314.
PMID
25006230
Source
epmc
Published In
mBio
Volume
5
Issue
4
Publish Date
2014
Start Page
e01390
End Page
e01314
DOI
10.1128/mbio.01390-14

Calcineurin controls hyphal growth, virulence, and drug tolerance of Candida tropicalis.

Candida tropicalis, a species closely related to Candida albicans, is an emerging fungal pathogen associated with high mortality rates of 40 to 70%. Like C. albicans and Candida dubliniensis, C. tropicalis is able to form germ tubes, pseudohyphae, and hyphae, but the genes involved in hyphal growth machinery and virulence remain unclear in C. tropicalis. Recently, echinocandin- and azole-resistant C. tropicalis isolates have frequently been isolated from various patients around the world, making treatment difficult. However, studies of the C. tropicalis genes involved in drug tolerance are limited. Here, we investigated the roles of calcineurin and its potential target, Crz1, for core stress responses and pathogenesis in C. tropicalis. We demonstrate that calcineurin and Crz1 are required for hyphal growth, micafungin tolerance, and virulence in a murine systemic infection model, while calcineurin but not Crz1 is essential for tolerance of azoles, caspofungin, anidulafungin, and cell wall-perturbing agents, suggesting that calcineurin has both Crz1-dependent and -independent functions in C. tropicalis. In addition, we found that calcineurin and Crz1 have opposite roles in controlling calcium tolerance. Calcineurin serves as a negative regulator, while Crz1 plays a positive role for calcium tolerance in C. tropicalis.

Authors
Chen, Y-L; Yu, S-J; Huang, H-Y; Chang, Y-L; Lehman, VN; Silao, FGS; Bigol, UG; Bungay, AAC; Averette, A; Heitman, J
MLA Citation
Chen, Y-L, Yu, S-J, Huang, H-Y, Chang, Y-L, Lehman, VN, Silao, FGS, Bigol, UG, Bungay, AAC, Averette, A, and Heitman, J. "Calcineurin controls hyphal growth, virulence, and drug tolerance of Candida tropicalis." Eukaryotic cell 13.7 (July 2014): 844-854.
PMID
24442892
Source
epmc
Published In
Eukaryotic cell
Volume
13
Issue
7
Publish Date
2014
Start Page
844
End Page
854
DOI
10.1128/ec.00302-13

Anatomy of an outbreak: recombining clonal clusters comprise the VGII C. gattii population

Authors
Billmyre, RB; Croll, D; Li, W; Mieczkowski, PA; Kronstad, J; Heitman, J
MLA Citation
Billmyre, RB, Croll, D, Li, W, Mieczkowski, PA, Kronstad, J, and Heitman, J. "Anatomy of an outbreak: recombining clonal clusters comprise the VGII C. gattii population." MYCOSES 57 (May 2014): 85-86.
Source
wos-lite
Published In
Mycoses
Volume
57
Publish Date
2014
Start Page
85
End Page
86

Evolutionary genomics of the Cryptococcus species complex

Authors
Heitman, J
MLA Citation
Heitman, J. "Evolutionary genomics of the Cryptococcus species complex." MYCOSES 57 (May 2014): 16-16.
Source
wos-lite
Published In
Mycoses
Volume
57
Publish Date
2014
Start Page
16
End Page
16

Structure-function analysis of the centromere-kinetochore and spindle assembly checkpoint machinery in Cryptococcus neoformans var grubii

Authors
Sanyal, K; Sridhar, S; Yadav, V; Heitman, J; Kozubowski, L
MLA Citation
Sanyal, K, Sridhar, S, Yadav, V, Heitman, J, and Kozubowski, L. "Structure-function analysis of the centromere-kinetochore and spindle assembly checkpoint machinery in Cryptococcus neoformans var grubii." MYCOSES 57 (May 2014): 71-72.
Source
wos-lite
Published In
Mycoses
Volume
57
Publish Date
2014
Start Page
71
End Page
72

When could loss be a gain: the two faces of genome instability in RNAi deficient Cryptococcus lineages

Authors
Chow, EWL; Clancey, S; Billmyre, RB; Heitman, J
MLA Citation
Chow, EWL, Clancey, S, Billmyre, RB, and Heitman, J. "When could loss be a gain: the two faces of genome instability in RNAi deficient Cryptococcus lineages." MYCOSES 57 (May 2014): 71-71.
Source
wos-lite
Published In
Mycoses
Volume
57
Publish Date
2014
Start Page
71
End Page
71

Unisexual reproduction reverses Muller's Ratchet

Authors
Roach, KC; Heitman, J
MLA Citation
Roach, KC, and Heitman, J. "Unisexual reproduction reverses Muller's Ratchet." MYCOSES 57 (May 2014): 69-69.
Source
wos-lite
Published In
Mycoses
Volume
57
Publish Date
2014
Start Page
69
End Page
69

The H99 family tree: variation in the common laboratory reference strains of Cryptococcus neoformans var. grubii characterised through whole-genome sequencing

Authors
Ormerod, KL; III, BEJ; Wood, IA; Lodge, JK; Heitman, J; Fraser, JA
MLA Citation
Ormerod, KL, III, BEJ, Wood, IA, Lodge, JK, Heitman, J, and Fraser, JA. "The H99 family tree: variation in the common laboratory reference strains of Cryptococcus neoformans var. grubii characterised through whole-genome sequencing." MYCOSES 57 (May 2014): 69-70.
Source
wos-lite
Published In
Mycoses
Volume
57
Publish Date
2014
Start Page
69
End Page
70

Analysis of the genome and transcriptome of Cryptococcus neoformans var. grubii reveals complex RNA expression and microevolution leading to virulence attenuation.

Cryptococcus neoformans is a pathogenic basidiomycetous yeast responsible for more than 600,000 deaths each year. It occurs as two serotypes (A and D) representing two varieties (i.e. grubii and neoformans, respectively). Here, we sequenced the genome and performed an RNA-Seq-based analysis of the C. neoformans var. grubii transcriptome structure. We determined the chromosomal locations, analyzed the sequence/structural features of the centromeres, and identified origins of replication. The genome was annotated based on automated and manual curation. More than 40,000 introns populating more than 99% of the expressed genes were identified. Although most of these introns are located in the coding DNA sequences (CDS), over 2,000 introns in the untranslated regions (UTRs) were also identified. Poly(A)-containing reads were employed to locate the polyadenylation sites of more than 80% of the genes. Examination of the sequences around these sites revealed a new poly(A)-site-associated motif (AUGHAH). In addition, 1,197 miscRNAs were identified. These miscRNAs can be spliced and/or polyadenylated, but do not appear to have obvious coding capacities. Finally, this genome sequence enabled a comparative analysis of strain H99 variants obtained after laboratory passage. The spectrum of mutations identified provides insights into the genetics underlying the micro-evolution of a laboratory strain, and identifies mutations involved in stress responses, mating efficiency, and virulence.

Authors
Janbon, G; Ormerod, KL; Paulet, D; Byrnes, EJ; Yadav, V; Chatterjee, G; Mullapudi, N; Hon, C-C; Billmyre, RB; Brunel, F; Bahn, Y-S; Chen, W; Chen, Y; Chow, EWL; Coppée, J-Y; Floyd-Averette, A; Gaillardin, C; Gerik, KJ; Goldberg, J; Gonzalez-Hilarion, S; Gujja, S; Hamlin, JL; Hsueh, Y-P; Ianiri, G; Jones, S; Kodira, CD; Kozubowski, L; Lam, W; Marra, M; Mesner, LD; Mieczkowski, PA; Moyrand, F; Nielsen, K; Proux, C; Rossignol, T; Schein, JE; Sun, S; Wollschlaeger, C; Wood, IA; Zeng, Q et al.
MLA Citation
Janbon, G, Ormerod, KL, Paulet, D, Byrnes, EJ, Yadav, V, Chatterjee, G, Mullapudi, N, Hon, C-C, Billmyre, RB, Brunel, F, Bahn, Y-S, Chen, W, Chen, Y, Chow, EWL, Coppée, J-Y, Floyd-Averette, A, Gaillardin, C, Gerik, KJ, Goldberg, J, Gonzalez-Hilarion, S, Gujja, S, Hamlin, JL, Hsueh, Y-P, Ianiri, G, Jones, S, Kodira, CD, Kozubowski, L, Lam, W, Marra, M, Mesner, LD, Mieczkowski, PA, Moyrand, F, Nielsen, K, Proux, C, Rossignol, T, Schein, JE, Sun, S, Wollschlaeger, C, Wood, IA, and Zeng, Q et al. "Analysis of the genome and transcriptome of Cryptococcus neoformans var. grubii reveals complex RNA expression and microevolution leading to virulence attenuation." PLoS genetics 10.4 (April 17, 2014): e1004261-.
Website
http://hdl.handle.net/10161/8468
PMID
24743168
Source
epmc
Published In
PLoS genetics
Volume
10
Issue
4
Publish Date
2014
Start Page
e1004261
DOI
10.1371/journal.pgen.1004261

Endolysosomal membrane trafficking complexes drive nutrient-dependent TORC1 signaling to control cell growth in Saccharomyces cerevisiae.

The rapamycin-sensitive and endomembrane-associated TORC1 pathway controls cell growth in response to nutrients in eukaryotes. Mutations in class C Vps (Vps-C) complexes are synthetically lethal with tor1 mutations and confer rapamycin hypersensitivity in Saccharomyces cerevisiae, suggesting a role for these complexes in TORC1 signaling. Vps-C complexes are required for vesicular trafficking and fusion and comprise four distinct complexes: HOPS and CORVET and their minor intermediaries (i)-CORVET and i-HOPS. We show that at least one Vps-C complex is required to promote TORC1 activity, with the HOPS complex having the greatest input. The vps-c mutants fail to recover from rapamycin-induced growth arrest and show low levels of TORC1 activity. TORC1 promotes cell growth via Sch9, a p70(S6) kinase ortholog. Constitutively active SCH9 or hyperactive TOR1 alleles restored rapamycin recovery and TORC1 activity of vps-c mutants, supporting a role for the Vps-C complexes upstream of TORC1. The EGO GTPase complex Exit from G0 Complex (EGOC) and its homologous Rag-GTPase complex convey amino acid signals to TORC1 in yeast and mammals, respectively. Expression of the activated EGOC GTPase subunits Gtr1(GTP) and Gtr2(GDP) partially suppressed vps-c mutant rapamycin recovery defects, and this suppression was enhanced by increased amino acid concentrations. Moreover, vps-c mutations disrupted EGOC-TORC1 interactions. TORC1 defects were more severe for vps-c mutants than those observed in EGOC mutants. Taken together, our results support a model in which distinct endolysosomal trafficking Vps-C complexes promote rapamycin-sensitive TORC1 activity via multiple inputs, one of which involves maintenance of amino acid homeostasis that is sensed and transmitted to TORC1 via interactions with EGOC.

Authors
Kingsbury, JM; Sen, ND; Maeda, T; Heitman, J; Cardenas, ME
MLA Citation
Kingsbury, JM, Sen, ND, Maeda, T, Heitman, J, and Cardenas, ME. "Endolysosomal membrane trafficking complexes drive nutrient-dependent TORC1 signaling to control cell growth in Saccharomyces cerevisiae." Genetics 196.4 (April 2014): 1077-1089.
PMID
24514902
Source
epmc
Published In
Genetics
Volume
196
Issue
4
Publish Date
2014
Start Page
1077
End Page
1089
DOI
10.1534/genetics.114.161646

Origins of eukaryotic sexual reproduction.

Sexual reproduction is a nearly universal feature of eukaryotic organisms. Given its ubiquity and shared core features, sex is thought to have arisen once in the last common ancestor to all eukaryotes. Using the perspectives of molecular genetics and cell biology, we consider documented and hypothetical scenarios for the instantiation and evolution of meiosis, fertilization, sex determination, uniparental inheritance of organelle genomes, and speciation.

Authors
Goodenough, U; Heitman, J
MLA Citation
Goodenough, U, and Heitman, J. "Origins of eukaryotic sexual reproduction." Cold Spring Harbor perspectives in biology 6.3 (March 2014). (Review)
PMID
24591519
Source
epmc
Published In
Cold Spring Harbor perspectives in biology
Volume
6
Issue
3
Publish Date
2014
DOI
10.1101/cshperspect.a016154

Estrogen receptor antagonists are anti-cryptococcal agents that directly bind EF hand proteins and synergize with fluconazole in vivo.

Cryptococcosis is an infectious disease of global significance for which new therapies are needed. Repurposing previously developed drugs for new indications can expedite the translation of new therapies from bench to beside. Here, we characterized the anti-cryptococcal activity and antifungal mechanism of estrogen receptor antagonists related to the breast cancer drugs tamoxifen and toremifene. Tamoxifen and toremifene are fungicidal and synergize with fluconazole and amphotericin B in vitro. In a mouse model of disseminated cryptococcosis, tamoxifen at concentrations achievable in humans combines with fluconazole to decrease brain burden by ~1 log10. In addition, these drugs inhibit the growth of Cryptococcus neoformans within macrophages, a niche not accessible by current antifungal drugs. Toremifene and tamoxifen directly bind to the essential EF hand protein calmodulin, as determined by thermal shift assays with purified C. neoformans calmodulin (Cam1), prevent Cam1 from binding to its well-characterized substrate calcineurin (Cna1), and block Cna1 activation. In whole cells, toremifene and tamoxifen block the calcineurin-dependent nuclear localization of the transcription factor Crz1. A large-scale chemical genetic screen with a library of C. neoformans deletion mutants identified a second EF hand-containing protein, which we have named calmodulin-like protein 1 (CNAG_05655), as a potential target, and further analysis showed that toremifene directly binds Cml1 and modulates its ability to bind and activate Cna1. Importantly, tamoxifen analogs (idoxifene and methylene-idoxifene) with increased calmodulin antagonism display improved anti-cryptococcal activity, indicating that calmodulin inhibition can be used to guide a systematic optimization of the anti-cryptococcal activity of the triphenylethylene scaffold.Worldwide, cryptococcosis affects approximately 1 million people annually and kills more HIV/AIDS patients per year than tuberculosis. The gold standard therapy for cryptococcosis is amphotericin B plus 5-flucytosine, but this regimen is not readily available in regions where resources are limited and where the burden of disease is highest. Herein, we show that molecules related to the breast cancer drug tamoxifen are fungicidal for Cryptococcus and display a number of pharmacological properties desirable for an anti-cryptococcal drug, including synergistic fungicidal activity with fluconazole in vitro and in vivo, oral bioavailability, and activity within macrophages. We have also demonstrated that this class of molecules targets calmodulin as part of their mechanism of action and that tamoxifen analogs with increased calmodulin antagonism have improved anti-cryptococcal activity. Taken together, these results indicate that tamoxifen is a pharmacologically attractive scaffold for the development of new anti-cryptococcal drugs and provide a mechanistic basis for its further optimization.

Authors
Butts, A; Koselny, K; Chabrier-Roselló, Y; Semighini, CP; Brown, JCS; Wang, X; Annadurai, S; DiDone, L; Tabroff, J; Childers, WE; Abou-Gharbia, M; Wellington, M; Cardenas, ME; Madhani, HD; Heitman, J; Krysan, DJ
MLA Citation
Butts, A, Koselny, K, Chabrier-Roselló, Y, Semighini, CP, Brown, JCS, Wang, X, Annadurai, S, DiDone, L, Tabroff, J, Childers, WE, Abou-Gharbia, M, Wellington, M, Cardenas, ME, Madhani, HD, Heitman, J, and Krysan, DJ. "Estrogen receptor antagonists are anti-cryptococcal agents that directly bind EF hand proteins and synergize with fluconazole in vivo." mBio 5.1 (February 11, 2014): e00765-e00713.
PMID
24520056
Source
epmc
Published In
mBio
Volume
5
Issue
1
Publish Date
2014
Start Page
e00765
End Page
e00713
DOI
10.1128/mbio.00765-13

Unseen sex in ancient virgin fungi

Authors
Lee, SC; Sun, S; Heitman, J
MLA Citation
Lee, SC, Sun, S, and Heitman, J. "Unseen sex in ancient virgin fungi." New Phytologist 201.1 (January 1, 2014): 3-5.
PMID
24274789
Source
scopus
Published In
New Phytologist
Volume
201
Issue
1
Publish Date
2014
Start Page
3
End Page
5
DOI
10.1111/nph.12564

Unisexual reproduction.

Sexual reproduction is ubiquitous throughout the eukaryotic kingdom, but the capacity of pathogenic fungi to undergo sexual reproduction has been a matter of intense debate. Pathogenic fungi maintained a complement of conserved meiotic genes but the populations appeared to be clonally derived. This debate was resolved first with the discovery of an extant sexual cycle and then unisexual reproduction. Unisexual reproduction is a distinct form of homothallism that dispenses with the requirement for an opposite mating type. Pathogenic and nonpathogenic fungi previously thought to be asexual are able to undergo robust unisexual reproduction. We review here recent advances in our understanding of the genetic and molecular basis of unisexual reproduction throughout fungi and the impact of unisex on the ecology and genomic evolution of fungal species.

Authors
Roach, KC; Feretzaki, M; Sun, S; Heitman, J
MLA Citation
Roach, KC, Feretzaki, M, Sun, S, and Heitman, J. "Unisexual reproduction." Advances in genetics 85 (January 2014): 255-305.
PMID
24880737
Source
epmc
Published In
Advances in genetics
Volume
85
Publish Date
2014
Start Page
255
End Page
305
DOI
10.1016/b978-0-12-800271-1.00005-6

Cryptococcus neoformans hyperfilamentous strain is hypervirulent in a murine model of cryptococcal meningoencephalitis.

Cryptococcus neoformans is a human fungal pathogen that causes lethal infections of the lung and central nervous system in immunocompromised individuals. C. neoformans has a defined bipolar sexual life cycle with a and α mating types. During the sexual cycle, which can occur between cells of opposite mating types (bisexual reproduction) or cells of one mating type (unisexual reproduction), a dimorphic transition from yeast to hyphal growth occurs. Hyphal development and meiosis generate abundant spores that, following inhalation, penetrate deep into the lung to enter the alveoli, germinate, and establish a pulmonary infection growing as budding yeast cells. Unisexual reproduction has been directly observed only in the Cryptococcus var. neoformans (serotype D) lineage under laboratory conditions. However, hyphal development has been previously associated with reduced virulence and the serotype D lineage exhibits limited pathogenicity in the murine model. In this study we show that the serotype D hyperfilamentous strain XL280α is hypervirulent in an animal model. It can grow inside the lung of the host, establish a pulmonary infection, and then disseminate to the brain to cause cryptococcal meningoencephalitis. Surprisingly, this hyperfilamentous strain triggers an immune response polarized towards Th2-type immunity, which is usually observed in the highly virulent sibling species C. gattii, responsible for the Pacific Northwest outbreak. These studies provide a technological advance that will facilitate analysis of virulence genes and attributes in C. neoformans var. neoformans, and reveal the virulence potential of serotype D as broader and more dynamic than previously appreciated.

Authors
Feretzaki, M; Hardison, SE; Wormley, FL; Heitman, J
MLA Citation
Feretzaki, M, Hardison, SE, Wormley, FL, and Heitman, J. "Cryptococcus neoformans hyperfilamentous strain is hypervirulent in a murine model of cryptococcal meningoencephalitis." PloS one 9.8 (January 2014): e104432-.
PMID
25093333
Source
epmc
Published In
PloS one
Volume
9
Issue
8
Publish Date
2014
Start Page
e104432
DOI
10.1371/journal.pone.0104432

Phylogenetic analysis of phenotypically characterized Cryptococcus laurentii isolates reveals high frequency of cryptic species.

Although Cryptococcus laurentii has been considered saprophytic and its taxonomy is still being described, several cases of human infections have already reported. This study aimed to evaluate molecular aspects of C. laurentii isolates from Brazil, Botswana, Canada, and the United States.In this study, 100 phenotypically identified C. laurentii isolates were evaluated by sequencing the 18S nuclear ribosomal small subunit rRNA gene (18S-SSU), D1/D2 region of 28S nuclear ribosomal large subunit rRNA gene (28S-LSU), and the internal transcribed spacer (ITS) of the ribosomal region.BLAST searches using 550-bp, 650-bp, and 550-bp sequenced amplicons obtained from the 18S-SSU, 28S-LSU, and the ITS region led to the identification of 75 C. laurentii strains that shared 99-100% identity with C. laurentii CBS 139. A total of nine isolates shared 99% identity with both Bullera sp. VY-68 and C. laurentii RY1. One isolate shared 99% identity with Cryptococcus rajasthanensis CBS 10406, and eight isolates shared 100% identity with Cryptococcus sp. APSS 862 according to the 28S-LSU and ITS regions and designated as Cryptococcus aspenensis sp. nov. (CBS 13867). While 16 isolates shared 99% identity with Cryptococcus flavescens CBS 942 according to the 18S-SSU sequence, only six were confirmed using the 28S-LSU and ITS region sequences. The remaining 10 shared 99% identity with Cryptococcus terrestris CBS 10810, which was recently described in Brazil. Through concatenated sequence analyses, seven sequence types in C. laurentii, three in C. flavescens, one in C. terrestris, and one in the C. aspenensis sp. nov. were identified.Sequencing permitted the characterization of 75% of the environmental C. laurentii isolates from different geographical areas and the identification of seven haplotypes of this species. Among sequenced regions, the increased variability of the ITS region in comparison to the 18S-SSU and 28S-LSU regions reinforces its applicability as a DNA barcode.

Authors
Ferreira-Paim, K; Ferreira, TB; Andrade-Silva, L; Mora, DJ; Springer, DJ; Heitman, J; Fonseca, FM; Matos, D; Melhem, MSC; Silva-Vergara, ML
MLA Citation
Ferreira-Paim, K, Ferreira, TB, Andrade-Silva, L, Mora, DJ, Springer, DJ, Heitman, J, Fonseca, FM, Matos, D, Melhem, MSC, and Silva-Vergara, ML. "Phylogenetic analysis of phenotypically characterized Cryptococcus laurentii isolates reveals high frequency of cryptic species." PloS one 9.9 (January 2014): e108633-.
PMID
25251413
Source
epmc
Published In
PloS one
Volume
9
Issue
9
Publish Date
2014
Start Page
e108633
DOI
10.1371/journal.pone.0108633

Unisexual reproduction of Cryptococcus gattii.

Cryptococcus gattii is a basidiomycetous human fungal pathogen that typically causes infection in tropical and subtropical regions and is responsible for an ongoing outbreak in immunocompetent individuals on Vancouver Island and in the Pacific Northwest of the US. Pathogenesis of this species may be linked to its sexual cycle that generates infectious propagules called basidiospores. A marked predominance of only one mating type (α) in clinical and environmental isolates suggests that a-α opposite-sex reproduction may be infrequent or geographically restricted, raising the possibility of an alternative unisexual cycle involving cells of only α mating type, as discovered previously in the related pathogenic species Cryptococcus neoformans. Here we report observation of hallmark features of unisexual reproduction in a clinical isolate of C. gattii (isolate 97/433) and describe genetic and environmental factors conducive to this sexual cycle. Our results are consistent with population genetic evidence of recombination in the largely unisexual populations of C. gattii and provide a useful genetic model for understanding how novel modes of sexual reproduction may contribute to evolution and virulence in this species.

Authors
Phadke, SS; Feretzaki, M; Clancey, SA; Mueller, O; Heitman, J
MLA Citation
Phadke, SS, Feretzaki, M, Clancey, SA, Mueller, O, and Heitman, J. "Unisexual reproduction of Cryptococcus gattii." PloS one 9.10 (January 2014): e111089-.
PMID
25337713
Source
epmc
Published In
PloS one
Volume
9
Issue
10
Publish Date
2014
Start Page
e111089
DOI
10.1371/journal.pone.0111089

Sex in the Mucoralean fungi

Summary: Sexual development is extant in virtually all eukaryotic species, including throughout the kingdom Fungi. Positioned within the opisthokonts along with metazoans, fungi serve as model systems to elucidate the genetics and impact of sexual development. Basal fungal lineages such as the Mucoralean fungi provide a unique basis to study sexual reproduction, in which common ancestral traits found in both animal and fungal lineages may be conserved. This review discusses the sexual development, sex loci, and evolution of the sex locus in the Mucoralean fungi, which sheds light on our understanding of the evolution and functions of sex.

Authors
Lee, SC; Heitman, J
MLA Citation
Lee, SC, and Heitman, J. "Sex in the Mucoralean fungi (Accepted)." Mycoses (2014).
Source
scopus
Published In
Mycoses
Publish Date
2014
DOI
10.1111/myc.12244

RNAi function, diversity, and loss in the fungal kingdom

RNAi is conserved and has been studied in a broad cross-section of the fungal kingdom, including Neurospora crassa, Schizosaccharomyces pombe, Cryptococcus neoformans, and Mucor circinelloides. And yet well known species, including the model yeast Saccharomyces cerevisiae and the plant pathogen Ustilago maydis, have lost RNAi, providing insights and opportunities to illuminate benefits conferred both by the presence of RNAi and its loss. Some of the earliest studies of RNAi were conducted in Neurospora, contemporaneously with the elucidation of RNAi in Caenorhabditis elegans. RNAi is a key epigenetic mechanism for maintaining genomic stability and integrity, as well as to defend against viruses, and given its ubiquity was likely present in the last eukaryotic common ancestor. In this review, we describe the diversity of RNAi mechanisms found in the fungi, highlighting recent work in Neurospora, S. pombe, and Cryptococcus. Finally, we consider frequent, independent losses of RNAi in diverse fungal lineages and both review and speculate on evolutionary forces that may drive the losses or result therefrom. © 2013 Springer Science+Business Media Dordrecht.

Authors
Billmyre, RB; Calo, S; Feretzaki, M; Wang, X; Heitman, J
MLA Citation
Billmyre, RB, Calo, S, Feretzaki, M, Wang, X, and Heitman, J. "RNAi function, diversity, and loss in the fungal kingdom." Chromosome Research 21.6-7 (December 1, 2013): 561-572. (Review)
Source
scopus
Published In
Chromosome Research
Volume
21
Issue
6-7
Publish Date
2013
Start Page
561
End Page
572
DOI
10.1007/s10577-013-9388-2

RNAi function, diversity, and loss in the fungal kingdom.

RNAi is conserved and has been studied in a broad cross-section of the fungal kingdom, including Neurospora crassa, Schizosaccharomyces pombe, Cryptococcus neoformans, and Mucor circinelloides. And yet well known species, including the model yeast Saccharomyces cerevisiae and the plant pathogen Ustilago maydis, have lost RNAi, providing insights and opportunities to illuminate benefits conferred both by the presence of RNAi and its loss. Some of the earliest studies of RNAi were conducted in Neurospora, contemporaneously with the elucidation of RNAi in Caenorhabditis elegans. RNAi is a key epigenetic mechanism for maintaining genomic stability and integrity, as well as to defend against viruses, and given its ubiquity was likely present in the last eukaryotic common ancestor. In this review, we describe the diversity of RNAi mechanisms found in the fungi, highlighting recent work in Neurospora, S. pombe, and Cryptococcus. Finally, we consider frequent, independent losses of RNAi in diverse fungal lineages and both review and speculate on evolutionary forces that may drive the losses or result therefrom.

Authors
Billmyre, RB; Calo, S; Feretzaki, M; Wang, X; Heitman, J
MLA Citation
Billmyre, RB, Calo, S, Feretzaki, M, Wang, X, and Heitman, J. "RNAi function, diversity, and loss in the fungal kingdom." Chromosome Res 21.6-7 (December 2013): 561-572. (Review)
PMID
24173579
Source
pubmed
Published In
Chromosome Research
Volume
21
Issue
6-7
Publish Date
2013
Start Page
561
End Page
572
DOI
10.1007/s10577-013-9388-2

Polyporales genomes reveal the genetic architecture underlying tetrapolar and bipolar mating systems.

The process of mating in Basidiomycota is regulated by homeodomain-encoding genes (HD) and pheromones and G protein-coupled pheromone receptor genes (P/R). Whether these genes are actually involved in determining mating type distinguishes mating systems that are considered tetrapolar (two locus) from bipolar (one locus). Polyporales are a diverse group of wood-decay basidiomycetes displaying high variability in mating and decay systems. Many of the bipolar species appear to be brown-rot fungi, and it has been hypothesized that there is a functional basis for this correlation. Here we characterize mating genes in recently sequenced Polyporales and other Agaricomycete genomes. All Agaricomycete genomes encode HD and pheromone receptor genes regardless of whether they are bipolar or tetrapolar. The HD genes are organized into a MAT-HD locus with a high degree of gene order conservation among neighboring genes, with the gene encoding mitochondrial intermediate peptidase consistently syntenic but no linkage to the P/R genes. To have a complete dataset of species with known mating systems we determined that Wolfiporia cocos appears to be bipolar, using the criterion that DNA polymorphism of MAT genes should be extreme. Testing the correlation of mating and decay systems while controlling for phylogenetic relatedness failed to identify a statistical association, likely due to the small number of taxa employed. Using a phylogenetic analysis of Ste3 proteins, we identified clades of sequences that contain no known mating type-specific receptors and therefore might have evolved novel functions. The data are consistent with multiple origins of bipolarity within the Agaricomycetes and Polyporales, although the alternative hypothesis that tetrapolarity and bipolarity are reversible states needs better testing.

Authors
James, TY; Sun, S; Li, W; Heitman, J; Kuo, H-C; Lee, Y-H; Asiegbu, FO; Olson, A
MLA Citation
James, TY, Sun, S, Li, W, Heitman, J, Kuo, H-C, Lee, Y-H, Asiegbu, FO, and Olson, A. "Polyporales genomes reveal the genetic architecture underlying tetrapolar and bipolar mating systems." Mycologia 105.6 (November 2013): 1374-1390.
PMID
23928418
Source
epmc
Published In
Mycologia
Volume
105
Issue
6
Publish Date
2013
Start Page
1374
End Page
1390
DOI
10.3852/13-162

Evolution of Virulence in Eukaryotic Microbes

© 2012 by Wiley-Blackwell. All rights reserved.A unique and timely review of the emergence of eukaryotic virulence in fungi, oomycetes, and protozoa, as they affect both animals and plants Evolution of Virulence in Eukaryotic Microbes addresses new developments in defining the molecular basis of virulence in eukaryotic pathogens. By examining how pathogenic determinants have evolved in concert with their hosts, often overcoming innate and adaptive immune mechanisms, the book takes a fresh look at the selective processes that have shaped their evolution. Introductory chapters ground the reader in principal evolutionary themes such as phylogenetics and genetic exchange, building a basis of knowledge for later chapters covering advances in genetic tools, how pathogens exchange genetic material in nature, and the common themes of evolutionary adaptation that lead to disease in different hosts. With the goal of linking the research findings of the many disparate scientific communities in the field, the book: Assembles for the first time a collection of chapters on the diversity of eukaryotic microorganisms and the influence of evolutionary forces on the origins and emergence of their virulent attributes Highlights examples from three important, divergent groups of eukaryotic microorganisms that cause disease in animals and plants: oomycetes, protozoan parasites, and fungi Covers how the development of genetic tools has fostered the identification and functional analyses of virulence determinants Addresses how pathogens exchange genetic material in nature via classical or modified meiotic processes, horizontal gene transfer, and sexual cycles including those that are cryptic or even unisexual Provides a broad framework for formulating future studies by illustrating themes common to different pathogenic microbes Evolution of Virulence in Eukaryotic Microbes is an ideal book for microbiologists, evolutionary biologists and medical professionals, as well as graduate students, postdoctoral fellows, and faculty members working on the evolution of pathogens.

Authors
Sibley, LD; Howlett, BJ; Heitman, J
MLA Citation
Sibley, LD, Howlett, BJ, and Heitman, J. Evolution of Virulence in Eukaryotic Microbes. October 28, 2013.
Source
scopus
Publish Date
2013
Start Page
1
End Page
606
DOI
10.1002/9781118308165

Evolution of Meiosis, Recombination, and Sexual Reproduction in Eukaryotic Microbes

Authors
Li, W; Savelkoul, E; Heitman, J; Logsdon, JM
MLA Citation
Li, W, Savelkoul, E, Heitman, J, and Logsdon, JM. "Evolution of Meiosis, Recombination, and Sexual Reproduction in Eukaryotic Microbes." Evolution of Virulence in Eukaryotic Microbes. October 28, 2013. 17-43.
Source
scopus
Publish Date
2013
Start Page
17
End Page
43
DOI
10.1002/9781118308165.ch2

Ecogenomics of Human and Animal Basidiomycetous Yeast Pathogens

Authors
Sun, S; Hagen, F; Xu, J; Dawson, T; Heitman, J; Kronstad, J; Saunders, C; Boekhout, T
MLA Citation
Sun, S, Hagen, F, Xu, J, Dawson, T, Heitman, J, Kronstad, J, Saunders, C, and Boekhout, T. "Ecogenomics of Human and Animal Basidiomycetous Yeast Pathogens." The Ecological Genomics of Fungi. September 20, 2013. 215-242.
Source
scopus
Publish Date
2013
Start Page
215
End Page
242
DOI
10.1002/9781118735893.ch10

Candida albicans yeast but not hyphae require pescadillo to respond to TOR

Authors
Koehler, J; Chowdhury, T; Jani, N; Van Werven, F; Bastidas, R; Heitman, J
MLA Citation
Koehler, J, Chowdhury, T, Jani, N, Van Werven, F, Bastidas, R, and Heitman, J. "Candida albicans yeast but not hyphae require pescadillo to respond to TOR." September 2013.
Source
wos-lite
Published In
Yeast
Volume
30
Publish Date
2013
Start Page
51
End Page
51

Unisexual and heterosexual meiotic reproduction generate aneuploidy and phenotypic diversity de novo in the yeast Cryptococcus neoformans.

Aneuploidy is known to be deleterious and underlies several common human diseases, including cancer and genetic disorders such as trisomy 21 in Down's syndrome. In contrast, aneuploidy can also be advantageous and in fungi confers antifungal drug resistance and enables rapid adaptive evolution. We report here that sexual reproduction generates phenotypic and genotypic diversity in the human pathogenic yeast Cryptococcus neoformans, which is globally distributed and commonly infects individuals with compromised immunity, such as HIV/AIDS patients, causing life-threatening meningoencephalitis. C. neoformans has a defined a-α opposite sexual cycle; however, >99% of isolates are of the α mating type. Interestingly, α cells can undergo α-α unisexual reproduction, even involving genotypically identical cells. A central question is: Why would cells mate with themselves given that sex is costly and typically serves to admix preexisting genetic diversity from genetically divergent parents? In this study, we demonstrate that α-α unisexual reproduction frequently generates phenotypic diversity, and the majority of these variant progeny are aneuploid. Aneuploidy is responsible for the observed phenotypic changes, as chromosome loss restoring euploidy results in a wild-type phenotype. Other genetic changes, including diploidization, chromosome length polymorphisms, SNPs, and indels, were also generated. Phenotypic/genotypic changes were not observed following asexual mitotic reproduction. Aneuploidy was also detected in progeny from a-α opposite-sex congenic mating; thus, both homothallic and heterothallic sexual reproduction can generate phenotypic diversity de novo. Our study suggests that the ability to undergo unisexual reproduction may be an evolutionary strategy for eukaryotic microbial pathogens, enabling de novo genotypic and phenotypic plasticity and facilitating rapid adaptation to novel environments.

Authors
Ni, M; Feretzaki, M; Li, W; Floyd-Averette, A; Mieczkowski, P; Dietrich, FS; Heitman, J
MLA Citation
Ni, M, Feretzaki, M, Li, W, Floyd-Averette, A, Mieczkowski, P, Dietrich, FS, and Heitman, J. "Unisexual and heterosexual meiotic reproduction generate aneuploidy and phenotypic diversity de novo in the yeast Cryptococcus neoformans." PLoS Biol 11.9 (September 2013): e1001653-.
PMID
24058295
Source
pubmed
Published In
PLoS biology
Volume
11
Issue
9
Publish Date
2013
Start Page
e1001653
DOI
10.1371/journal.pbio.1001653

Synthesis and Antifungal Activities of Miltefosine Analogs

Authors
Ravu, RR; Chen, YL; Jacob, MR; Pan, X; Agarwal, AK; Khan, SI; Heitman, J; Clark, AM; Li, XC
MLA Citation
Ravu, RR, Chen, YL, Jacob, MR, Pan, X, Agarwal, AK, Khan, SI, Heitman, J, Clark, AM, and Li, XC. "Synthesis and Antifungal Activities of Miltefosine Analogs." July 2013.
Source
wos-lite
Published In
Planta Medica
Volume
79
Issue
10
Publish Date
2013
Start Page
828
End Page
828

Molecular and genetic evidence for a tetrapolar mating system in the basidiomycetous yeast Kwoniella mangrovensis and two novel sibling species.

Kwoniella mangrovensis has been described as a sexual species with a bipolar mating system. Phylogenetic analysis of multiple genes places this species together with Kwoniella heveanensis in the Kwoniella clade, a sister clade to that containing two pathogenic species of global importance, Cryptococcus neoformans and Cryptococcus gattii, within the Tremellales. Recent studies defining the mating type loci (MAT) of species in these clades showed that, with the exception of C. neoformans and C. gattii, which are bipolar with a single biallelic multigene MAT locus, several other species feature a tetrapolar mating system with two unlinked loci (homeodomain [HD] and pheromone/receptor [P/R] loci). We characterized several strains from the original study describing K. mangrovensis; two MAT regions were amplified and sequenced: the STE20 gene (P/R locus) and the divergently transcribed SXI1 and SXI2 genes (HD locus). We identified five different mating types with different STE20/SXI allele combinations that together with results of mating experiments demonstrate that K. mangrovensis is not bipolar but instead has a tetrapolar mating system. Sequence and gene analysis for a 43-kb segment of the K. mangrovensis type strain MAT locus revealed remarkable synteny with the homologous K. heveanensis MAT P/R region, providing new insights into slower evolution of MAT loci in the Kwoniella compared to the Cryptococcus clade of the Tremellales. The study of additional isolates from plant substrates in Europe and Botswana using a combination of multilocus sequencing with MAT gene analysis revealed two novel sibling species that we name Kwoniella europaea and Kwoniella botswanensis and which appear to also have tetrapolar mating systems.

Authors
Guerreiro, MA; Springer, DJ; Rodrigues, JA; Rusche, LN; Findley, K; Heitman, J; Fonseca, A
MLA Citation
Guerreiro, MA, Springer, DJ, Rodrigues, JA, Rusche, LN, Findley, K, Heitman, J, and Fonseca, A. "Molecular and genetic evidence for a tetrapolar mating system in the basidiomycetous yeast Kwoniella mangrovensis and two novel sibling species." Eukaryot Cell 12.5 (May 2013): 746-760.
PMID
23524993
Source
pubmed
Published In
Eukaryotic cell
Volume
12
Issue
5
Publish Date
2013
Start Page
746
End Page
760
DOI
10.1128/EC.00065-13

Sex-induced silencing operates during opposite-sex and unisexual reproduction in Cryptococcus neoformans.

Cryptococcus neoformans is a human fungal pathogen that undergoes a dimorphic transition from yeast to hyphae during a-α opposite-sex mating and α-α unisexual reproduction (same-sex mating). Infectious spores are generated during both processes. We previously identified a sex-induced silencing (SIS) pathway in the C. neoformans serotype A var. grubii lineage, in which tandem transgene arrays trigger RNAi-dependent gene silencing at a high frequency during a-α opposite-sex mating, but at an ∼250-fold lower frequency during asexual mitotic vegetative growth. Here we report that SIS also operates during α-α unisexual reproduction. A self-fertile strain containing either SXI2a-URA5 or NEO-URA5 transgene arrays exhibited an elevated silencing frequency during solo and unisexual mating compared with mitotic vegetative growth. We also found that SIS operates at a similar efficiency on transgene arrays of the same copy number during either α-α unisexual reproduction or a-α opposite-sex mating. URA5-derived small RNAs were detected in the silenced progeny of α-α unisexual reproduction and RNAi core components were required, providing evidence that SIS induced by same-sex mating is also mediated by RNAi via sequence-specific small RNAs. In addition, our data show that the SIS RNAi pathway also operates to defend the genome via squelching transposon activity during same-sex mating as it does during opposite-sex mating. Taken together, our results confirm that SIS is conserved between the divergent C. neoformans serotype A and serotype D cryptic sibling species.

Authors
Wang, X; Darwiche, S; Heitman, J
MLA Citation
Wang, X, Darwiche, S, and Heitman, J. "Sex-induced silencing operates during opposite-sex and unisexual reproduction in Cryptococcus neoformans." Genetics 193.4 (April 2013): 1163-1174.
PMID
23378067
Source
pubmed
Published In
Genetics
Volume
193
Issue
4
Publish Date
2013
Start Page
1163
End Page
1174
DOI
10.1534/genetics.113.149443

Cryptococcus neoformans copper detoxification machinery is critical for fungal virulence.

Copper (Cu) is an essential metal that is toxic at high concentrations. Thus, pathogens often rely on host Cu for growth, but host cells can hyperaccumulate Cu to exert antimicrobial effects. The human fungal pathogen Cryptococcus neoformans encodes many Cu-responsive genes, but their role in infection is unclear. We determined that pulmonary C. neoformans infection results in Cu-specific induction of genes encoding the Cu-detoxifying metallothionein (Cmt) proteins. Mutant strains lacking CMTs or expressing Cmt variants defective in Cu-coordination exhibit severely attenuated virulence and reduced pulmonary colonization. Consistent with the upregulation of Cmt proteins, C. neoformans pulmonary infection results in increased serum Cu concentrations and increases and decreases alveolar macrophage expression of the Cu importer (Ctr1) and ATP7A, a transporter implicated in phagosomal Cu compartmentalization, respectively. These studies indicate that the host mobilizes Cu as an innate antifungal defense but C. neoformans senses and neutralizes toxic Cu to promote infection.

Authors
Ding, C; Festa, RA; Chen, Y-L; Espart, A; Palacios, Ò; Espín, J; Capdevila, M; Atrian, S; Heitman, J; Thiele, DJ
MLA Citation
Ding, C, Festa, RA, Chen, Y-L, Espart, A, Palacios, Ò, Espín, J, Capdevila, M, Atrian, S, Heitman, J, and Thiele, DJ. "Cryptococcus neoformans copper detoxification machinery is critical for fungal virulence." Cell Host Microbe 13.3 (March 13, 2013): 265-276.
PMID
23498952
Source
pubmed
Published In
Cell Host and Microbe
Volume
13
Issue
3
Publish Date
2013
Start Page
265
End Page
276
DOI
10.1016/j.chom.2013.02.002

Identification of the mating-type (MAT) locus that controls sexual reproduction of Blastomyces dermatitidis.

Blastomyces dermatitidis is a dimorphic fungal pathogen that primarily causes blastomycosis in the midwestern and northern United States and Canada. While the genes controlling sexual development have been known for a long time, the genes controlling sexual reproduction of B. dermatitidis (teleomorph, Ajellomyces dermatitidis) are unknown. We identified the mating-type (MAT) locus in the B. dermatitidis genome by comparative genomic approaches. The B. dermatitidis MAT locus resembles those of other dimorphic fungi, containing either an alpha-box (MAT1-1) or an HMG domain (MAT1-2) gene linked to the APN2, SLA2, and COX13 genes. However, in some strains of B. dermatitidis, the MAT locus harbors transposable elements (TEs) that make it unusually large compared to the MAT locus of other dimorphic fungi. Based on the MAT locus sequences of B. dermatitidis, we designed specific primers for PCR determination of the mating type. Two B. dermatitidis isolates of opposite mating types were cocultured on mating medium. Immature sexual structures were observed starting at 3 weeks of coculture, with coiled-hyphae-containing cleistothecia developing over the next 3 to 6 weeks. Genetic recombination was detected in potential progeny by mating-type determination, PCR-restriction fragment length polymorphism (PCR-RFLP), and random amplification of polymorphic DNA (RAPD) analyses, suggesting that a meiotic sexual cycle might have been completed. The F1 progeny were sexually fertile when tested with strains of the opposite mating type. Our studies provide a model for the evolution of the MAT locus in the dimorphic and closely related fungi and open the door to classic genetic analysis and studies on the possible roles of mating and mating type in infection and virulence.

Authors
Li, W; Sullivan, TD; Walton, E; Averette, AF; Sakthikumar, S; Cuomo, CA; Klein, BS; Heitman, J
MLA Citation
Li, W, Sullivan, TD, Walton, E, Averette, AF, Sakthikumar, S, Cuomo, CA, Klein, BS, and Heitman, J. "Identification of the mating-type (MAT) locus that controls sexual reproduction of Blastomyces dermatitidis." Eukaryot Cell 12.1 (January 2013): 109-117.
PMID
23143684
Source
pubmed
Published In
Eukaryotic cell
Volume
12
Issue
1
Publish Date
2013
Start Page
109
End Page
117
DOI
10.1128/EC.00249-12

Reconstructing genome evolution in historic samples of the Irish potato famine pathogen.

Responsible for the Irish potato famine of 1845-49, the oomycete pathogen Phytophthora infestans caused persistent, devastating outbreaks of potato late blight across Europe in the 19th century. Despite continued interest in the history and spread of the pathogen, the genome of the famine-era strain remains entirely unknown. Here we characterize temporal genomic changes in introduced P. infestans. We shotgun sequence five 19th-century European strains from archival herbarium samples--including the oldest known European specimen, collected in 1845 from the first reported source of introduction. We then compare their genomes to those of extant isolates. We report multiple distinct genotypes in historical Europe and a suite of infection-related genes different from modern strains. At virulence-related loci, several now-ubiquitous genotypes were absent from the historical gene pool. At least one of these genotypes encodes a virulent phenotype in modern strains, which helps explain the 20th century's episodic replacements of European P. infestans lineages.

Authors
Martin, MD; Cappellini, E; Samaniego, JA; Zepeda, ML; Campos, PF; Seguin-Orlando, A; Wales, N; Orlando, L; Ho, SYW; Dietrich, FS; Mieczkowski, PA; Heitman, J; Willerslev, E; Krogh, A; Ristaino, JB; Gilbert, MTP
MLA Citation
Martin, MD, Cappellini, E, Samaniego, JA, Zepeda, ML, Campos, PF, Seguin-Orlando, A, Wales, N, Orlando, L, Ho, SYW, Dietrich, FS, Mieczkowski, PA, Heitman, J, Willerslev, E, Krogh, A, Ristaino, JB, and Gilbert, MTP. "Reconstructing genome evolution in historic samples of the Irish potato famine pathogen." Nature communications 4 (January 2013): 2172-.
PMID
23863894
Source
epmc
Published In
Nature Communications
Volume
4
Publish Date
2013
Start Page
2172
DOI
10.1038/ncomms3172

Ordered kinetochore assembly in the human-pathogenic basidiomycetous yeast Cryptococcus neoformans

Kinetochores facilitate interaction between chromosomes and the spindle apparatus. The formation of a metazoan trilayered kinetochore is an ordered event in which inner, middle, and outer layers assemble during disassembly of the nuclear envelope during mitosis. The existence of a similar strong correlation between kinetochore assembly and nuclear envelope breakdown in unicellular eukaryotes is unclear. Studies in the hemiascomycetous budding yeasts Saccharomyces cerevisiae and Candida albicans suggest that an ordered kinetochore assembly may not be evolutionarily conserved. Here, we utilized high-resolution time-lapse microscopy to analyze the localization patterns of a series of putative kinetochore proteins in the basidiomycetous budding yeast Cryptococcus neoformans, a human pathogen. Strikingly, similar to most metazoa but atypical of yeasts, the centromeres are not clustered but positioned adjacent to the nuclear envelope in premitotic C. neoformans cells. The centromeres gradually coalesce to a single cluster as cells progress toward mitosis. The mitotic clustering of centromeres seems to be dependent on the integrity of the mitotic spindle. To study the dynamics of the nuclear envelope, we followed the localization of two marker proteins, Ndc1 and Nup107. Fluorescence microscopy of the nuclear envelope and components of the kinetochore, along with ultrastructure analysis by transmission electron microscopy, reveal that in C. neoformans, the kinetochore assembles in an ordered manner prior to mitosis in concert with a partial opening of the nuclear envelope. Taken together, the results of this study demonstrate that kinetochore dynamics in C. neoformans is reminiscent of that of metazoans and shed new light on the evolution of mitosis in eukaryotes. © 2013 Kozubowski et al.

Authors
Kozubowski, L; Yadav, V; Chatterjee, G; Sridhar, S; Yamaguchi, M; Kawamoto, S; Bose, I; Heitman, J; Sanyal, K
MLA Citation
Kozubowski, L, Yadav, V, Chatterjee, G, Sridhar, S, Yamaguchi, M, Kawamoto, S, Bose, I, Heitman, J, and Sanyal, K. "Ordered kinetochore assembly in the human-pathogenic basidiomycetous yeast Cryptococcus neoformans." mBio 4.5 (2013).
PMID
24085781
Source
scival
Published In
mBio
Volume
4
Issue
5
Publish Date
2013
DOI
10.1128/mBio.00614-13

Evolution of sex: Mating rituals of a pre-metazoan

Sex is pervasive throughout eukaryotes, yet many species have not been caught in the act. Now, a sexual cycle has been discovered in the choanoflagellate Salpingoeca rosetta, a pre-metazoan evolutionary model, opening a window on metazoan sexual evolution. © 2013 Elsevier Ltd.

Authors
Umen, J; Heitman, J
MLA Citation
Umen, J, and Heitman, J. "Evolution of sex: Mating rituals of a pre-metazoan." Current Biology 23.22 (2013): R1006-R1008.
PMID
24262825
Source
scival
Published In
Current Biology
Volume
23
Issue
22
Publish Date
2013
Start Page
R1006
End Page
R1008
DOI
10.1016/j.cub.2013.10.009

Unisexual Reproduction Drives Evolution of Eukaryotic Microbial Pathogens

Authors
Feretzaki, M; Heitman, J
MLA Citation
Feretzaki, M, and Heitman, J. "Unisexual Reproduction Drives Evolution of Eukaryotic Microbial Pathogens." PLoS Pathogens 9.10 (2013).
PMID
24204257
Source
scival
Published In
PLoS pathogens
Volume
9
Issue
10
Publish Date
2013
DOI
10.1371/journal.ppat.1003674

Posaconazole exhibits in vitro and in vivo synergistic antifungal activity with caspofungin or FK506 against Candida albicans.

The object of this study was to test whether posaconazole, a broad-spectrum antifungal agent inhibiting ergosterol biosynthesis, exhibits synergy with the β-1,3 glucan synthase inhibitor caspofungin or the calcineurin inhibitor FK506 against the human fungal pathogen Candida albicans. Although current drug treatments for Candida infection are often efficacious, the available antifungal armamentarium may not be keeping pace with the increasing incidence of drug resistant strains. The development of drug combinations or novel antifungal drugs to address emerging drug resistance is therefore of general importance. Combination drug therapies are employed to treat patients with HIV, cancer, or tuberculosis, and has considerable promise in the treatment of fungal infections like cryptococcal meningitis and C. albicans infections. Our studies reported here demonstrate that posaconazole exhibits in vitro synergy with caspofungin or FK506 against drug susceptible or resistant C. albicans strains. Furthermore, these combinations also show in vivo synergy against C. albicans strain SC5314 and its derived echinocandin-resistant mutants, which harbor an S645Y mutation in the CaFks1 β-1,3 glucan synthase drug target, suggesting potential therapeutic applicability for these combinations in the future.

Authors
Chen, Y-L; Lehman, VN; Averette, AF; Perfect, JR; Heitman, J
MLA Citation
Chen, Y-L, Lehman, VN, Averette, AF, Perfect, JR, and Heitman, J. "Posaconazole exhibits in vitro and in vivo synergistic antifungal activity with caspofungin or FK506 against Candida albicans." PLoS One 8.3 (2013): e57672-.
PMID
23472097
Source
pubmed
Published In
PloS one
Volume
8
Issue
3
Publish Date
2013
Start Page
e57672
DOI
10.1371/journal.pone.0057672

Evolution of fungal sexual reproduction

We review here recent advances in our understanding of the genetic, molecular and genomic basis of sex determination and sexual reproduction in the fungal kingdom as a window on the evolution of sex in eukaryotes more generally. In particular, we focus on the evolution of the mating-type locus and transitions in modes of sexual reproduction using examples from throughout the kingdom. These examples illustrate general principles of the origins of mating-type loci/sex chromosomes and the balance between inbreeding and outcrossing afforded by different modes of sexual reproduction involving tetrapolar, bipolar and unipolar sexual cycles. © 2013 by The Mycological Society of America, Lawrence, KS 66044-8897.

Authors
Heitman, J; Sun, S; James, TY
MLA Citation
Heitman, J, Sun, S, and James, TY. "Evolution of fungal sexual reproduction." Mycologia 105.1 (2013): 1-27.
PMID
23099518
Source
scival
Published In
Mycologia
Volume
105
Issue
1
Publish Date
2013
Start Page
1
End Page
27
DOI
10.3852/12-253

Generators of Phenotypic Diversity in the Evolution of Pathogenic Microorganisms

Authors
Calo, S; Billmyre, RB; Heitman, J
MLA Citation
Calo, S, Billmyre, RB, and Heitman, J. "Generators of Phenotypic Diversity in the Evolution of Pathogenic Microorganisms." PLoS Pathogens 9.3 (2013).
PMID
23555239
Source
scival
Published In
PLoS pathogens
Volume
9
Issue
3
Publish Date
2013
DOI
10.1371/journal.ppat.1003181

Genomic insights into the atopic eczema-associated skin commensal yeast Malassezia sympodialis

Malassezia commensal yeasts are associated with a number of skin disorders, such as atopic eczema/dermatitis and dandruff, and they also can cause systemic infections. Here we describe the 7.67-Mbp genome of Malassezia sympodialis, a species associated with atopic eczema, and contrast its genome repertoire with that of Malassezia globosa, associated with dandruff, as well as those of other closely related fungi. Ninety percent of the predicted M. sympodialis protein coding genes were experimentally verified by mass spectrometry at the protein level.Weidentified a relatively limited number of genes related to lipid biosynthesis, and both species lack the fatty acid synthase gene, in line with the known requirement of these yeasts to assimilate lipids from the host. Malassezia species do not appear to have many cell wall-localized glycosylphosphatidylinositol (GPI) proteins and lack other cell wall proteins previously identified in other fungi. This is surprising given that in other fungi these proteins have been shown to mediate interactions (e.g., adhesion and biofilm formation) with the host. The genome revealed a complex evolutionary history for an allergen of unknown function, Mala s 7, shown to be encoded by a member of an amplified gene family of secreted proteins. Based on genetic and biochemical studies with the basidiomycete human fungal pathogen Cryptococcus neoformans, we characterized the allergen Mala s 6 as the cytoplasmic cyclophilin A.Wefurther present evidence that M. sympodialis may have the capacity to undergo sexual reproduction and present a model for a pseudobipolar mating system that allows limited recombination between two linkedMATloci. Importance Malassezia commensal yeasts are associated with a number of skin disorders. The previously published genome of M. globosa provided some of the first insights into Malassezia biology and its involvement in dandruff. Here, we present the genome of M. sympodialis, frequently isolated from patients with atopic eczema and healthy individuals. We combined comparative genomics with sequencing and functional characterization of specific genes in a population of clinical isolates and in closely related model systems. Our analyses provide insights into the evolution of allergens related to atopic eczema and the evolutionary trajectory of the machinery for sexual reproduction and meiosis. We hypothesize that M. sympodialis may undergo sexual reproduction, which has important implications for the understanding of the life cycle and virulence potential of this medically important yeast. Our findings provide a foundation for the development of genetic and genomic tools to elucidate host-microbe interactions that occur on the skin and to identify potential therapeutic targets. © 2013 Gioti et al.

Authors
Gioti, A; Nystedt, B; Li, W; Xu, J; Andersson, A; Averette, AF; Münch, K; Wang, X; Kappauf, C; Kingsbury, JM; Kraak, B; Walker, LA; Johansson, HJ; Holm, T; Lehtiö, J; Stajich, JE; Mieczkowski, P; Kahmann, R; Kennell, JC; Cardenas, ME; Lundeberg, J; Saunders, CW; Boekhout, T; Dawson, TL; Munro, CA; Groot, PWJD; Butler, G; Heitman, J; Scheynius, A
MLA Citation
Gioti, A, Nystedt, B, Li, W, Xu, J, Andersson, A, Averette, AF, Münch, K, Wang, X, Kappauf, C, Kingsbury, JM, Kraak, B, Walker, LA, Johansson, HJ, Holm, T, Lehtiö, J, Stajich, JE, Mieczkowski, P, Kahmann, R, Kennell, JC, Cardenas, ME, Lundeberg, J, Saunders, CW, Boekhout, T, Dawson, TL, Munro, CA, Groot, PWJD, Butler, G, Heitman, J, and Scheynius, A. "Genomic insights into the atopic eczema-associated skin commensal yeast Malassezia sympodialis." mBio 4.1 (2013).
PMID
23341551
Source
scival
Published In
mBio
Volume
4
Issue
1
Publish Date
2013
DOI
10.1128/mBio.00572-12

Development of an aerosol model of Cryptococcus reveals humidity as an important factor affecting the viability of Cryptococcus during aerosolization.

Cryptococcus is an emerging global health threat that is annually responsible for over 1,000,000 infections and one third of all AIDS patient deaths. There is an ongoing outbreak of cryptococcosis in the western United States and Canada. Cryptococcosis is a disease resulting from the inhalation of the infectious propagules from the environment. The current and most frequently used animal infection models initiate infection via liquid suspension through intranasal instillation or intravenous injection. These models do not replicate the typically dry nature of aerosol exposure and may hinder our ability to decipher the initial events that lead to clearance or the establishment of infection. We have established a standardized aerosol model of murine infection for the human fungal pathogen Cryptococcus. Aerosolized cells were generated utilizing a Collison nebulizer in a whole-body Madison Chamber at different humidity conditions. The aerosols inside the chamber were sampled using a BioSampler to determine viable aerosol concentration and spray factor (ratio of viable aerosol concentration to total inoculum concentration). We have effectively delivered yeast and yeast-spore mixtures to the lungs of mice and observed the establishment of disease. We observed that growth conditions prior to exposure and humidity within the Madison Chamber during exposure can alter Cryptococcus survival and dose retained in mice.

Authors
Springer, DJ; Saini, D; Byrnes, EJ; Heitman, J; Frothingham, R
MLA Citation
Springer, DJ, Saini, D, Byrnes, EJ, Heitman, J, and Frothingham, R. "Development of an aerosol model of Cryptococcus reveals humidity as an important factor affecting the viability of Cryptococcus during aerosolization. (Published online)" PLoS One 8.7 (2013): e69804-.
PMID
23894542
Source
pubmed
Published In
PloS one
Volume
8
Issue
7
Publish Date
2013
Start Page
e69804
DOI
10.1371/journal.pone.0069804

Synthesis and antifungal activities of miltefosine analogs

Miltefosine is an alkylphosphocholine that shows broad-spectrum in vitro antifungal activities and limited in vivo efficacy in mouse models of cryptococcosis. To further explore the potential of this class of compounds for the treatment of systemic mycoses, nine analogs (3a-3i) were synthesized by modifying the choline structural moiety and the alkyl chain length of miltefosine. In vitro testing of these compounds against the opportunistic fungal pathogens Candida albicans, Candida glabrata, Candida krusei, Aspergillus fumigatus, and Cryptococcus neoformans revealed that N-benzyl-N,N-dimethyl-2-{[(hexadecyloxy)hydroxyphosphinyl]oxy}ethanaminium inner salt (3a), N,N-dimethyl-N-(4-nitrobenzyl)-2-{[(hexadecyloxy)hydroxyphosphinyl]oxy}ethanaminium inner salt (3d), and N-(4-methoxybenzyl)-N,N-dimethyl-2-{[(hexadecyloxy)hydroxyphosphinyl]oxy}ethanaminium inner salt (3e) exhibited minimum inhibitory concentrations (MIC) of 2.5-5.0 μg/mL against all tested pathogens, when compared to miltefosine with MICs of 2.5-3.3 μg/mL. Compound 3a showed low in vitro cytotoxicity against three mammalian cell lines similar to miltefosine. In vivo testing of 3a and miltefosine against C. albicans in a mouse model of systemic infection did not demonstrate efficacy. The results of this study indicate that further investigation will be required to determine the potential usefulness of the alkylphosphocholines in the treatment of invasive fungal infections. © 2013 Elsevier Ltd. All rights reserved.

Authors
Ravu, RR; Chen, Y-L; Jacob, MR; Pan, X; Agarwal, AK; Khan, SI; Heitman, J; Clark, AM; Li, X-C
MLA Citation
Ravu, RR, Chen, Y-L, Jacob, MR, Pan, X, Agarwal, AK, Khan, SI, Heitman, J, Clark, AM, and Li, X-C. "Synthesis and antifungal activities of miltefosine analogs." Bioorganic and Medicinal Chemistry Letters (2013).
PMID
23891181
Source
scival
Published In
Bioorganic & Medicinal Chemistry Letters
Publish Date
2013
DOI
10.1016/j.bmcl.2013.06.096

Genomic insights into the atopic eczema-associated skin commensal yeast Malassezia sympodialis.

ABSTRACT: Malassezia commensal yeasts are associated with a number of skin disorders, such as atopic eczema/dermatitis and dandruff, and they also can cause systemic infections. Here we describe the 7.67-Mbp genome of Malassezia sympodialis, a species associated with atopic eczema, and contrast its genome repertoire with that of Malassezia globosa, associated with dandruff, as well as those of other closely related fungi. Ninety percent of the predicted M. sympodialis protein coding genes were experimentally verified by mass spectrometry at the protein level. We identified a relatively limited number of genes related to lipid biosynthesis, and both species lack the fatty acid synthase gene, in line with the known requirement of these yeasts to assimilate lipids from the host. Malassezia species do not appear to have many cell wall-localized glycosylphosphatidylinositol (GPI) proteins and lack other cell wall proteins previously identified in other fungi. This is surprising given that in other fungi these proteins have been shown to mediate interactions (e.g., adhesion and biofilm formation) with the host. The genome revealed a complex evolutionary history for an allergen of unknown function, Mala s 7, shown to be encoded by a member of an amplified gene family of secreted proteins. Based on genetic and biochemical studies with the basidiomycete human fungal pathogen Cryptococcus neoformans, we characterized the allergen Mala s 6 as the cytoplasmic cyclophilin A. We further present evidence that M. sympodialis may have the capacity to undergo sexual reproduction and present a model for a pseudobipolar mating system that allows limited recombination between two linked MAT loci. Malassezia commensal yeasts are associated with a number of skin disorders. The previously published genome of M. globosa provided some of the first insights into Malassezia biology and its involvement in dandruff. Here, we present the genome of M. sympodialis, frequently isolated from patients with atopic eczema and healthy individuals. We combined comparative genomics with sequencing and functional characterization of specific genes in a population of clinical isolates and in closely related model systems. Our analyses provide insights into the evolution of allergens related to atopic eczema and the evolutionary trajectory of the machinery for sexual reproduction and meiosis. We hypothesize that M. sympodialis may undergo sexual reproduction, which has important implications for the understanding of the life cycle and virulence potential of this medically important yeast. Our findings provide a foundation for the development of genetic and genomic tools to elucidate host-microbe interactions that occur on the skin and to identify potential therapeutic targets.

Authors
Gioti, A; Nystedt, B; Li, W; Xu, J; Andersson, A; Averette, AF; Münch, K; Wang, X; Kappauf, C; Kingsbury, JM; Kraak, B; Walker, LA; Johansson, HJ; Holm, T; Lehtiö, J; Stajich, JE; Mieczkowski, P; Kahmann, R; Kennell, JC; Cardenas, ME; Lundeberg, J; Saunders, CW; Boekhout, T; Dawson, TL; Munro, CA; Groot, PWJD; Butler, G; Heitman, J; Scheynius, A
MLA Citation
Gioti, A, Nystedt, B, Li, W, Xu, J, Andersson, A, Averette, AF, Münch, K, Wang, X, Kappauf, C, Kingsbury, JM, Kraak, B, Walker, LA, Johansson, HJ, Holm, T, Lehtiö, J, Stajich, JE, Mieczkowski, P, Kahmann, R, Kennell, JC, Cardenas, ME, Lundeberg, J, Saunders, CW, Boekhout, T, Dawson, TL, Munro, CA, Groot, PWJD, Butler, G, Heitman, J, and Scheynius, A. "Genomic insights into the atopic eczema-associated skin commensal yeast Malassezia sympodialis." mBio 4.1 (2013): e00572-e00512.
Source
scival
Published In
mBio
Volume
4
Issue
1
Publish Date
2013
Start Page
e00572
End Page
e00512
DOI
10.1128/mBio.00572-12

Synthesis and antifungal activities of miltefosine analogs

Miltefosine is an alkylphosphocholine that shows broad-spectrum in vitro antifungal activities and limited in vivo efficacy in mouse models of cryptococcosis. To further explore the potential of this class of compounds for the treatment of systemic mycoses, nine analogs (3a-3i) were synthesized by modifying the choline structural moiety and the alkyl chain length of miltefosine. In vitro testing of these compounds against the opportunistic fungal pathogens Candida albicans, Candida glabrata, Candida krusei, Aspergillus fumigatus, and Cryptococcus neoformans revealed that N-benzyl-N,N-dimethyl-2- {[(hexadecyloxy)hydroxyphosphinyl]oxy}ethanaminium inner salt (3a), N,N-dimethyl-N-(4-nitrobenzyl)-2-{[(hexadecyloxy)hydroxyphosphinyl]oxy} ethanaminium inner salt (3d), and N-(4-methoxybenzyl)-N,N-dimethyl-2- {[(hexadecyloxy)hydroxyphosphinyl]oxy}ethanaminium inner salt (3e) exhibited minimum inhibitory concentrations (MIC) of 2.5-5.0 μg/mL against all tested pathogens, when compared to miltefosine with MICs of 2.5-3.3 μg/mL. Compound 3a showed low in vitro cytotoxicity against three mammalian cell lines similar to miltefosine. In vivo testing of 3a and miltefosine against C. albicans in a mouse model of systemic infection did not demonstrate efficacy. The results of this study indicate that further investigation will be required to determine the potential usefulness of the alkylphosphocholines in the treatment of invasive fungal infections. © 2013 Elsevier Ltd. All rights reserved.

Authors
Ravu, RR; Chen, YL; Jacob, MR; Pan, X; Agarwal, AK; Khan, SI; Heitman, J; Clark, AM; Li, XC
MLA Citation
Ravu, RR, Chen, YL, Jacob, MR, Pan, X, Agarwal, AK, Khan, SI, Heitman, J, Clark, AM, and Li, XC. "Synthesis and antifungal activities of miltefosine analogs." Bioorganic and Medicinal Chemistry Letters 23.17 (2013): 4828-4831.
Source
scival
Published In
Bioorganic & Medicinal Chemistry Letters
Volume
23
Issue
17
Publish Date
2013
Start Page
4828
End Page
4831
DOI
10.1016/j.bmcl.2013.06.096

Unisexual reproduction enhances fungal competitiveness by promoting habitat exploration via hyphal growth and sporulation

Unisexual reproduction is a novel homothallic sexual cycle recently discovered in both ascomycetous and basidiomycetous pathogenic fungi. It is a form of selfing that induces the yeast-to-hyphal dimorphic transition in isolates of the mating type of the human fungal pathogen Cryptococcus neoformans. Unisexual reproduction may benefit the pathogen by facilitating sexual reproduction in the absence of the opposite a mating type and by generating infectious propagules called basidiospores. Here, we report an independent potential selective advantage of unisexual reproduction beyond genetic exchange and recombination. We competed a wild-type strain capable of undergoing unisexual reproduction with mutants defective in this developmental pathway and found that unisexual reproduction provides a considerable dispersal advantage through hyphal growth and sporulation. Our results show that unisexual reproduction may serve to facilitate access to both nutrients and potential mating partners and may provide a means to maintain the capacity for dimorphic transitions in the environment.© 2013, American Society for Microbiology. All Rights Reserved.

Authors
Phadke, SS; Feretzaki, M; Heitman, J
MLA Citation
Phadke, SS, Feretzaki, M, and Heitman, J. "Unisexual reproduction enhances fungal competitiveness by promoting habitat exploration via hyphal growth and sporulation." Eukaryotic Cell 12.8 (2013): 1155-1159.
PMID
23794511
Source
scival
Published In
Eukaryotic cell
Volume
12
Issue
8
Publish Date
2013
Start Page
1155
End Page
1159
DOI
10.1128/EC.00147-13

Calcineurin governs thermotolerance and virulence of cryptococcus gattii

The pathogenic yeast Cryptococcus gattii, which is causing an outbreak in the Pacific Northwest region of North America, causes life-threatening pulmonary infections and meningoencephalitis in healthy individuals, unlike Cryptococcus neoformans, which commonly infects immunocompromised patients. In addition to a greater predilection for C. gattii to infect healthy hosts, the C. gattii genome sequence project revealed extensive chromosomal rearrangements compared with C. neoformans, showing genomic differences between the two Cryptococcus species. We investigated the roles of C. gattii calcineurin in three molecular types: VGIIa (R265), VGIIb (R272), and VGI (WM276). We found that calcineurin exhibits a differential requirement for growth on solid medium at 37°, as calcineurin mutants generated from R265 were more thermotolerant than mutants from R272 and WM276. We demonstrated that tolerance to calcineurin inhibitors (FK506, CsA) at 37° is linked with the VGIIa molecular type. The calcineurin mutants from the R272 background showed the most extensive growth and morphological defects (multivesicle and larger ring-like cells), as well as increased fluconazole susceptibility. Our cellular architecture examination showed that C. gattii and C. neoformans calcineurin mutants exhibit plasma membrane disruptions. Calcineurin in the C. gattii VGII molecular type plays a greater role in controlling cation homeostasis compared with that in C. gattii VGI and C. neoformans H99. Importantly, we demonstrate that C. gattii calcineurin is essential for virulence in a murine inhalation model, supporting C. gattii calcineurin as an attractive antifungal drug target. © 2013 Chen et al.

Authors
Chen, Y-L; Lehman, VN; Lewit, Y; Averette, AF; Heitman, J
MLA Citation
Chen, Y-L, Lehman, VN, Lewit, Y, Averette, AF, and Heitman, J. "Calcineurin governs thermotolerance and virulence of cryptococcus gattii." G3: Genes, Genomes, Genetics 3.3 (2013): 527-539.
PMID
23450261
Source
scival
Published In
G3 (Bethesda, Md.)
Volume
3
Issue
3
Publish Date
2013
Start Page
527
End Page
539
DOI
10.1534/g3.112.004242

Transmission of Hypervirulence Traits via Sexual Reproduction within and between Lineages of the Human Fungal Pathogen Cryptococcus gattii

Since 1999 a lineage of the pathogen Cryptococcus gattii has been infecting humans and other animals in Canada and the Pacific Northwest of the USA. It is now the largest outbreak of a life-threatening fungal infection in a healthy population in recorded history. The high virulence of outbreak strains is closely linked to the ability of the pathogen to undergo rapid mitochondrial tubularisation and proliferation following engulfment by host phagocytes. Most outbreaks spread by geographic expansion across suitable niches, but it is known that genetic re-assortment and hybridisation can also lead to rapid range and host expansion. In the context of C. gattii, however, the likelihood of virulence traits associated with the outbreak lineages spreading to other lineages via genetic exchange is currently unknown. Here we address this question by conducting outgroup crosses between distantly related C. gattii lineages (VGII and VGIII) and ingroup crosses between isolates from the same molecular type (VGII). Systematic phenotypic characterisation shows that virulence traits are transmitted to outgroups infrequently, but readily inherited during ingroup crosses. In addition, we observed higher levels of biparental (as opposed to uniparental) mitochondrial inheritance during VGII ingroup sexual mating in this species and provide evidence for mitochondrial recombination following mating. Taken together, our data suggest that hypervirulence can spread among the C. gattii lineages VGII and VGIII, potentially creating novel hypervirulent genotypes, and that current models of uniparental mitochondrial inheritance in the Cryptococcus genus may not be universal. © 2013 Voelz et al.

Authors
Voelz, K; Ma, H; Phadke, S; Byrnes, EJ; Zhu, P; Mueller, O; Farrer, RA; Henk, DA; Lewit, Y; Hsueh, Y-P; Fisher, MC; Idnurm, A; Heitman, J; May, RC
MLA Citation
Voelz, K, Ma, H, Phadke, S, Byrnes, EJ, Zhu, P, Mueller, O, Farrer, RA, Henk, DA, Lewit, Y, Hsueh, Y-P, Fisher, MC, Idnurm, A, Heitman, J, and May, RC. "Transmission of Hypervirulence Traits via Sexual Reproduction within and between Lineages of the Human Fungal Pathogen Cryptococcus gattii." PLoS Genetics 9.9 (2013).
PMID
24039607
Source
scival
Published In
PLoS genetics
Volume
9
Issue
9
Publish Date
2013
DOI
10.1371/journal.pgen.1003771

Calcineurin Plays Key Roles in the Dimorphic Transition and Virulence of the Human Pathogenic Zygomycete Mucor circinelloides

Many pathogenic fungi are dimorphic and switch between yeast and filamentous states. This switch alters host-microbe interactions and is critical for pathogenicity. However, in zygomycetes, whether dimorphism contributes to virulence is a central unanswered question. The pathogenic zygomycete Mucor circinelloides exhibits hyphal growth in aerobic conditions but switches to multi-budded yeast growth under anaerobic/high CO2 conditions. We found that in the presence of the calcineurin inhibitor FK506, Mucor exhibits exclusively multi-budded yeast growth. We also found that M. circinelloides encodes three calcineurin catalytic A subunits (CnaA, CnaB, and CnaC) and one calcineurin regulatory B subunit (CnbR). Mutations in the latch region of CnbR and in the FKBP12-FK506 binding domain of CnaA result in hyphal growth of Mucor in the presence of FK506. Disruption of the cnbR gene encoding the sole calcineurin B subunit necessary for calcineurin activity yielded mutants locked in permanent yeast phase growth. These findings reveal that the calcineurin pathway plays key roles in the dimorphic transition from yeast to hyphae. The cnbR yeast-locked mutants are less virulent than the wild-type strain in a heterologous host system, providing evidence that hyphae or the yeast-hyphal transition are linked to virulence. Protein kinase A activity (PKA) is elevated during yeast growth under anaerobic conditions, in the presence of FK506, or in the yeast-locked cnbR mutants, suggesting a novel connection between PKA and calcineurin. cnaA mutants lacking the CnaA catalytic subunit are hypersensitive to calcineurin inhibitors, display a hyphal polarity defect, and produce a mixture of yeast and hyphae in aerobic culture. The cnaA mutants also produce spores that are larger than wild-type, and spore size is correlated with virulence potential. Our results demonstrate that the calcineurin pathway orchestrates the yeast-hyphal and spore size dimorphic transitions that contribute to virulence of this common zygomycete fungal pathogen. © 2013 Lee et al.

Authors
Lee, SC; Li, A; Calo, S; Heitman, J
MLA Citation
Lee, SC, Li, A, Calo, S, and Heitman, J. "Calcineurin Plays Key Roles in the Dimorphic Transition and Virulence of the Human Pathogenic Zygomycete Mucor circinelloides." PLoS Pathogens 9.9 (2013).
PMID
24039585
Source
scival
Published In
PLoS pathogens
Volume
9
Issue
9
Publish Date
2013
DOI
10.1371/journal.ppat.1003625

Genetic Circuits that Govern Bisexual and Unisexual Reproduction in Cryptococcus neoformans

Cryptococcus neoformans is a human fungal pathogen with a defined sexual cycle. Nutrient-limiting conditions and pheromones induce a dimorphic transition from unicellular yeast to multicellular hyphae and the production of infectious spores. Sexual reproduction involves cells of either opposite (bisexual) or one (unisexual) mating type. Bisexual and unisexual reproduction are governed by shared components of the conserved pheromone-sensing Cpk1 MAPK signal transduction cascade and by Mat2, the major transcriptional regulator of the pathway. However, the downstream targets of the pathway are largely unknown, and homology-based approaches have failed to yield downstream transcriptional regulators or other targets. In this study, we applied insertional mutagenesis via Agrobacterium tumefaciens transkingdom DNA delivery to identify mutants with unisexual reproduction defects. In addition to elements known to be involved in sexual development (Crg1, Ste7, Mat2, and Znf2), three key regulators of sexual development were identified by our screen: Znf3, Spo11, and Ubc5. Spo11 and Ubc5 promote sporulation during both bisexual and unisexual reproduction. Genetic and phenotypic analyses provide further evidence implicating both genes in the regulation of meiosis. Phenotypic analysis of sexual development showed that Znf3 is required for hyphal development during unisexual reproduction and also plays a central role during bisexual reproduction. Znf3 promotes cell fusion and pheromone production through a pathway parallel to and independent of the pheromone signaling cascade. Surprisingly, Znf3 participates in transposon silencing during unisexual reproduction and may serve as a link between RNAi silencing and sexual development. Our studies illustrate the power of unbiased genetic screens to reveal both novel and conserved circuits that operate sexual reproduction. © 2013 Feretzaki and Heitman.

Authors
Feretzaki, M; Heitman, J
MLA Citation
Feretzaki, M, and Heitman, J. "Genetic Circuits that Govern Bisexual and Unisexual Reproduction in Cryptococcus neoformans." PLoS Genetics 9.8 (2013).
PMID
23966871
Source
scival
Published In
PLoS genetics
Volume
9
Issue
8
Publish Date
2013
DOI
10.1371/journal.pgen.1003688

Surfactant protein D facilitates Cryptococcus neoformans infection.

Concurrent with the global escalation of the AIDS pandemic, cryptococcal infections are increasing and are of significant medical importance. Furthermore, Cryptococcus neoformans has become a primary human pathogen, causing infection in seemingly healthy individuals. Although numerous studies have elucidated the virulence properties of C. neoformans, less is understood regarding lung host immune factors during early stages of fungal infection. Based on our previous studies documenting that pulmonary surfactant protein D (SP-D) protects C. neoformans cells against macrophage-mediated defense mechanisms in vitro (S. Geunes-Boyer et al., Infect. Immun. 77:2783-2794, 2009), we postulated that SP-D would facilitate fungal infection in vivo. To test this hypothesis, we examined the role of SP-D in response to C. neoformans using SP-D⁻/⁻ mice. Here, we demonstrate that mice lacking SP-D were partially protected during C. neoformans infection; they displayed a longer mean time to death and decreased fungal burden at several time points postinfection than wild-type mice. This effect was reversed by the administration of exogenous SP-D. Furthermore, we show that SP-D bound to the surface of the yeast cells and protected the pathogenic microbes against macrophage-mediated defense mechanisms and hydrogen peroxide (H₂O₂)-induced oxidative stress in vitro and in vivo. These findings indicate that C. neoformans is capable of coopting host SP-D to increase host susceptibility to the yeast. This study establishes a new paradigm for the role played by SP-D during host responses to C. neoformans and consequently imparts insight into potential future preventive and/or treatment strategies for cryptococcosis.

Authors
Geunes-Boyer, S; Beers, MF; Perfect, JR; Heitman, J; Wright, JR
MLA Citation
Geunes-Boyer, S, Beers, MF, Perfect, JR, Heitman, J, and Wright, JR. "Surfactant protein D facilitates Cryptococcus neoformans infection." Infect Immun 80.7 (July 2012): 2444-2453.
PMID
22547543
Source
pubmed
Published In
Infection and immunity
Volume
80
Issue
7
Publish Date
2012
Start Page
2444
End Page
2453
DOI
10.1128/IAI.05613-11

Convergent Evolution of Calcineurin Pathway Roles in Thermotolerance and Virulence in Candida glabrata

Authors
Chen, Y-L; Konieczka, JH; Springer, DJ; Bowen, SE; Zhang, J; Silao, FGS; Bungay, AAC; Bigol, UG; Nicolas, MG; Abraham, SN; Thompson, DA; Regev, A; Heitman, J
MLA Citation
Chen, Y-L, Konieczka, JH, Springer, DJ, Bowen, SE, Zhang, J, Silao, FGS, Bungay, AAC, Bigol, UG, Nicolas, MG, Abraham, SN, Thompson, DA, Regev, A, and Heitman, J. "Convergent Evolution of Calcineurin Pathway Roles in Thermotolerance and Virulence in Candida glabrata." G3-GENES GENOMES GENETICS 2.6 (June 1, 2012): 675-691.
PMID
22690377
Source
wos-lite
Published In
G3 (Bethesda, Md.)
Volume
2
Issue
6
Publish Date
2012
Start Page
675
End Page
691
DOI
10.1534/g3.112.002279

Structure, function, and evolution of mating type loci

Authors
Heitman, J
MLA Citation
Heitman, J. "Structure, function, and evolution of mating type loci." MYCOSES 55 (June 2012): 15-16.
Source
wos-lite
Published In
Mycoses
Volume
55
Publish Date
2012
Start Page
15
End Page
16

Discussion Round: Ethics in scientific publishing Aim high in navigating the journal cascade: Opportunities and challenges

Authors
Heitman, J
MLA Citation
Heitman, J. "Discussion Round: Ethics in scientific publishing Aim high in navigating the journal cascade: Opportunities and challenges." MYCOSES 55 (June 2012): 55-55.
Source
wos-lite
Published In
Mycoses
Volume
55
Publish Date
2012
Start Page
55
End Page
55

Sexual reproduction and virulence of Cryptococcus

Authors
Heitman, J
MLA Citation
Heitman, J. "Sexual reproduction and virulence of Cryptococcus." MYCOSES 55 (June 2012): 22-22.
Source
wos-lite
Published In
Mycoses
Volume
55
Publish Date
2012
Start Page
22
End Page
22

Know your enemy: how to build and vanquish a global fungal scourge.

The 8th International Conference on Cryptococcus and Cryptococcosis, chaired by Maurizio Del Poeta (Medical University of South Carolina), and organized together with June Kwon-Chung (National Institute of Allergy and Infectious Diseases), Stuart Levitz (University of Massachusetts Medical School), and John Perfect (Duke University), occurred in May 2011. This meeting brought together the world's leading researchers on Cryptococcus and cryptococcosis, including basic scientists, epidemiologists, and clinicians, to discuss new developments in Cryptococcus biology. With more than 60 oral presentations and 180 posters, this meeting enhanced our understanding of pathogenicity of Cryptococcus and served as a robust forum that facilitated cross-disciplinary discussions, research, and clinical collaborations. Due to space constraints, this brief overview highlights only a few of the topics discussed in this meeting, focusing on the evolution of virulence, host and pathogen interactions, fungal and host signaling, new advances of genomics studies on Cryptococcus, and the current status of the outbreak caused by C. gattii. The 8th International Conference on Cryptococcus and Cryptococcosis brought together scientists from across the globe in the beautiful historical downtown setting of Charleston to share their latest findings and highlight advances in Cryptococcus research. With more than 250 participants, this meeting was the largest gathering of the Cryptococcus international community in the 24-year history. Here, we review the advances presented and the current state of knowledge in the field.

Authors
Wang, X; Li, W; Sun, S; Kozubowski, L; Lee, SC; Feretzaki, M; Heitman, J
MLA Citation
Wang, X, Li, W, Sun, S, Kozubowski, L, Lee, SC, Feretzaki, M, and Heitman, J. "Know your enemy: how to build and vanquish a global fungal scourge." Mycopathologia 173.5-6 (June 2012): 295-301.
PMID
21997858
Source
pubmed
Published In
Mycopathologia
Volume
173
Issue
5-6
Publish Date
2012
Start Page
295
End Page
301
DOI
10.1007/s11046-011-9484-9

The basidiomycete Malassezia species complex - a genomic platform to elucidate host-fungal interactions involved in common skin disorders

Authors
Gioti, A; Nysted, B; Li, W; Andersson, A; Lundeberg, J; Boekhout, T; Heitman, J; Scheynius, A
MLA Citation
Gioti, A, Nysted, B, Li, W, Andersson, A, Lundeberg, J, Boekhout, T, Heitman, J, and Scheynius, A. "The basidiomycete Malassezia species complex - a genomic platform to elucidate host-fungal interactions involved in common skin disorders." MYCOSES 55 (June 2012): 131-131.
Source
wos-lite
Published In
Mycoses
Volume
55
Publish Date
2012
Start Page
131
End Page
131

Cryptococcus gattii, no longer an accidental pathogen?

Cryptococcus gattii is an environmentally occurring pathogen that is responsible for causing cryptococcosis marked by pneumonia and meningoencephalitis in humans and animals. C. Gattii can form long-term associations with trees and soil resulting in the production of infectious propagules (spores and desiccated yeast). The ever-expanding number of reports of clinical and environmental isolation of C. Gattii in temperate climates strongly imply that C. Gattii occurs worldwide. The key ability of yeast and spores to enter, survive, multiply, and exit host cells, and to infect immunocompetent hosts distinguishes C. Gattii as a primary pathogen and suggests evolution of C. Gattii pathogenesis as a result of interaction with plants and other organisms in its environmental niche. Here we summarize the historical literature on C. Gattii and recent literature supporting the worldwide occurrence of the primary pathogen C. Gattii. © 2012 Springer Science+Business Media New York.

Authors
Springer, DJ; Phadke, S; Billmyre, RB; Heitman, J
MLA Citation
Springer, DJ, Phadke, S, Billmyre, RB, and Heitman, J. "Cryptococcus gattii, no longer an accidental pathogen?." Current Fungal Infection Reports 6.4 (2012): 245-256.
PMID
23243480
Source
scival
Published In
Current Fungal Infection Reports
Volume
6
Issue
4
Publish Date
2012
Start Page
245
End Page
256
DOI
10.1007/s12281-012-0111-0

Should Y stay or should Y go: The evolution of non-recombining sex chromosomes

Gradual degradation seems inevitable for non-recombining sex chromosomes. This has been supported by the observation of degenerated non-recombining sex chromosomes in a variety of species. The human Y chromosome has also degenerated significantly during its evolution, and theories have been advanced that the Y chromosome could disappear within the next ~5 million years, if the degeneration rate it has experienced continues. However, recent studies suggest that this is unlikely. Conservative evolutionary forces such as strong purifying selection and intrachromosomal repair through gene conversion balance the degeneration tendency of the Y chromosome and maintain its integrity after an initial period of faster degeneration. We discuss the evidence both for and against the extinction of the Y chromosome. We also discuss potential insights gained on the evolution of sex-determining chromosomes by studying simpler sex-determining chromosomal regions of unicellular and multicellular microorganisms. © 2012 WILEY Periodicals, Inc..

Authors
Sun, S; Heitman, J
MLA Citation
Sun, S, and Heitman, J. "Should Y stay or should Y go: The evolution of non-recombining sex chromosomes." BioEssays 34.11 (2012): 938-942.
PMID
22948853
Source
scival
Published In
Bioessays
Volume
34
Issue
11
Publish Date
2012
Start Page
938
End Page
942
DOI
10.1002/bies.201200064

Rapamycin exerts antifungal activity in vitro and in vivo against mucor circinelloides via FKBP12-dependent inhibition of tor

The zygomycete Mucor circinelloides is an opportunistic fungal pathogen that commonly infects patients with malignancies, diabetes mellitus, and solid organ transplants. Despite the widespread use of antifungal therapy in the management of zygomycosis, the incidence of infections continues to rise among immunocompromised individuals. In this study, we established that the target and mechanism of antifungal action of the immunosuppressant rapamycin in M. circinelloides are mediated via conserved complexes with FKBP12 and a Tor homolog. We found that spontaneous mutations that disrupted conserved residues in FKBP12 conferred rapamycin and FK506 resistance. Disruption of the FKBP12-encoding gene, fkbA, also conferred rapamycin and FK506 resistance. Expression of M. circinelloides FKBP12 (McFKBP12) complemented a Saccharomyces cerevisiae mutant strain lacking FKBP12 to restore rapamycin sensitivity. Expression of the McTor FKBP12-rapamycin binding (FRB) domain conferred rapamycin resistance in S. cerevisiae, and McFKBP12 interacted in a rapamycin-dependent fashion with the McTor FRB domain in a yeast two-hybrid assay, validating McFKBP12 and McTor as conserved targets of rapamycin. We showed that in vitro, rapamycin exhibited potent growth inhibitory activity against M. circinelloides. In a Galleria mellonella model of systemic mucormycosis, rapamycin improved survival by 50%, suggesting that rapamycin and nonimmunosuppressive analogs have the potential to be developed as novel antifungal therapies for treatment of patients with mucormycosis. © 2012, American Society for Microbiology.

Authors
Bastidas, RJ; Shertz, CA; Lee, SC; Heitman, J; Cardenas, ME
MLA Citation
Bastidas, RJ, Shertz, CA, Lee, SC, Heitman, J, and Cardenas, ME. "Rapamycin exerts antifungal activity in vitro and in vivo against mucor circinelloides via FKBP12-dependent inhibition of tor." Eukaryotic Cell 11.3 (2012): 270-281.
PMID
22210828
Source
scival
Published In
Eukaryotic cell
Volume
11
Issue
3
Publish Date
2012
Start Page
270
End Page
281
DOI
10.1128/EC.05284-11

Discovery of a modified tetrapolar sexual cycle in Cryptococcus amylolentus and the evolution of MAT in the Cryptococcus species complex

Sexual reproduction in fungi is governed by a specialized genomic region called the mating-type locus (MAT). The human fungal pathogenic and basidiomycetous yeast Cryptococcus neoformans has evolved a bipolar mating system (a, α) in which the MAT locus is unusually large (>100 kb) and encodes >20 genes including homeodomain (HD) and pheromone/receptor (P/R) genes. To understand how this unique bipolar mating system evolved, we investigated MAT in the closely related species Tsuchiyaea wingfieldii and Cryptococcus amylolentus and discovered two physically unlinked loci encoding the HD and P/R genes. Interestingly, the HD (B) locus sex-specific region is restricted (~2 kb) and encodes two linked and divergently oriented homeodomain genes in contrast to the solo HD genes (SXI1α, SXI2a) of C. neoformans and Cryptococcus gattii. The P/R (A) locus contains the pheromone and pheromone receptor genes but has expanded considerably compared to other outgroup species (Cryptococcus heveanensis) and is linked to many of the genes also found in the MAT locus of the pathogenic Cryptococcus species. Our discovery of a heterothallic sexual cycle for C. amylolentus allowed us to establish the biological roles of the sex-determining regions. Matings between two strains of opposite mating-types (A1B1×A2B2) produced dikaryotic hyphae with fused clamp connections, basidia, and basidiospores. Genotyping progeny using markers linked and unlinked to MAT revealed that meiosis and uniparental mitochondrial inheritance occur during the sexual cycle of C. amylolentus. The sexual cycle is tetrapolar and produces fertile progeny of four mating-types (A1B1, A1B2, A2B1, and A2B2), but a high proportion of progeny are infertile, and fertility is biased towards one parental mating-type (A1B1). Our studies reveal insights into the plasticity and transitions in both mechanisms of sex determination (bipolar versus tetrapolar) and sexual reproduction (outcrossing versus inbreeding) with implications for similar evolutionary transitions and processes in fungi, plants, and animals.

Authors
Findley, K; Sun, S; Fraser, JA; Hsueh, Y-P; Averette, AF; Li, W; Dietrich, FS; Heitman, J
MLA Citation
Findley, K, Sun, S, Fraser, JA, Hsueh, Y-P, Averette, AF, Li, W, Dietrich, FS, and Heitman, J. "Discovery of a modified tetrapolar sexual cycle in Cryptococcus amylolentus and the evolution of MAT in the Cryptococcus species complex." PLoS Genetics 8.2 (2012).
PMID
22359516
Source
scival
Published In
PLoS genetics
Volume
8
Issue
2
Publish Date
2012
DOI
10.1371/journal.pgen.1002528

Pleiotropic roles of the msi1-like protein msl1 in Cryptococcus neoformans

Msi1-like (MSIL) proteins contain WD40 motifs and have a pleiotropic cellular function as negative regulators of the Ras/cyclic AMP (cAMP) pathway and components of chromatin assembly factor 1 (CAF-1), yet they have not been studied in fungal pathogens. Here we identified and characterized an MSIL protein, Msl1, in Cryptococcus neoformans, which causes life-threatening meningoencephalitis in humans. Notably, Msl1 plays pleiotropic roles in C. neoformans in both cAMP-dependent and -independent manners largely independent of Ras. Msl1 negatively controls antioxidant melanin production and sexual differentiation, and this was repressed by the inhibition of the cAMP-signaling pathway. In contrast, Msl1 controls thermotolerance, diverse stress responses, and antifungal drug resistance in a Ras/cAMP-independent manner. Cac2, which is the second CAF-1 component, appears to play both redundant and distinct functions compared to the functions of Msl1. Msl1 is required for the full virulence of C. neoformans. Transcriptome analysis identified a group of Msl1-regulated genes, which include stress-related genes such as HSP12 and HSP78. In conclusion, this study demonstrates pleiotropic roles of Msl1 in the human fungal pathogen C. neoformans, providing insight into a potential novel antifungal therapeutic target. © 2012, American Society for Microbiology. All Rights Reserved.

Authors
Yang, D-H; Maeng, S; Strain, AK; Floyd, A; Nielsen, K; Heitman, J; Bahn, Y-S
MLA Citation
Yang, D-H, Maeng, S, Strain, AK, Floyd, A, Nielsen, K, Heitman, J, and Bahn, Y-S. "Pleiotropic roles of the msi1-like protein msl1 in Cryptococcus neoformans." Eukaryotic Cell 11.12 (2012): 1482-1495.
PMID
23042129
Source
scival
Published In
Eukaryotic cell
Volume
11
Issue
12
Publish Date
2012
Start Page
1482
End Page
1495
DOI
10.1128/EC.00261-12

Calcineurin Is Required for Pseudohyphal Growth, Virulence, and Drug Resistance in Candida lusitaniae

Candida lusitaniae is an emerging fungal pathogen that infects immunocompromised patients including HIV/AIDS, cancer, and neonatal pediatric patients. Though less prevalent than other Candida species, C. lusitaniae is unique in its ability to develop resistance to amphotericin B. We investigated the role of the calcium-activated protein phosphatase calcineurin in several virulence attributes of C. lusitaniae including pseudohyphal growth, serum survival, and growth at 37°C. We found that calcineurin and Crz1, a C. albicans Crz1 homolog acting as a downstream target of calcineurin, are required for C. lusitaniae pseudohyphal growth, a process for which the underlying mechanism remains largely unknown in C. lusitaniae but hyphal growth is fundamental to C. albicans virulence. We demonstrate that calcineurin is required for cell wall integrity, ER stress response, optimal growth in serum, virulence in a murine systemic infection model, and antifungal drug tolerance in C. lusitaniae. To further examine the potential of targeting the calcineurin signaling cascade for antifungal drug development, we examined the activity of a calcineurin inhibitor FK506 in combination with caspofungin against echinocandin resistant C. lusitaniae clinical isolates. Broth microdilution and drug disk diffusion assays demonstrate that FK506 has synergistic fungicidal activity with caspofungin against echinocandin resistant isolates. Our findings reveal that pseudohyphal growth is controlled by the calcineurin signaling cascade, and highlight the potential use of calcineurin inhibitors and caspofungin for emerging drug-resistant C. lusitaniae infections. © 2012 Zhang et al.

Authors
Zhang, J; Silao, FGS; Bigol, UG; Bungay, AAC; Nicolas, MG; Heitman, J; Chen, Y-L
MLA Citation
Zhang, J, Silao, FGS, Bigol, UG, Bungay, AAC, Nicolas, MG, Heitman, J, and Chen, Y-L. "Calcineurin Is Required for Pseudohyphal Growth, Virulence, and Drug Resistance in Candida lusitaniae." PLoS ONE 7.8 (2012).
PMID
22952924
Source
scival
Published In
PloS one
Volume
7
Issue
8
Publish Date
2012
DOI
10.1371/journal.pone.0044192

Calcofluor white combination antifungal treatments for Trichophyton rubrum and Candida albicans.

Superficial mycoses caused by dermatophyte fungi are among the most common infections worldwide, yet treatment is restricted by limited effective drugs available, drug toxicity, and emergence of drug resistance. The stilbene fluorescent brightener calcofluor white (CFW) inhibits fungi by binding chitin in the cell wall, disrupting cell wall integrity, and thus entails a different mechanism of inhibition than currently available antifungal drugs. To identify novel therapeutic options for the treatment of skin infections, we compared the sensitivity of representative strains of the dermatophyte Trichophyton rubrum and Candida albicans to CFW and a panel of fluorescent brighteners and phytoalexin compounds. We identified the structurally related stilbene fluorescent brighteners 71, 85, 113 and 134 as fungicidal to both T. rubrum and C. albicans to a similar degree as CFW, and the stilbene phytoalexins pinosylvan monomethyl ether and pterostilbene inhibited to a lesser degree, allowing us to develop a structure-activity relationship for fungal inhibition. Given the abilities of CFW to absorb UV(365 nm) and bind specifically to fungal cell walls, we tested whether CFW combined with UV(365 nm) irradiation would be synergistic to fungi and provide a novel photodynamic treatment option. However, while both treatments individually were cytocidal, UV(365 nm) irradiation reduced sensitivity to CFW, which we attribute to CFW photoinactivation. We also tested combination treatments of CFW with other fungal inhibitors and identified synergistic interactions between CFW and some ergosterol biosynthesis inhibitors in C. albicans. Therefore, our studies identify novel fungal inhibitors and drug interactions, offering promise for combination topical treatment regimes for superficial mycoses.

Authors
Kingsbury, JM; Heitman, J; Pinnell, SR
MLA Citation
Kingsbury, JM, Heitman, J, and Pinnell, SR. "Calcofluor white combination antifungal treatments for Trichophyton rubrum and Candida albicans." PLoS One 7.7 (2012): e39405-.
PMID
22792174
Source
pubmed
Published In
PloS one
Volume
7
Issue
7
Publish Date
2012
Start Page
e39405
DOI
10.1371/journal.pone.0039405

Transgene induced co-suppression during vegetative growth in Cryptococcus neoformans.

Introduction of DNA sequences into the genome often results in homology-dependent gene silencing in organisms as diverse as plants, fungi, flies, nematodes, and mammals. We previously showed in Cryptococcus neoformans that a repeat transgene array can induce gene silencing at a high frequency during mating (∼50%), but at a much lower frequency during vegetative growth (∼0.2%). Here we report a robust asexual co-suppression phenomenon triggered by the introduction of a cpa1::ADE2 transgene. Multiple copies of the cpa1::ADE2 transgene were ectopically integrated into the genome, leading to silencing of the endogenous CPA1 and CPA2 genes encoding the cyclosporine A target protein cyclophilin A. Given that CPA1-derived antisense siRNAs were detected in the silenced isolates, and that RNAi components (Rdp1, Ago1, and Dcr2) are required for silencing, we hypothesize that an RNAi pathway is involved, in which siRNAs function as trans factors to silence both the CPA1 and the CPA2 genes. The silencing efficiency of the CPA1 and CPA2 genes is correlated with the transgene copy number and reached ∼90% in the presence of >25 copies of the transgene. We term this transgene silencing phenomenon asexual co-suppression to distinguish it from the related sex-induced silencing (SIS) process. We further show that replication protein A (RPA), a single-stranded DNA binding complex, is required for transgene silencing, suggesting that RPA might play a similar role in aberrant RNA production as observed for quelling in Neurospora crassa. Interestingly, we also observed that silencing of the ADE2 gene occurred at a much lower frequency than the CPA1/2 genes even though it is present in the same transgene array, suggesting that factors in addition to copy number influence silencing. Taken together, our results illustrate that a transgene induced co-suppression process operates during C. neoformans vegetative growth that shares mechanistic features with quelling.

Authors
Wang, X; Wang, P; Sun, S; Darwiche, S; Idnurm, A; Heitman, J
MLA Citation
Wang, X, Wang, P, Sun, S, Darwiche, S, Idnurm, A, and Heitman, J. "Transgene induced co-suppression during vegetative growth in Cryptococcus neoformans." PLoS Genet 8.8 (2012): e1002885-.
PMID
22916030
Source
pubmed
Published In
PLoS genetics
Volume
8
Issue
8
Publish Date
2012
Start Page
e1002885
DOI
10.1371/journal.pgen.1002885

Gene conversion occurs within the mating-type locus of Cryptococcus neoformans during sexual reproduction

Meiotic recombination of sex chromosomes is thought to be repressed in organisms with heterogametic sex determination (e.g. mammalian X/Y chromosomes), due to extensive divergence and chromosomal rearrangements between the two chromosomes. However, proper segregation of sex chromosomes during meiosis requires crossing-over occurring within the pseudoautosomal regions (PAR). Recent studies reveal that recombination, in the form of gene conversion, is widely distributed within and may have played important roles in the evolution of some chromosomal regions within which recombination was thought to be repressed, such as the centromere cores of maize. Cryptococcus neoformans, a major human pathogenic fungus, has an unusually large mating-type locus (MAT, >100 kb), and the MAT alleles from the two opposite mating-types show extensive nucleotide sequence divergence and chromosomal rearrangements, mirroring characteristics of sex chromosomes. Meiotic recombination was assumed to be repressed within the C. neoformans MAT locus. A previous study identified recombination hot spots flanking the C. neoformans MAT, and these hot spots are associated with high GC content. Here, we investigated a GC-rich intergenic region located within the MAT locus of C. neoformans to establish if this region also exhibits unique recombination behavior during meiosis. Population genetics analysis of natural C. neoformans isolates revealed signals of homogenization spanning this GC-rich intergenic region within different C. neoformans lineages, consistent with a model in which gene conversion of this region during meiosis prevents it from diversifying within each lineage. By analyzing meiotic progeny from laboratory crosses, we found that meiotic recombination (gene conversion) occurs around the GC-rich intergenic region at a frequency equal to or greater than the meiotic recombination frequency observed in other genomic regions. We discuss the implications of these findings with regards to the possible functional and evolutionary importance of gene conversion within the C. neoformans MAT locus and, more generally, in fungi. © 2012 Sun et al.

Authors
Sun, S; Hsueh, Y-P; Heitman, J
MLA Citation
Sun, S, Hsueh, Y-P, and Heitman, J. "Gene conversion occurs within the mating-type locus of Cryptococcus neoformans during sexual reproduction." PLoS Genetics 8.7 (2012).
PMID
22792079
Source
scival
Published In
PLoS genetics
Volume
8
Issue
7
Publish Date
2012
DOI
10.1371/journal.pgen.1002810

Malassezia fungi are specialized to live on skin and associated with dandruff, eczema, and other skin diseases

Authors
Saunders, CW; Scheynius, A; Heitman, J
MLA Citation
Saunders, CW, Scheynius, A, and Heitman, J. "Malassezia fungi are specialized to live on skin and associated with dandruff, eczema, and other skin diseases." PLoS Pathogens 8.6 (2012).
PMID
22737067
Source
scival
Published In
PLoS pathogens
Volume
8
Issue
6
Publish Date
2012
DOI
10.1371/journal.ppat.1002701

The paleozoic origin of enzymatic lignin decomposition reconstructed from 31 fungal genomes

Wood is a major pool of organic carbon that is highly resistant to decay, owing largely to the presence of lignin. The only organisms capable of substantial lignin decay are white rot fungi in the Agaricomycetes, which also contains non-lignin-degrading brown rot and ectomycorrhizal species. Comparative analyses of 31 fungal genomes (12 generated for this study) suggest that lignin-degrading peroxidases expanded in the lineage leading to the ancestor of the Agaricomycetes, which is reconstructed as a white rot species, and then contracted in parallel lineages leading to brown rot and mycorrhizal species. Molecular clock analyses suggest that the origin of lignin degradation might have coincided with the sharp decrease in the rate of organic carbon burial around the end of the Carboniferous period.

Authors
Floudas, D; Binder, M; Riley, R; Barry, K; Blanchette, RA; Henrissat, B; Martínez, AT; Otillar, R; Spatafora, JW; Yadav, JS; Aerts, A; Benoit, I; Boyd, A; Carlson, A; Copeland, A; Coutinho, PM; Vries, RPD; Ferreira, P; Findley, K; Foster, B; Gaskell, J; Glotzer, D; Górecki, P; Heitman, J; Hesse, C; Hori, C; Igarashi, K; Jurgens, JA; Kallen, N; Kersten, P; Kohler, A; Kües, U; Kumar, TKA; Kuo, A; LaButti, K; Larrondo, LF; Lindquist, E; Ling, A; Lombard, V; Lucas, S; Lundell, T; Martin, R et al.
MLA Citation
Floudas, D, Binder, M, Riley, R, Barry, K, Blanchette, RA, Henrissat, B, Martínez, AT, Otillar, R, Spatafora, JW, Yadav, JS, Aerts, A, Benoit, I, Boyd, A, Carlson, A, Copeland, A, Coutinho, PM, Vries, RPD, Ferreira, P, Findley, K, Foster, B, Gaskell, J, Glotzer, D, Górecki, P, Heitman, J, Hesse, C, Hori, C, Igarashi, K, Jurgens, JA, Kallen, N, Kersten, P, Kohler, A, Kües, U, Kumar, TKA, Kuo, A, LaButti, K, Larrondo, LF, Lindquist, E, Ling, A, Lombard, V, Lucas, S, Lundell, T, and Martin, R et al. "The paleozoic origin of enzymatic lignin decomposition reconstructed from 31 fungal genomes." Science 336.6089 (2012): 1715-1719.
PMID
22745431
Source
scival
Published In
Science
Volume
336
Issue
6089
Publish Date
2012
Start Page
1715
End Page
1719
DOI
10.1126/science.1221748

Function of Cryptococcus neoformans KAR7 (SEC66) in karyogamy during unisexual and opposite-sex mating

The human basidiomycetous fungal pathogen Cryptococcus neoformans serves as a model fungus to study sexual development and produces infectious propagules, basidiospores, via the sexual cycle. Karyogamy is the process of nuclear fusion and an essential step to complete mating. Therefore, regulation of nuclear fusion is central to understanding sexual development of C. neoformans. However, our knowledge of karyogamy genes was limited. In this study, using a BLAST search with the Saccharomyces cerevisiae KAR genes, we identified five C. neoformans karyogamy gene orthologs: CnKAR2, CnKAR3, CnKAR4, CnKAR7 (or CnSEC66), and CnKAR8. There are no apparent orthologs of the S. cerevisiae genes ScKAR1, ScKAR5, and ScKar9 in C. neoformans. Karyogamy involves the congression of two nuclei followed by nuclear membrane fusion, which results in diploidization. ScKar7 (or ScSec66) is known to be involved in nuclear membrane fusion. In C. neoformans, kar7 mutants display significant defects in hyphal growth and basidiospore chain formation during both a-α opposite and α-α unisexual reproduction. Fluorescent nuclear imaging revealed that during kar7 × kar7 bilateral mutant matings, the nuclei congress but fail to fuse in the basidia. These results demonstrate that the KAR7 gene plays an integral role in both opposite-sex and unisexual mating, indicating that proper control of nuclear dynamics is important. CnKAR2 was found to be essential for viability, and its function in mating is not known. No apparent phenotypes were observed during mating of kar3, kar4, or kar8 mutants, suggesting that the role of these genes may be dispensable for C. neoformans mating, which demonstrates a different evolutionary trajectory for the KAR genes in C. neoformans compared to those in S. cerevisiae. © 2012, American Society for Microbiology. All Rights Reserved.

Authors
Lee, SC; Heitman, J
MLA Citation
Lee, SC, and Heitman, J. "Function of Cryptococcus neoformans KAR7 (SEC66) in karyogamy during unisexual and opposite-sex mating." Eukaryotic Cell 11.6 (2012): 783-794.
PMID
22544906
Source
scival
Published In
Eukaryotic cell
Volume
11
Issue
6
Publish Date
2012
Start Page
783
End Page
794
DOI
10.1128/EC.00066-12

Global analysis of the evolution and mechanism of echinocandin resistance in Candida glabrata

The evolution of drug resistance has a profound impact on human health. Candida glabrata is a leading human fungal pathogen that can rapidly evolve resistance to echinocandins, which target cell wall biosynthesis and are front-line therapeutics for Candida infections. Here, we provide the first global analysis of mutations accompanying the evolution of fungal drug resistance in a human host utilizing a series of C. glabrata isolates that evolved echinocandin resistance in a patient treated with the echinocandin caspofungin for recurring bloodstream candidemia. Whole genome sequencing identified a mutation in the drug target, FKS2, accompanying a major resistance increase, and 8 additional non-synonymous mutations. The FKS2-T1987C mutation was sufficient for echinocandin resistance, and associated with a fitness cost that was mitigated with further evolution, observed in vitro and in a murine model of systemic candidemia. A CDC6-A511G(K171E) mutation acquired before FKS2-T1987C(S663P), conferred a small resistance increase. Elevated dosage of CDC55, which acquired a C463T(P155S) mutation after FKS2-T1987C(S663P), ameliorated fitness. To discover strategies to abrogate echinocandin resistance, we focused on the molecular chaperone Hsp90 and downstream effector calcineurin. Genetic or pharmacological compromise of Hsp90 or calcineurin function reduced basal tolerance and resistance. Hsp90 and calcineurin were required for caspofungin-dependent FKS2 induction, providing a mechanism governing echinocandin resistance. A mitochondrial respiration-defective petite mutant in the series revealed that the petite phenotype does not confer echinocandin resistance, but renders strains refractory to synergy between echinocandins and Hsp90 or calcineurin inhibitors. The kidneys of mice infected with the petite mutant were sterile, while those infected with the HSP90-repressible strain had reduced fungal burden. We provide the first global view of mutations accompanying the evolution of fungal drug resistance in a human host, implicate the premier compensatory mutation mitigating the cost of echinocandin resistance, and suggest a new mechanism of echinocandin resistance with broad therapeutic potential. © 2012 Singh-Babak et al.

Authors
Singh-Babak, SD; Babak, T; Diezmann, S; Hill, JA; Xie, JL; Chen, Y-L; Poutanen, SM; Rennie, RP; Heitman, J; Cowen, LE
MLA Citation
Singh-Babak, SD, Babak, T, Diezmann, S, Hill, JA, Xie, JL, Chen, Y-L, Poutanen, SM, Rennie, RP, Heitman, J, and Cowen, LE. "Global analysis of the evolution and mechanism of echinocandin resistance in Candida glabrata." PLoS Pathogens 8.5 (2012).
PMID
22615574
Source
scival
Published In
PLoS pathogens
Volume
8
Issue
5
Publish Date
2012
DOI
10.1371/journal.ppat.1002718

A unique chromosomal rearrangement in the Cryptococcus neoformans var. grubii type strain enhances key phenotypes associated with virulence

The accumulation of genomic structural variation between closely related populations over time can lead to reproductive isolation and speciation. The fungal pathogen Cryptococcus is thought to have recently diversified, forming a species complex containing members with distinct morphologies, distributions, and pathologies of infection. We have investigated structural changes in genomic architecture such as inversions and translocations that distinguish the most pathogenic variety, Cryptococcus neoformans var. grubii, from the less clinically prevalent Cryptococcus neoformans var. neoformans and Cryptococcus gattii. Synteny analysis between the genomes of the three Cryptococcus species/varieties (strains H99, JEC21, and R265) reveals that C. neoformans var. grubii possesses surprisingly few unique genomic rearrangements. All but one are relatively small and are shared by all molecular subtypes of C. neoformans var. grubii. In contrast, the large translocation peculiar to the C. neoformans var. grubii type strain is found in all tested subcultures from multiple laboratories, suggesting that it has possessed this rearrangement since its isolation from a human clinical sample. Furthermore, we find that the translocation directly disrupts two genes. The first of these encodes a novel protein involved in metabolism of glucose at human body temperature and affects intracellular levels of trehalose. The second encodes a homeodomain-containing transcription factor that modulates melanin production. Both mutations would be predicted to increase pathogenicity; however, when recreated in an alternate genetic background, these mutations do not affect virulence in animal models. The type strain of C. neoformans var. grubii in which the majority of molecular studies have been performed is therefore atypical for carbon metabolism and key virulence attributes. © 2012 Morrow et al.

Authors
Morrow, CA; Lee, R; Chow, EWL; Ormerod, KL; Goldinger, A; Byrnes, EJ; Nielsen, K; Heitman, J; Schirra, HJ; Fraser, JA
MLA Citation
Morrow, CA, Lee, R, Chow, EWL, Ormerod, KL, Goldinger, A, Byrnes, EJ, Nielsen, K, Heitman, J, Schirra, HJ, and Fraser, JA. "A unique chromosomal rearrangement in the Cryptococcus neoformans var. grubii type strain enhances key phenotypes associated with virulence." mBio 3.2 (2012).
PMID
22375073
Source
scival
Published In
mBio
Volume
3
Issue
2
Publish Date
2012
DOI
10.1128/mBio.00310-11

Pseudohyphal growth of Cryptococcus neoformans is a reversible dimorphic transition in response to ammonium that requires Amt1 and Amt2 ammonium permeases

Cryptococcus neoformans is a human-pathogenic basidiomycete that commonly infects HIV/AIDS patients to cause meningoencephalitis (7, 19). C. neoformans grows as a budding yeast during vegetative growth or as hyphae during sexual reproduction. Pseudohyphal growth of C. neoformans has been observed rarely during murine and human infections but frequently during coculture with amoeba; however, the genetics underlying pseudohyphal growth are largely unknown. Our studies found that C. neoformans displays pseudohyphal growth under nitrogen-limiting conditions, especially when a small amount of ammonium is available as a sole nitrogen source. Pseudohyphal growth was observed with Cryptococcus neoformans serotypes A and D and Cryptococcus gattii. C. neoformans pseudohyphae bud to produce yeast cells and normal smooth hemispherical colonies when transferred to complete media, indicating that pseudohyphal growth is a conditional developmental stage. Subsequent analysis revealed that two ammonium permeases encoded by the AMT1 and AMT2 genes are required for pseudohyphal growth. Both amt1 and amt2 mutants are capable of forming pseudohyphae; however, amt1 amt2 double mutants do not form pseudohyphae. Interestingly, C. gattii pseudohypha formation is irreversible and involves a RAM pathway mutation that drives pseudohyphal development. We also found that pseudohyphal growth is related to the invasive growth into the medium. These results demonstrate that pseudohyphal growth is a common reversible growth pattern in C. neoformans but a mutational genetic event in C. gattii and provide new insights into understanding pseudohyphal growth of Cryptococcus. © 2012, American Society for Microbiology. All Rights Reserved.

Authors
Lee, SC; Phadke, S; Sun, S; Heitman, J
MLA Citation
Lee, SC, Phadke, S, Sun, S, and Heitman, J. "Pseudohyphal growth of Cryptococcus neoformans is a reversible dimorphic transition in response to ammonium that requires Amt1 and Amt2 ammonium permeases." Eukaryotic Cell 11.11 (2012): 1391-1398.
PMID
23002105
Source
scival
Published In
Eukaryotic cell
Volume
11
Issue
11
Publish Date
2012
Start Page
1391
End Page
1398
DOI
10.1128/EC.00242-12

Cryptococcus gattii, No Longer an Accidental Pathogen?

Cryptococcus gattii is an environmentally occurring pathogen that is responsible for causing cryptococcosis marked by pneumonia and meningoencephalitis in humans and animals. C. gattii can form long-term associations with trees and soil resulting in the production of infectious propagules (spores and desiccated yeast). The ever-expanding number of reports of clinical and environmental isolation of C. gattii in temperate climates strongly imply that C. gattii occurs worldwide. The key ability of yeast and spores to enter, survive, multiply, and exit host cells, and to infect immunocompetent hosts distinguishes C. gattii as a primary pathogen and suggests evolution of C. gattii pathogenesis as a result of interaction with plants and other organisms in its environmental niche. Here we summarize the historical literature on C. gattii and recent literature supporting the worldwide occurrence of the primary pathogen C. gattii. © 2012 Springer Science+Business Media New York.

Authors
Springer, DJ; Phadke, S; Billmyer, RB; Heitman, J
MLA Citation
Springer, DJ, Phadke, S, Billmyer, RB, and Heitman, J. "Cryptococcus gattii, No Longer an Accidental Pathogen?." Current Fungal Infection Reports (2012): 1-12.
Source
scival
Published In
Current Fungal Infection Reports
Publish Date
2012
Start Page
1
End Page
12
DOI
10.1007/s12281-012-0111-0

A flucytosine-responsive mbp1/swi4-like protein, mbs1, plays pleiotropic roles in antifungal drug resistance, stress response, and virulence of cryptococcus neoformans

Cryptococcosis, caused by the basidiomycetous fungus Cryptococcus neoformans, is responsible for more than 600,000 deaths annually in AIDS patients. Flucytosine is one of the most commonly used antifungal drugs for its treatment, but its resistance and regulatory mechanisms have never been investigated at the genome scale in C. neoformans. In the present study, we performed comparative transcriptome analysis by employing two-component system mutants (tco1 and tco2) exhibiting opposing flucytosine susceptibility. As a result, a total of 177 flucytosine-responsive genes were identified, and many of them were found to be regulated by Tco1 or Tco2. Among these, we discovered an APSES-like transcription factor, Mbs1 (Mbp1and Swi4like protein 1). Expression analysis revealed that MBS1 was regulated in response to flucytosine in a Tco2/Hog1-dependent manner. Supporting this, C. neoformans with the deletion of MBS1 exhibited increased susceptibility to flucytosine. Intriguingly, Mbs1 played pleiotropic roles in diverse cellular processes of C. neoformans. Mbs1 positively regulated ergosterol biosynthesis and thereby affected polyene and azole drug susceptibility. Mbs1 was also involved in genotoxic and oxidative stress responses. Furthermore, Mbs1 promoted production of melanin and capsule and thereby was required for full virulence of C. neoformans. In conclusion, Mbs1 is considered to be a novel antifungal therapeutic target for treatment of cryptococcosis. © 2012, American Society for Microbiology. All Rights Reserved.

Authors
Song, M-H; Lee, J-W; Kim, MS; Yoon, J-K; White, TC; Floyd, A; Heitman, J; Strain, AK; Nielsen, JN; Nielsen, K; Bahn, Y-S
MLA Citation
Song, M-H, Lee, J-W, Kim, MS, Yoon, J-K, White, TC, Floyd, A, Heitman, J, Strain, AK, Nielsen, JN, Nielsen, K, and Bahn, Y-S. "A flucytosine-responsive mbp1/swi4-like protein, mbs1, plays pleiotropic roles in antifungal drug resistance, stress response, and virulence of cryptococcus neoformans." Eukaryotic Cell 11.1 (2012): 53-67.
PMID
22080454
Source
scival
Published In
Eukaryotic cell
Volume
11
Issue
1
Publish Date
2012
Start Page
53
End Page
67
DOI
10.1128/EC.05236-11

Profiling a killer, the development of Cryptococcus neoformans

The ability of fungi to transition between unicellular and multicellular growth has a profound impact on our health and the economy. Many important fungal pathogens of humans, animals, and plants are dimorphic, and the ability to switch between morphological states has been associated with their virulence. Cryptococcus neoformans is a human fungal pathogen that causes life-threatening meningoencephalitis in immunocompromised and, in some cases, immunocompetent hosts. Cryptococcus neoformans grows vegetatively as a budding yeast and switches to hyphal growth during the sexual cycle, which is important in the study of cryptococcal pathogenicity because spores resulting from sexual development are infectious propagules and can colonize the lungs of a host. In addition, sexual reproduction contributes to the genotypic variability of Cryptococcus species, which may lead to increased fitness and virulence. Despite significant advances in our understanding of the mechanisms behind the development of C. neoformans, our knowledge is still incomplete. Recent studies have led to the emergence of many intriguing questions and hypotheses. In this review, we describe and discuss the most interesting aspects of C. neoformans development and address their impact on pathogenicity. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd.

Authors
Kozubowski, L; Heitman, J
MLA Citation
Kozubowski, L, and Heitman, J. "Profiling a killer, the development of Cryptococcus neoformans." FEMS Microbiology Reviews 36.1 (2012): 78-94.
PMID
21658085
Source
scival
Published In
Fems Microbiology Reviews
Volume
36
Issue
1
Publish Date
2012
Start Page
78
End Page
94
DOI
10.1111/j.1574-6976.2011.00286.x

Parallels in Intercellular Communication in Oomycete and Fungal Pathogens of Plants and Humans

Authors
Lee, SC; Ristaino, JB; Heitman, J
MLA Citation
Lee, SC, Ristaino, JB, and Heitman, J. "Parallels in Intercellular Communication in Oomycete and Fungal Pathogens of Plants and Humans." PLoS Pathogens 8.12 (2012).
PMID
23271965
Source
scival
Published In
PLoS pathogens
Volume
8
Issue
12
Publish Date
2012
DOI
10.1371/journal.ppat.1003028

Comparative genome analysis of Trichophyton rubrum and related dermatophytes reveals candidate genes involved in infection.

The major cause of athlete's foot is Trichophyton rubrum, a dermatophyte or fungal pathogen of human skin. To facilitate molecular analyses of the dermatophytes, we sequenced T. rubrum and four related species, Trichophyton tonsurans, Trichophyton equinum, Microsporum canis, and Microsporum gypseum. These species differ in host range, mating, and disease progression. The dermatophyte genomes are highly colinear yet contain gene family expansions not found in other human-associated fungi. Dermatophyte genomes are enriched for gene families containing the LysM domain, which binds chitin and potentially related carbohydrates. These LysM domains differ in sequence from those in other species in regions of the peptide that could affect substrate binding. The dermatophytes also encode novel sets of fungus-specific kinases with unknown specificity, including nonfunctional pseudokinases, which may inhibit phosphorylation by competing for kinase sites within substrates, acting as allosteric effectors, or acting as scaffolds for signaling. The dermatophytes are also enriched for a large number of enzymes that synthesize secondary metabolites, including dermatophyte-specific genes that could synthesize novel compounds. Finally, dermatophytes are enriched in several classes of proteases that are necessary for fungal growth and nutrient acquisition on keratinized tissues. Despite differences in mating ability, genes involved in mating and meiosis are conserved across species, suggesting the possibility of cryptic mating in species where it has not been previously detected. These genome analyses identify gene families that are important to our understanding of how dermatophytes cause chronic infections, how they interact with epithelial cells, and how they respond to the host immune response.

Authors
Martinez, DA; Oliver, BG; Gräser, Y; Goldberg, JM; Li, W; Martinez-Rossi, NM; Monod, M; Shelest, E; Barton, RC; Birch, E; Brakhage, AA; Chen, Z; Gurr, SJ; Heiman, D; Heitman, J; Kosti, I; Rossi, A; Saif, S; Samalova, M; Saunders, CW; Shea, T; Summerbell, RC; Xu, J; Young, S; Zeng, Q; Birren, BW; Cuomo, CA; White, TC
MLA Citation
Martinez, DA, Oliver, BG, Gräser, Y, Goldberg, JM, Li, W, Martinez-Rossi, NM, Monod, M, Shelest, E, Barton, RC, Birch, E, Brakhage, AA, Chen, Z, Gurr, SJ, Heiman, D, Heitman, J, Kosti, I, Rossi, A, Saif, S, Samalova, M, Saunders, CW, Shea, T, Summerbell, RC, Xu, J, Young, S, Zeng, Q, Birren, BW, Cuomo, CA, and White, TC. "Comparative genome analysis of Trichophyton rubrum and related dermatophytes reveals candidate genes involved in infection." mBio 3.5 (2012): e00259-e00212.
PMID
22951933
Source
scival
Published In
mBio
Volume
3
Issue
5
Publish Date
2012
Start Page
e00259
End Page
e00212
DOI
10.1128/mBio.00259-12

Comparative genome analysis of Trichophyton rubrum and related dermatophytes reveals candidate genes involved in infection

The major cause of athlete's foot is Trichophyton rubrum, a dermatophyte or fungal pathogen of human skin. To facilitate molecular analyses of the dermatophytes, we sequenced T. rubrum and four related species, Trichophyton tonsurans, Trichophyton equinum, Microsporum canis, and Microsporum gypseum. These species differ in host range, mating, and disease progression. The dermatophyte genomes are highly colinear yet contain gene family expansions not found in other human-associated fungi. Dermatophyte genomes are enriched for gene families containing the LysM domain, which binds chitin and potentially related carbohydrates. These LysM domains differ in sequence from those in other species in regions of the peptide that could affect substrate binding. The dermatophytes also encode novel sets of fungus-specific kinases with unknown specificity, including nonfunctional pseudokinases, which may inhibit phosphorylation by competing for kinase sites within substrates, acting as allosteric effectors, or acting as scaffolds for signaling. The dermatophytes are also enriched for a large number of enzymes that synthesize secondary metabolites, including dermatophyte-specific genes that could synthesize novel compounds. Finally, dermatophytes are enriched in several classes of proteases that are necessary for fungal growth and nutrient acquisition on keratinized tissues. Despite differences in mating ability, genes involved in mating and meiosis are conserved across species, suggesting the possibility of cryptic mating in species where it has not been previously detected. These genome analyses identify gene families that are important to our understanding of how dermatophytes cause chronic infections, how they interact with epithelial cells, and how they respond to the host immune response. © 2012 Martinez et al.

Authors
Martinez, DA; Oliver, BG; Gräser, Y; Goldberg, JM; Li, W; Martinez-Rossi, NM; Monod, M; Shelest, E; Barton, RC; Birch, E; Brakhage, AA; Chen, Z; Gurr, SJ; Heiman, D; Heitman, J; Kosti, I; Rossi, A; Saif, S; Samalova, M; Saunders, CW; Shea, T; Summerbell, RC; Xu, J; Young, S; Zeng, Q; Birren, BW; Cuomo, CA; White, TC
MLA Citation
Martinez, DA, Oliver, BG, Gräser, Y, Goldberg, JM, Li, W, Martinez-Rossi, NM, Monod, M, Shelest, E, Barton, RC, Birch, E, Brakhage, AA, Chen, Z, Gurr, SJ, Heiman, D, Heitman, J, Kosti, I, Rossi, A, Saif, S, Samalova, M, Saunders, CW, Shea, T, Summerbell, RC, Xu, J, Young, S, Zeng, Q, Birren, BW, Cuomo, CA, and White, TC. "Comparative genome analysis of Trichophyton rubrum and related dermatophytes reveals candidate genes involved in infection." mBio 3.5 (2012).
Source
scival
Published In
mBio
Volume
3
Issue
5
Publish Date
2012
DOI
10.1128/mBio.00259-12

Genetic diversity and genomic plasticity of cryptococcus neoformans AD hybrid strains

Natural hybridization between two strains, varieties, or species is a common phenomenon in both plants and animals. Although hybridization may skew established gene pools, it generates population diversity efficiently and sometimes results in the emergence of newly adapted genotypes. Cryptococcus neoformans, which causes the most frequent opportunistic fungal infection in immunocompromised hosts, has three serotypes: A, D, and AD. Serotype-specific multilocus sequence typing and serotype-specific comparative genome hybridization were applied to investigate the genetic variability and genomic organization of C. neoformans serotype AD isolates. We confirm that C. neoformans serotype AD isolates are hybrids of serotype A and D strains. Compared with haploid strains, most AD hybrid isolates exhibit unique multilocus sequence typing genotypes, suggesting that multiple independent hybridization events punctuated the origin and evolutionary trajectory of AD hybrids. The MATa alleles from both haploid and AD hybrid isolates group closely to form a cluster or subcluster in both the serotype A and D populations. The rare and unique distribution of MATa alleles may restrict sexual reproduction between isolates of opposite mating types. The genetic diversity of the serotype D population, including haploid strains and serotype D genomes of the AD hybrid, is significantly greater than that of serotype A, and there are signatures of recombination within the serotype D population. Given that MATa isolates are relatively rare, both opposite-sex and same-sex mating may contribute to genetic recombination of serotype D in nature. Extensive chromosome loss was observed in AD hybrid isolates, which results in loss of heterozygosity in the otherwise-heterozygous AD hybrid genome. Most AD hybrid isolates exhibit hybrid vigor and are resistant to the antifungal drug FK506. In addition, the C. neoformans AD hybrid genome is highly dynamic, with continuous chromosome loss, which may be a facile route for pathogen evolution through which genotypic and phenotypic variation is generated. © 2012 Li et al.

Authors
Li, W; Averette, AF; Desnos-Ollivier, M; Ni, M; Dromer, F; Heitman, J
MLA Citation
Li, W, Averette, AF, Desnos-Ollivier, M, Ni, M, Dromer, F, and Heitman, J. "Genetic diversity and genomic plasticity of cryptococcus neoformans AD hybrid strains." G3: Genes, Genomes, Genetics 2.1 (2012): 83-97.
PMID
22384385
Source
scival
Published In
G3 (Bethesda, Md.)
Volume
2
Issue
1
Publish Date
2012
Start Page
83
End Page
97
DOI
10.1534/g3.111.001255

Comparative analysis of calcineurin signaling between Candida dubliniensis and Candida albicans

Candida dubliniensis, an emerging fungal pathogen, is the closest known species to the established pathogenic species Candida albicans. Despite the fact that these two species share.80% genome sequence identity, they exhibit distinct properties such as less hyphal growth, reduced pathogenicity and increased sensitivity to sodium stress and elevated temperatures in C. dubliniensis compared with C. albicans. It is, however, largely unknown whether signaling pathways are conserved in the two Candida species. Calcineurin signaling is known to be required for hyphal growth in Cryptococcus neoformans and Aspergillus fumigatus but remains elusive in C. albicans. Our recent study showed that calcineurin plays a clearly demonstrable role in controlling hyphal growth, drug tolerance and virulence in C. dubliniensis. Here, we extend our studies and show that calcineurin is conserved in controlling endoplasmic reticulum stress but distinct in governing pH homeostasis. Furthermore, we demonstrate that azole or echinocandin drugs in combination with the calcineurin inhibitor FK506 exhibit a synergistic effect against C. dubliniensis wild-type and echinocandin-resistant strains. The involvement of calcineurin in a variety of fungal virulence attributes and as a target for fungicidal synergism with azoles and echinocandins highlights the potential of combination therapy with calcineurin inhibitors for treating Candida infections. © 2012 Landes Bioscience.

Authors
Zhang, J; Heitman, J; Chen, Y-L
MLA Citation
Zhang, J, Heitman, J, and Chen, Y-L. "Comparative analysis of calcineurin signaling between Candida dubliniensis and Candida albicans." Communicative and Integrative Biology 5.2 (2012): 122-126.
Source
scival
Published In
Communicative & integrative biology
Volume
5
Issue
2
Publish Date
2012
Start Page
122
End Page
126
DOI
10.4161/cib.18833

The C2 domain protein Cts1 functions in the calcineurin signaling circuit during high-temperature stress responses in Cryptococcus neoformans.

Calcineurin is a conserved calcium/calmodulin-dependent serine/threonine-specific protein phosphatase that acts in cell stress responses. Calcineurin is essential for growth at 37°C and for virulence of the human fungal pathogen Cryptococcus neoformans, but its substrates remain unknown. The C2 domain-containing, phospholipid-binding protein Cts1 was previously identified as a multicopy suppressor of a calcineurin mutation in C. neoformans. Here we further characterize the function of Cts1 and the links between Cts1 and calcineurin. GFP-Cts1 localizes to cytoplasmic puncta and colocalizes with the endosomal marker FM4-64. The cts1Δ mutant shows a distinct FM4-64 staining pattern, suggesting involvement of Cts1 in endocytic trafficking. In large budded cells, GFP-Cts1 localizes transiently at the mother bud neck, as a single ring that undergoes contraction. mCherry-Cts1 colocalizes with the GFP-tagged calcineurin catalytic subunit Cna1 at sites of mRNA processing at 37°C, suggesting that Cts1 and calcineurin function coordinately during thermal stress. GFP-Cts1 exhibits slower electrophoretic mobility for cells grown at 37°C than for cells grown at 24°C, and the shift to a higher molecular weight is more pronounced in the presence of the calcineurin inhibitor FK506. In vitro treatment with calf intestinal alkaline phosphatase (CIP) restores faster electrophoretic mobility to GFP-Cts1, suggesting that Cts1 is phosphorylated at 37°C and may be dephosphorylated in a calcineurin-dependent manner. mCherry-Cts1 also coimmunoprecipitates with GFP-Cna1, with greater complex formation at 37°C than at 24°C. Taken together, these findings support potential roles for Cts1 in endocytic trafficking, mRNA processing, and cytokinesis and suggest that Cts1 is a substrate of calcineurin during high-temperature stress responses.

Authors
Aboobakar, EF; Wang, X; Heitman, J; Kozubowski, L
MLA Citation
Aboobakar, EF, Wang, X, Heitman, J, and Kozubowski, L. "The C2 domain protein Cts1 functions in the calcineurin signaling circuit during high-temperature stress responses in Cryptococcus neoformans." Eukaryot Cell 10.12 (December 2011): 1714-1723.
PMID
22002655
Source
pubmed
Published In
Eukaryotic cell
Volume
10
Issue
12
Publish Date
2011
Start Page
1714
End Page
1723
DOI
10.1128/EC.05148-11

Cryptococcus gattii genotype VGI infection in New England.

Cryptococcus gattii is a known, emerging infectious disease pathogen predominantly in the Pacific Northwest, the United States, and British Columbia, Canada. We report a case of an immunocompetent adolescent from New England who had severe pulmonary and central nervous system infection caused by the VGI genotype of C. gattii.

Authors
McCulloh, RJ; Phillips, R; Perfect, JR; Byrnes, EJ; Heitman, J; Dufort, E
MLA Citation
McCulloh, RJ, Phillips, R, Perfect, JR, Byrnes, EJ, Heitman, J, and Dufort, E. "Cryptococcus gattii genotype VGI infection in New England." Pediatr Infect Dis J 30.12 (December 2011): 1111-1114.
PMID
21814154
Source
pubmed
Published In
Pediatric Infectious Disease Journal
Volume
30
Issue
12
Publish Date
2011
Start Page
1111
End Page
1114
DOI
10.1097/INF.0b013e31822d14fd

Deletion of Cryptococcus neoformans AIF ortholog promotes chromosome aneuploidy and fluconazole-resistance in a metacaspase-independent manner.

Apoptosis is a form of programmed cell death critical for development and homeostasis in multicellular organisms. Apoptosis-like cell death (ALCD) has been described in several fungi, including the opportunistic human pathogen Cryptococcus neoformans. In addition, capsular polysaccharides of C. neoformans are known to induce apoptosis in host immune cells, thereby contributing to its virulence. Our goals were to characterize the apoptotic signaling cascade in C. neoformans as well as its unique features compared to the host machinery to exploit the endogenous fungal apoptotic pathways as a novel antifungal strategy in the future. The dissection of apoptotic pathways revealed that apoptosis-inducing factor (Aif1) and metacaspases (Mca1 and Mca2) are independently required for ALCD in C. neoformans. We show that the apoptotic pathways are required for cell fusion and sporulation during mating, indicating that apoptosis may occur during sexual development. Previous studies showed that antifungal drugs induce ALCD in fungi and that C. neoformans adapts to high concentrations of the antifungal fluconazole (FLC) by acquisition of aneuploidy, especially duplication of chromosome 1 (Chr1). Disruption of aif1, but not the metacaspases, stimulates the emergence of aneuploid subpopulations with Chr1 disomy that are resistant to fluconazole (FLC(R)) in vitro and in vivo. FLC(R) isolates in the aif1 background are stable in the absence of the drug, while those in the wild-type background readily revert to FLC sensitivity. We propose that apoptosis orchestrated by Aif1 might eliminate aneuploid cells from the population and defects in this pathway contribute to the selection of aneuploid FLC(R) subpopulations during treatment. Aneuploid clinical isolates with disomies for chromosomes other than Chr1 exhibit reduced AIF1 expression, suggesting that inactivation of Aif1 might be a novel aneuploidy-tolerating mechanism in fungi that facilitates the selection of antifungal drug resistance.

Authors
Semighini, CP; Averette, AF; Perfect, JR; Heitman, J
MLA Citation
Semighini, CP, Averette, AF, Perfect, JR, and Heitman, J. "Deletion of Cryptococcus neoformans AIF ortholog promotes chromosome aneuploidy and fluconazole-resistance in a metacaspase-independent manner." PLoS Pathog 7.11 (November 2011): e1002364-.
PMID
22114551
Source
pubmed
Published In
PLoS pathogens
Volume
7
Issue
11
Publish Date
2011
Start Page
e1002364
DOI
10.1371/journal.ppat.1002364

Cryptococcus gattii: an emerging fungal pathogen infecting humans and animals.

Infectious fungi are among a broad group of microbial pathogens that has and continues to emerge concomitantly due to the global AIDS pandemic as well as an overall increase of patients with compromised immune systems. In addition, many pathogens have been emerging and re-emerging, causing disease in both individuals who have an identifiable immune defect and those who do not. The fungal pathogen Cryptococcus gattii can infect individuals with and without an identifiable immune defect, with a broad geographic range including both endemic areas and emerging outbreak regions. Infections in patients and animals can be severe and often fatal if untreated. We review the molecular epidemiology, population structure, clinical manifestations, and ecological niche of this emerging pathogen.

Authors
Byrnes, EJ; Bartlett, KH; Perfect, JR; Heitman, J
MLA Citation
Byrnes, EJ, Bartlett, KH, Perfect, JR, and Heitman, J. "Cryptococcus gattii: an emerging fungal pathogen infecting humans and animals." Microbes Infect 13.11 (October 2011): 895-907. (Review)
PMID
21684347
Source
pubmed
Published In
Microbes and Infection: a journal on infectious agents and host defenses
Volume
13
Issue
11
Publish Date
2011
Start Page
895
End Page
907
DOI
10.1016/j.micinf.2011.05.009

A diverse population of Cryptococcus gattii molecular type VGIII in southern Californian HIV/AIDS patients.

Cryptococcus gattii infections in southern California have been reported in patients with HIV/AIDS. In this study, we examined the molecular epidemiology, population structure, and virulence attributes of isolates collected from HIV/AIDS patients in Los Angeles County, California. We show that these isolates consist almost exclusively of VGIII molecular type, in contrast to the VGII molecular type isolates causing the North American Pacific Northwest outbreak. The global VGIII population structure can be divided into two molecular groups, VGIIIa and VGIIIb. Isolates from the Californian patients are virulent in murine and macrophage models of infection, with VGIIIa significantly more virulent than VGIIIb. Several VGIII isolates are highly fertile and produce abundant sexual spores that may serve as infectious propagules. The a and α VGIII MAT locus alleles are largely syntenic with limited rearrangements compared to the known VGI (a/α) and VGII (α) MAT loci, but each has unique characteristics including a distinct deletion flanking the 5' VGIII MATa alleles and the α allele is more heterogeneous than the a allele. Our studies indicate that C. gattii VGIII is endemic in southern California, with other isolates originating from the neighboring regions of Mexico, and in rarer cases from Oregon and Washington state. Given that >1,000,000 cases of cryptococcal infection and >620,000 attributable mortalities occur annually in the context of the global AIDS pandemic, our findings suggest a significant burden of C. gattii may be unrecognized, with potential prognostic and therapeutic implications. These results signify the need to classify pathogenic Cryptococcus cases and highlight possible host differences among the C. gattii molecular types influencing infection of immunocompetent (VGI/VGII) vs. immunocompromised (VGIII/VGIV) hosts.

Authors
Byrnes, EJ; Li, W; Ren, P; Lewit, Y; Voelz, K; Fraser, JA; Dietrich, FS; May, RC; Chaturvedi, S; Chaturvedi, V; Heitman, J
MLA Citation
Byrnes, EJ, Li, W, Ren, P, Lewit, Y, Voelz, K, Fraser, JA, Dietrich, FS, May, RC, Chaturvedi, S, Chaturvedi, V, and Heitman, J. "A diverse population of Cryptococcus gattii molecular type VGIII in southern Californian HIV/AIDS patients." PLoS Pathog 7.9 (September 2011): e1002205-.
PMID
21909264
Source
pubmed
Published In
PLoS pathogens
Volume
7
Issue
9
Publish Date
2011
Start Page
e1002205
DOI
10.1371/journal.ppat.1002205

Calcineurin colocalizes with P-bodies and stress granules during thermal stress in cryptococcus neoformans

Calcineurin is a calcium-calmodulin-activated serine/threonine-specific phosphatase that operates during cellular responses to stress and plays a prominent role in transcriptional control, whereas regulatory events beyond transcription are less well characterized. This study reveals a novel transcription-independent role of calcineurin during the temperature stress response in the human fungal pathogen Cryptococcus neoformans. The diffusely cytoplasmic calcineurin catalytic subunit Cna1 relocates to endoplasmic reticulum (ER)-associated puncta and the mother-bud neck when cells are subjected to 37°C. More than 50% of Cna1 puncta contain the P-body constituent decapping enzyme Dcp1 and the stress granule constituent poly(A)-binding protein Pub1. These results support a model in which calcineurin orchestrates thermal stress responses by associating with sites of mRNA processing. © 2011, American Society for Microbiology. All Rights Reserved.

Authors
Kozubowski, L; Aboobakar, EF; Cardenas, ME; Heitman, J
MLA Citation
Kozubowski, L, Aboobakar, EF, Cardenas, ME, and Heitman, J. "Calcineurin colocalizes with P-bodies and stress granules during thermal stress in cryptococcus neoformans." Eukaryotic Cell 10.11 (2011): 1396-1402.
PMID
21724937
Source
scival
Published In
Eukaryotic cell
Volume
10
Issue
11
Publish Date
2011
Start Page
1396
End Page
1402
DOI
10.1128/EC.05087-11

Association of calcineurin with the COPI protein Sec28 and the COPII protein Sec13 revealed by quantitative proteomics.

Calcineurin is a calcium-calmodulin-dependent serine/threonine specific protein phosphatase operating in key cellular processes governing responses to extracellular cues. Calcineurin is essential for growth at high temperature and virulence of the human fungal pathogen Cryptococcus neoformans but the underlying mechanism is unknown. We performed a mass spectrometry analysis to identify proteins that associate with the calcineurin A catalytic subunit (Cna1) in C. neoformans cells grown under non-stress and high temperature stress conditions. A novel prioritization strategy for mass spectrometry data from immunoprecipitation experiments identified putative substrates and proteins potentially operating with calcineurin in common pathways. Cna1 co-purified with proteins involved in membrane trafficking including the COPI component Sec28 and the COPII component Sec13. The association of Cna1 with Sec28 and Sec13 was confirmed by co-immunoprecipitation. Cna1 exhibited a dramatic change in subcellular localization during high temperature stress from diffuse cytoplasmic to ER-associated puncta and the mother-bud neck and co-localized with Sec28 and Sec13.

Authors
Kozubowski, L; Thompson, JW; Cardenas, ME; Moseley, MA; Heitman, J
MLA Citation
Kozubowski, L, Thompson, JW, Cardenas, ME, Moseley, MA, and Heitman, J. "Association of calcineurin with the COPI protein Sec28 and the COPII protein Sec13 revealed by quantitative proteomics." PLoS One 6.10 (2011): e25280-.
PMID
21984910
Source
pubmed
Published In
PloS one
Volume
6
Issue
10
Publish Date
2011
Start Page
e25280
DOI
10.1371/journal.pone.0025280

Diversity in the realm of eukaryotic microbe form and function

Authors
Heitman, J
MLA Citation
Heitman, J. "Diversity in the realm of eukaryotic microbe form and function." Current Opinion in Microbiology 14.6 (2011): 631-633.
PMID
22074862
Source
scival
Published In
Current Opinion in Microbiology
Volume
14
Issue
6
Publish Date
2011
Start Page
631
End Page
633
DOI
10.1016/j.mib.2011.10.010

Unique evolution of the UPR pathway with a novel bZIP transcription factor, HxL1, for controlling pathogenicity of cryptococcus neoformans

In eukaryotic cells, the unfolded protein response (UPR) pathway plays a crucial role in cellular homeostasis of the endoplasmic reticulum (ER) during exposure to diverse environmental conditions that cause ER stress. Here we report that the human fungal pathogen Cryptococcus neoformans has evolved a unique UPR pathway composed of an evolutionarily conserved Ire1 protein kinase and a novel bZIP transcription factor encoded by HXL1 (HAC1 and XBP1-Like gene 1). C. neoformans HXL1 encodes a protein lacking sequence homology to any known fungal or mammalian Hac1/Xbp1 protein yet undergoes the UPR-induced unconventional splicing in an Ire1-dependent manner upon exposure to various stresses. The structural organization of HXL1 and its unconventional splicing is widely conserved in C. neoformans strains of divergent serotypes. Notably, both C. neoformans ire1 and hxl1 mutants exhibited extreme growth defects at 37°C and hypersensitivity to ER stress and cell wall destabilization. All of the growth defects of the ire1 mutant were suppressed by the spliced active form of Hxl1, supporting that HXL1 mRNA is a downstream target of Ire1. Interestingly, however, the ire1 and hxl1 mutants showed differences in thermosensitivity, expression patterns for a subset of genes, and capsule synthesis, indicating that Ire1 has both Hxl1-dependent and -independent functions in C. neoformans. Finally, Ire1 and Hxl1 were shown to be critical for virulence of C. neoformans, suggesting UPR signaling as a novel antifungal therapeutic target. © 2011 Cheon et al.

Authors
Cheon, SA; Jung, K-W; Chen, Y-L; Heitman, J; Bahn, Y-S; Kang, HA
MLA Citation
Cheon, SA, Jung, K-W, Chen, Y-L, Heitman, J, Bahn, Y-S, and Kang, HA. "Unique evolution of the UPR pathway with a novel bZIP transcription factor, HxL1, for controlling pathogenicity of cryptococcus neoformans." PLoS Pathogens 7.8 (2011).
PMID
21852949
Source
scival
Published In
PLoS pathogens
Volume
7
Issue
8
Publish Date
2011
DOI
10.1371/journal.ppat.1002177

Is sex necessary?

Fungal sexual reproductive modes have markedly high diversity and plasticity, and asexual species have been hypothesized to arise frequently from sexual fungal species. A recent study on the red yeasts provides further support for the notion that sexual ancestors may give rise to shorter-lived asexual species. However, presumed asexual species may also be cryptically sexual, as revealed by other recent studies. © 2011 Sun and Heitman; licensee BioMed Central Ltd.

Authors
Sun, S; Heitman, J
MLA Citation
Sun, S, and Heitman, J. "Is sex necessary?." BMC Biology 9 (2011).
PMID
21880159
Source
scival
Published In
BMC Biology
Volume
9
Publish Date
2011
DOI
10.1186/1741-7007-9-56

Calcineurin controls drug tolerance, hyphal growth, and virulence in Candida dubliniensis

Candida dubliniensis is an emerging pathogenic yeast species closely related to Candida albicans and frequently found colonizing or infecting the oral cavities of HIV/AIDS patients. Drug resistance during C. dubliniensis infection is common and constitutes a significant therapeutic challenge. The calcineurin inhibitor FK506 exhibits synergistic fungicidal activity with azoles or echinocandins in the fungal pathogens C. albicans, Cryptococcus neoformans, and Aspergillus fumigatus. In this study, we show that calcineurin is required for cell wall integrity and wild-type tolerance of C. dubliniensis to azoles and echinocandins; hence, these drugs are candidates for combination therapy with calcineurin inhibitors. In contrast to C. albicans, in which the roles of calcineurin and Crz1 in hyphal growth are unclear, here we show that calcineurin and Crz1 play a clearly demonstrable role in hyphal growth in response to nutrient limitation in C. dubliniensis. We further demonstrate that thigmotropism is controlled by Crz1, but not calcineurin, in C. dubliniensis. Similar to C. albicans, C. dubliniensis calcineurin enhances survival in serum. C. dubliniensis calcineurin and crz1/crz1 mutants exhibit attenuated virulence in a murine systemic infection model, likely attributable to defects in cell wall integrity, hyphal growth, and serum survival. Furthermore, we show that C. dubliniensis calcineurin mutants are unable to establish murine ocular infection or form biofilms in a rat denture model. That calcineurin is required for drug tolerance and virulence makes fungus-specific calcineurin inhibitors attractive candidates for combination therapy with azoles or echinocandins against emerging C. dubliniensis infections. © 2011, American Society for Microbiology.

Authors
Chen, Y-L; Brand, A; Morrison, EL; Silao, FGS; Bigol, UG; Jr, FFM; Nett, JE; Andes, DR; Solis, NV; Filler, SG; Averette, A; Heitman, J
MLA Citation
Chen, Y-L, Brand, A, Morrison, EL, Silao, FGS, Bigol, UG, Jr, FFM, Nett, JE, Andes, DR, Solis, NV, Filler, SG, Averette, A, and Heitman, J. "Calcineurin controls drug tolerance, hyphal growth, and virulence in Candida dubliniensis." Eukaryotic Cell 10.6 (2011): 803-819.
PMID
21531874
Source
scival
Published In
Eukaryotic cell
Volume
10
Issue
6
Publish Date
2011
Start Page
803
End Page
819
DOI
10.1128/EC.00310-10

Sporangiospore size dimorphism is linked to virulence of mucor circinelloides

Mucor circinelloides is a zygomycete fungus and an emerging opportunistic pathogen in immunocompromised patients, especially transplant recipients and in some cases otherwise healthy individuals. We have discovered a novel example of size dimorphism linked to virulence. M. circinelloides is a heterothallic fungus: (+) sex allele encodes SexP and (-) sex allele SexM, both of which are HMG domain protein sex determinants. M. circinelloides f. lusitanicus (Mcl) (-) mating type isolates produce larger asexual sporangiospores that are more virulent in the wax moth host compared to (+) isolates that produce smaller less virulent sporangiospores. The larger sporangiospores germinate inside and lyse macrophages, whereas the smaller sporangiospores do not. sexMΔ mutants are sterile and still produce larger virulent sporangiospores, suggesting that either the sex locus is not involved in virulence/spore size or the sexP allele plays an inhibitory role. Phylogenetic analysis supports that at least three extant subspecies populate the M. circinelloides complex in nature: Mcl, M. circinelloides f. griseocyanus, and M. circinelloides f. circinelloides (Mcc). Mcc was found to be more prevalent among clinical Mucor isolates, and more virulent than Mcl in a diabetic murine model in contrast to the wax moth host. The M. circinelloides sex locus encodes an HMG domain protein (SexP for plus and SexM for minus mating types) flanked by genes encoding triose phosphate transporter (TPT) and RNA helicase homologs. The borders of the sex locus between the three subspecies differ: the Mcg sex locus includes the promoters of both the TPT and the RNA helicase genes, whereas the Mcl and Mcc sex locus includes only the TPT gene promoter. Mating between subspecies was restricted compared to mating within subspecies. These findings demonstrate that spore size dimorphism is linked to virulence of M. circinelloides species and that plasticity of the sex locus and adaptations in pathogenicity have occurred during speciation of the M. circinelloides complex. © 2011 Li et al.

Authors
Li, CH; Cervantes, M; Springer, DJ; Boekhout, T; Ruiz-Vazquez, RM; Torres-Martinez, SR; Heitman, J; Lee, SC
MLA Citation
Li, CH, Cervantes, M, Springer, DJ, Boekhout, T, Ruiz-Vazquez, RM, Torres-Martinez, SR, Heitman, J, and Lee, SC. "Sporangiospore size dimorphism is linked to virulence of mucor circinelloides." PLoS Pathogens 7.6 (2011).
PMID
21698218
Source
scival
Published In
PLoS pathogens
Volume
7
Issue
6
Publish Date
2011
DOI
10.1371/journal.ppat.1002086

Microbial pathogens in the fungal kingdom

The fungal kingdom is vast, spanning ∼1.5 to as many as 5 million species diverse as unicellular yeasts, filamentous fungi, mushrooms, lichens, and both plant and animal pathogens. The fungi are closely aligned with animals in one of the six to eight supergroups of eukaryotes, the opisthokonts. The animal and fungal kingdoms last shared a common ancestor ∼1 billion years ago, more recently than other groups of eukaryotes. As a consequence of their close evolutionary history and shared cellular machinery with metazoans, fungi are exceptional models for mammalian biology, but prove more difficult to treat in infected animals. The last common ancestor to the fungal/metazoan lineages is thought to have been unicellular, aquatic, and motile with a posterior flagellum, and certain extant species closely resemble this hypothesized ancestor. Species within the fungal kingdom were traditionally assigned to four phyla, including the basal fungi (Chytridiomycota, Zygomycota) and the more recently derived monophyletic lineage, the dikarya (Ascomycota, Basidiomycota). The fungal tree of life project has revealed that the basal lineages are polyphyletic, and thus there are as many as eight to ten fungal phyla. Fungi that infect vertebrates are found in all of the major lineages, and virulence arose multiple times independently. A sobering recent development involves the species Batrachochytrium dendrobatidis from the basal fungal phylum, the Chytridiomycota, which has emerged to cause global amphibian declines and extinctions. Genomics is revolutionizing our view of the fungal kingdom, and genome sequences for zygomycete pathogens (Rhizopus, Mucor), skin-associated fungi (dermatophytes, Malassezia), and the Candida pathogenic species clade promise to provide insights into the origins of virulence. Here we survey the diversity of fungal pathogens and illustrate key principles revealed by genomics involving sexual reproduction and sex determination, loss of conserved pathways in derived fungal lineages that are retained in basal fungi, and shared and divergent virulence strategies of successful human pathogens, including dimorphic and trimorphic transitions in form. The overarching conclusion is that fungal pathogens of animals have arisen repeatedly and independently throughout the fungal tree of life, and while they share general properties, there are also unique features to the virulence strategies of each successful microbial pathogen. © 2011 The British Mycological Society.

Authors
Heitman, J
MLA Citation
Heitman, J. "Microbial pathogens in the fungal kingdom." Fungal Biology Reviews 25.1 (2011): 48-60.
PMID
21528015
Source
scival
Published In
Fungal Biology Reviews
Volume
25
Issue
1
Publish Date
2011
Start Page
48
End Page
60
DOI
10.1016/j.fbr.2011.01.003

Validation of kwoniella heveanensis, teleomorph of the basidiomycetous yeast Cryptococcus heveanensis

Kwoniella heveanensis, recently published in an electronic journal and without a designated holotype, is validated as the name of the newly discovered teleomorph of Cryptococcus heveanensis. © 2011. Mycotaxon, Ltd.

Authors
Sun, S; Metin, B; Findley, K; Fonseca, A; Heitman, J
MLA Citation
Sun, S, Metin, B, Findley, K, Fonseca, A, and Heitman, J. "Validation of kwoniella heveanensis, teleomorph of the basidiomycetous yeast Cryptococcus heveanensis." Mycotaxon 116 (2011): 227-229.
Source
scival
Published In
Mycotaxon
Volume
116
Publish Date
2011
Start Page
227
End Page
229
DOI
10.5248/116.227

Sex in fungi.

Sexual reproduction enables genetic exchange in eukaryotic organisms as diverse as fungi, animals, plants, and ciliates. Given its ubiquity, sex is thought to have evolved once, possibly concomitant with or shortly after the origin of eukaryotic organisms themselves. The basic principles of sex are conserved, including ploidy changes, the formation of gametes via meiosis, mate recognition, and cell-cell fusion leading to the production of a zygote. Although the basic tenants are shared, sex determination and sexual reproduction occur in myriad forms throughout nature, including outbreeding systems with more than two mating types or sexes, unisexual selfing, and even examples in which organisms switch mating type. As robust and diverse genetic models, fungi provide insights into the molecular nature of sex, sexual specification, and evolution to advance our understanding of sexual reproduction and its impact throughout the eukaryotic tree of life.

Authors
Ni, M; Feretzaki, M; Sun, S; Wang, X; Heitman, J
MLA Citation
Ni, M, Feretzaki, M, Sun, S, Wang, X, and Heitman, J. "Sex in fungi." Annu Rev Genet 45 (2011): 405-430. (Review)
PMID
21942368
Source
pubmed
Published In
Annual Review of Genetics
Volume
45
Publish Date
2011
Start Page
405
End Page
430
DOI
10.1146/annurev-genet-110410-132536

Comparative and functional genomics provide insights into the pathogenicity of dermatophytic fungi

Background: Millions of humans and animals suffer from superficial infections caused by a group of highly specialized filamentous fungi, the dermatophytes, which exclusively infect keratinized host structures. To provide broad insights into the molecular basis of the pathogenicity-associated traits, we report the first genome sequences of two closely phylogenetically related dermatophytes, Arthroderma benhamiae and Trichophyton verrucosum, both of which induce highly inflammatory infections in humans.Results: 97% of the 22.5 megabase genome sequences of A. benhamiae and T. verrucosum are unambiguously alignable and collinear. To unravel dermatophyte-specific virulence-associated traits, we compared sets of potentially pathogenicity-associated proteins, such as secreted proteases and enzymes involved in secondary metabolite production, with those of closely related onygenales (Coccidioides species) and the mould Aspergillus fumigatus. The comparisons revealed expansion of several gene families in dermatophytes and disclosed the peculiarities of the dermatophyte secondary metabolite gene sets. Secretion of proteases and other hydrolytic enzymes by A. benhamiae was proven experimentally by a global secretome analysis during keratin degradation. Molecular insights into the interaction of A. benhamiae with human keratinocytes were obtained for the first time by global transcriptome profiling. Given that A. benhamiae is able to undergo mating, a detailed comparison of the genomes further unraveled the genetic basis of sexual reproduction in this species.Conclusions: Our results enlighten the genetic basis of fundamental and putatively virulence-related traits of dermatophytes, advancing future research on these medically important pathogens. © 2011 Burmester et al.; licensee BioMed Central Ltd.

Authors
Burmester, A; Shelest, E; Glöckner, G; Heddergott, C; Schindler, S; Staib, P; Heidel, A; Felder, M; Petzold, A; Szafranski, K; Feuermann, M; Pedruzzi, I; Priebe, S; Groth, M; Winkler, R; Li, W; Kniemeyer, O; Schroeckh, V; Hertweck, C; Hube, B; White, TC; Platzer, M; Guthke, R; Heitman, J; Wöstemeyer, J; Zipfel, PF; Monod, M; Brakhage, AA
MLA Citation
Burmester, A, Shelest, E, Glöckner, G, Heddergott, C, Schindler, S, Staib, P, Heidel, A, Felder, M, Petzold, A, Szafranski, K, Feuermann, M, Pedruzzi, I, Priebe, S, Groth, M, Winkler, R, Li, W, Kniemeyer, O, Schroeckh, V, Hertweck, C, Hube, B, White, TC, Platzer, M, Guthke, R, Heitman, J, Wöstemeyer, J, Zipfel, PF, Monod, M, and Brakhage, AA. "Comparative and functional genomics provide insights into the pathogenicity of dermatophytic fungi." Genome Biology 12.1 (2011).
PMID
21247460
Source
scival
Published In
Genome Biology: biology for the post-genomic era
Volume
12
Issue
1
Publish Date
2011
DOI
10.1186/gb-2011-12-1-r7

Genome variation in Cryptococcus gattii, an emerging pathogen of immunocompetent hosts

Cryptococcus gattii recently emerged as the causative agent of cryptococcosis in healthy individuals in western North America, despite previous characterization of the fungus as a pathogen in tropical or subtropical regions. As a foundation to study the genetics of virulence in this pathogen, we sequenced the genomes of a strain (WM276) representing the predominant global molecular type (VGI) and a clinical strain (R265) of the major genotype (VGIIa) causing disease in North America. We compared these C. gattii genomes with each other and with the genomes of representative strains of the two varieties of Cryptococcus neoformans that generally cause disease in immunocompromised people. Our comparisons included chromosome alignments, analysis of gene content and gene family evolution, and comparative genome hybridization (CGH). These studies revealed that the genomes of the two representative C. gattii strains (genotypes VGI and VGIIa) are colinear for the majority of chromosomes, with some minor rearrangements. However, multiortholog phylogenetic analysis and an evaluation of gene/sequence conservation support the existence of speciation within the C. gattii complex. More extensive chromosome rearrangements were observed upon comparison of the C. gattii and the C. neoformans genomes. Finally, CGH revealed considerable variation in clinical and environmental isolates as well as changes in chromosome copy numbers in C. gattii isolates displaying fluconazole heteroresistance. © 2011 D'Souza et al.

Authors
D'Souza, CA; Kronstad, JW; Taylor, G; Warren, R; Yuen, M; Hu, G; Jung, WH; Sham, A; Kidd, SE; Tangen, K; Lee, N; Zeilmaker, T; Sawkins, J; McVicker, G; Shah, S; Gnerre, S; Griggs, A; Zeng, Q; Bartlett, K; Li, W; Wang, X; Heitman, J; Stajich, JE; Fraser, JA; Meyer, W; Carter, D; Schein, J; Krzywinski, M; Kwon-Chung, KJ; Varma, A; Wang, J; Brunham, R; Fyfe, M; Ouellette, BFF; Siddiqui, A; Marra, M; Jones, S; Holt, R; Birren, BW; Galagan, JE; Cuomoe, CA
MLA Citation
D'Souza, CA, Kronstad, JW, Taylor, G, Warren, R, Yuen, M, Hu, G, Jung, WH, Sham, A, Kidd, SE, Tangen, K, Lee, N, Zeilmaker, T, Sawkins, J, McVicker, G, Shah, S, Gnerre, S, Griggs, A, Zeng, Q, Bartlett, K, Li, W, Wang, X, Heitman, J, Stajich, JE, Fraser, JA, Meyer, W, Carter, D, Schein, J, Krzywinski, M, Kwon-Chung, KJ, Varma, A, Wang, J, Brunham, R, Fyfe, M, Ouellette, BFF, Siddiqui, A, Marra, M, Jones, S, Holt, R, Birren, BW, Galagan, JE, and Cuomoe, CA. "Genome variation in Cryptococcus gattii, an emerging pathogen of immunocompetent hosts." mBio 2.1 (2011).
PMID
21304167
Source
scival
Published In
mBio
Volume
2
Issue
1
Publish Date
2011
DOI
10.1128/mBio.00342-10

Epidemiology and evolution of fungal pathogens in plants and animals

This chapter discusses the main pathogenic fungi parasitizing humans, animals, and plants, and having important consequences on human health or human activities. It discusses the modern molecular tools used for epidemiology and population genetics of fungal pathogens. The two major groups, Ascomycota and Basidiomycota, are the most common causes of invasive fungal infections in humans. Fungal pathogens can cause severe disease in wildlife species and plants. They reduce crop yield and lower product quality by attacking cultivated plants and their products. Our primary food production is at risk due to emerging crop diseases. Epidemics caused by invasive pathogens have been repeatedly reported to alter natural ecosystems. Molecular epidemiologic studies to study fungal pathogens use techniques such as MLST, multilocus strain typing and MLMT, multilocus microsatellite typing. Gene-sequence data from studies using genealogical concordance phylogenetic recognition (GCPSR) can easily be converted to strain sequence types (STs) and stored in web databases. Population genetics is also needed to understand fungal diseases. It has provided important insights for some fungal pathogens on their mating systems, dispersal, and population structure. It offers the opportunity to forecast the emergence of genotypes, populations, or species with detrimental characteristics for human affairs. This chapter reveals that comparative genomic studies in plant pathogenic and symbiotic fungi have brought many insights into the evolution of the pathogenic lifestyle, in particular into the mechanisms of virulence and host adaptations. Regarding epidemiology, it is concluded that molecular methods have much to offer to the study of fungal pathogens, allowing elucidation of ecological and microevolutionary processes. The chapter states that microsatellite markers in particular are very powerful tools and should be more widely used for population studies in fungi, despite the technical challenges of their isolation in this kingdom. © 2011 Elsevier Inc. All rights reserved.

Authors
Gladieux, P; Byrnes, EJ; Aguileta, G; Fisher, MC; Heitman, J; Giraud, T
MLA Citation
Gladieux, P, Byrnes, EJ, Aguileta, G, Fisher, MC, Heitman, J, and Giraud, T. "Epidemiology and evolution of fungal pathogens in plants and animals." Genetics and Evolution of Infectious Diseases (2011): 59-132.
Source
scival
Published In
Genetics and Evolution of Infectious Diseases
Publish Date
2011
Start Page
59
End Page
132
DOI
10.1016/B978-0-12-384890-1.00004-2

Sex-induced silencing defends the genome of Cryptococcus neoformans via RNAi.

Cosuppression is a silencing phenomenon triggered by the introduction of homologous DNA sequences into the genomes of organisms as diverse as plants, fungi, flies, and nematodes. Here we report sex-induced silencing (SIS), which is triggered by tandem integration of a transgene array in the human fungal pathogen Cryptococcus neoformans. A SXI2a-URA5 transgene array was found to be post-transcriptionally silenced during sexual reproduction. More than half of the progeny that inherited the SXI2a-URA5 transgene became uracil-auxotrophic due to silencing of the URA5 gene. In vegetative mitotic growth, silencing of this transgene array occurred at an ∼250-fold lower frequency, indicating that silencing is induced during the sexual cycle. Central components of the RNAi pathway-including genes encoding Argonaute, Dicer, and an RNA-dependent RNA polymerase-are all required for both meiotic and mitotic transgene silencing. URA5-derived ∼22-nucleotide (nt) small RNAs accumulated in the silenced isolates, suggesting that SIS is mediated by RNAi via sequence-specific small RNAs. Through deep sequencing of the small RNA population in C. neoformans, we also identified abundant small RNAs mapping to repetitive transposable elements, and these small RNAs were absent in rdp1 mutant strains. Furthermore, a group of retrotransposons was highly expressed during mating of rdp1 mutant strains, and an increased transposition/mutation rate was detected in their progeny, indicating that the RNAi pathway squelches transposon activity during the sexual cycle. Interestingly, Ago1, Dcr1, Dcr2, and Rdp1 are translationally induced in mating cells, and Ago1, Dcr1, and Dcr2 localize to processing bodies (P bodies), whereas Rdp1 appears to be nuclear, providing mechanistic insights into the elevated silencing efficiency during sexual reproduction. We hypothesize that the SIS RNAi pathway operates to defend the genome during sexual development.

Authors
Wang, X; Hsueh, Y-P; Li, W; Floyd, A; Skalsky, R; Heitman, J
MLA Citation
Wang, X, Hsueh, Y-P, Li, W, Floyd, A, Skalsky, R, and Heitman, J. "Sex-induced silencing defends the genome of Cryptococcus neoformans via RNAi." Genes Dev 24.22 (November 15, 2010): 2566-2582.
PMID
21078820
Source
pubmed
Published In
Genes & development
Volume
24
Issue
22
Publish Date
2010
Start Page
2566
End Page
2582
DOI
10.1101/gad.1970910

Conservation, duplication, and loss of the Tor signaling pathway in the fungal kingdom.

BACKGROUND: The nutrient-sensing Tor pathway governs cell growth and is conserved in nearly all eukaryotic organisms from unicellular yeasts to multicellular organisms, including humans. Tor is the target of the immunosuppressive drug rapamycin, which in complex with the prolyl isomerase FKBP12 inhibits Tor functions. Rapamycin is a gold standard drug for organ transplant recipients that was approved by the FDA in 1999 and is finding additional clinical indications as a chemotherapeutic and antiproliferative agent. Capitalizing on the plethora of recently sequenced genomes we have conducted comparative genomic studies to annotate the Tor pathway throughout the fungal kingdom and related unicellular opisthokonts, including Monosiga brevicollis, Salpingoeca rosetta, and Capsaspora owczarzaki. RESULTS: Interestingly, the Tor signaling cascade is absent in three microsporidian species with available genome sequences, the only known instance of a eukaryotic group lacking this conserved pathway. The microsporidia are obligate intracellular pathogens with highly reduced genomes, and we hypothesize that they lost the Tor pathway as they adapted and streamlined their genomes for intracellular growth in a nutrient-rich environment. Two TOR paralogs are present in several fungal species as a result of either a whole genome duplication or independent gene/segmental duplication events. One such event was identified in the amphibian pathogen Batrachochytrium dendrobatidis, a chytrid responsible for worldwide global amphibian declines and extinctions. CONCLUSIONS: The repeated independent duplications of the TOR gene in the fungal kingdom might reflect selective pressure acting upon this kinase that populates two proteinaceous complexes with different cellular roles. These comparative genomic analyses illustrate the evolutionary trajectory of a central nutrient-sensing cascade that enables diverse eukaryotic organisms to respond to their natural environments.

Authors
Shertz, CA; Bastidas, RJ; Li, W; Heitman, J; Cardenas, ME
MLA Citation
Shertz, CA, Bastidas, RJ, Li, W, Heitman, J, and Cardenas, ME. "Conservation, duplication, and loss of the Tor signaling pathway in the fungal kingdom. (Published online)" BMC Genomics 11 (September 23, 2010): 510-.
Website
http://hdl.handle.net/10161/4347
PMID
20863387
Source
pubmed
Published In
BMC Genomics
Volume
11
Publish Date
2010
Start Page
510
DOI
10.1186/1471-2164-11-510

Surfactant protein D binding to Aspergillus fumigatus hyphae is calcineurin-sensitive.

Surfactant protein D (SP-D) plays a central role in pulmonary innate immune responses to microbes and allergens, often enhancing clearance of inhaled material. Although SP-D functions during bacterial and viral infections are well established, much less is known about its possible roles during invasive fungal infections. Aspergillus fumigatus is a prominent fungal pathogen in immunocompromised individuals, and can cause allergic or invasive aspergillosis. SP-D has been shown to be protective against both of these disease modalities. The moieties present on the fungal surface responsible for SP-D binding remain largely unclear, although cell wall 1,3-beta-D-glucan is bound by SP-D in other fungal species. There is little information regarding the interaction of SP-D with A. fumigatus hyphae which are responsible for the invasive form of disease. Here, we show that SP-D binding to A. fumigatus hyphae is sensitive to the activity of the calcium-activated protein phosphatase calcineurin. Deletion of the catalytic subunit calcineurin A (DeltacnaA) or pharmacologic inhibition of calcineurin through FK506 abrogated SP-D binding. In contrast, SP-D binding to Cruptococcus neoformans was calcineurin-independent. Pharmacologic inhibition of A. fumigatus cell wall components by caspofungin (inhibits 1,3-beta-D-glucan synthesis) and nikkomycin Z (inhibits chitin synthesis) increased SP-D binding to the wild-type strain. In contrast, SP-D binding increased in the DeltacnaA strain only after nikkomycin Z treatment. We conclude that SP-D binding to A. fumigatus hyphae is calcineurin-sensitive, presumably as a consequence of calcineurin's role in regulating production of key cell wall binding partners, such as 1,3-beta-D-glucan. Elucidation of the interaction between lung innate immune factors and A. fumigatus could lead to the development of novel therapeutic interventions.

Authors
Geunes-Boyer, S; Heitman, J; Wright, JR; Steinbach, WJ
MLA Citation
Geunes-Boyer, S, Heitman, J, Wright, JR, and Steinbach, WJ. "Surfactant protein D binding to Aspergillus fumigatus hyphae is calcineurin-sensitive." Med Mycol 48.4 (June 2010): 580-588.
PMID
20141481
Source
pubmed
Published In
Medical Mycology (Informa)
Volume
48
Issue
4
Publish Date
2010
Start Page
580
End Page
588
DOI
10.3109/13693780903401682

The evolution of sex: a perspective from the fungal kingdom.

Sex is shrouded in mystery. Not only does it preferentially occur in the dark for both fungi and many animals, but evolutionary biologists continue to debate its benefits given costs in light of its pervasive nature. Experimental studies of the benefits and costs of sexual reproduction with fungi as model systems have begun to provide evidence that the balance between sexual and asexual reproduction shifts in response to selective pressures. Given their unique evolutionary history as opisthokonts, along with metazoans, fungi serve as exceptional models for the evolution of sex and sex-determining regions of the genome (the mating type locus) and for transitions that commonly occur between outcrossing/self-sterile and inbreeding/self-fertile modes of reproduction. We review here the state of the understanding of sex and its evolution in the fungal kingdom and also areas where the field has contributed and will continue to contribute to illuminating general principles and paradigms of sexual reproduction.

Authors
Lee, SC; Ni, M; Li, W; Shertz, C; Heitman, J
MLA Citation
Lee, SC, Ni, M, Li, W, Shertz, C, and Heitman, J. "The evolution of sex: a perspective from the fungal kingdom." Microbiol Mol Biol Rev 74.2 (June 2010): 298-340. (Review)
PMID
20508251
Source
pubmed
Published In
Microbiology and molecular biology reviews : MMBR
Volume
74
Issue
2
Publish Date
2010
Start Page
298
End Page
340
DOI
10.1128/MMBR.00005-10

Role of an expanded inositol transporter repertoire in Cryptococcus neoformans sexual reproduction and virulence.

Cryptococcus neoformans and Cryptococcus gattii are globally distributed human fungal pathogens and the leading causes of fungal meningitis. Recent studies reveal that myo-inositol is an important factor for fungal sexual reproduction. That C. neoformans can utilize myo-inositol as a sole carbon source and the existence of abundant inositol in the human central nervous system suggest that inositol is important for Cryptococcus development and virulence. In accord with this central importance of inositol, an expanded myo-inositol transporter (ITR) gene family has been identified in Cryptococcus. This gene family contains two phylogenetically distinct groups, with a total of 10 or more members in C. neoformans and at least six members in the sibling species C. gattii. These inositol transporter genes are differentially expressed under inositol-inducing conditions based on quantitative real-time PCR analyses. Expression of ITR genes in a Saccharomyces cerevisiae itr1 itr2 mutant lacking inositol transport can complement the slow-growth phenotype of this strain, confirming that ITR genes are bona fide inositol transporters. Gene mutagenesis studies reveal that the Itr1 and Itr1A transporters are important for myo-inositol stimulation of mating and that functional redundancies among the myo-inositol transporters likely exist. Deletion of the inositol 1-phosphate synthase gene INO1 in an itr1 or itr1a mutant background compromised virulence in a murine inhalation model, indicating the importance of inositol sensing and acquisition for fungal infectivity. Our study provides a platform for further understanding the roles of inositol in fungal physiology and virulence.

Authors
Xue, C; Liu, T; Chen, L; Li, W; Liu, I; Kronstad, JW; Seyfang, A; Heitman, J
MLA Citation
Xue, C, Liu, T, Chen, L, Li, W, Liu, I, Kronstad, JW, Seyfang, A, and Heitman, J. "Role of an expanded inositol transporter repertoire in Cryptococcus neoformans sexual reproduction and virulence. (Published online)" MBio 1.1 (May 18, 2010).
PMID
20689743
Source
pubmed
Published In
mBio
Volume
1
Issue
1
Publish Date
2010
DOI
10.1128/mBio.00084-10

Evolution of the sex-related locus and genomic features shared in microsporidia and fungi.

BACKGROUND: Microsporidia are obligate intracellular, eukaryotic pathogens that infect a wide range of animals from nematodes to humans, and in some cases, protists. The preponderance of evidence as to the origin of the microsporidia reveals a close relationship with the fungi, either within the kingdom or as a sister group to it. Recent phylogenetic studies and gene order analysis suggest that microsporidia share a particularly close evolutionary relationship with the zygomycetes. METHODOLOGY/PRINCIPAL FINDINGS: Here we expanded this analysis and also examined a putative sex-locus for variability between microsporidian populations. Whole genome inspection reveals a unique syntenic gene pair (RPS9-RPL21) present in the vast majority of fungi and the microsporidians but not in other eukaryotic lineages. Two other unique gene fusions (glutamyl-prolyl tRNA synthetase and ubiquitin-ribosomal subunit S30) that are present in metazoans, choanoflagellates, and filasterean opisthokonts are unfused in the fungi and microsporidians. One locus previously found to be conserved in many microsporidian genomes is similar to the sex locus of zygomycetes in gene order and architecture. Both sex-related and sex loci harbor TPT, HMG, and RNA helicase genes forming a syntenic gene cluster. We sequenced and analyzed the sex-related locus in 11 different Encephalitozoon cuniculi isolates and the sibling species E. intestinalis (3 isolates) and E. hellem (1 isolate). There was no evidence for an idiomorphic sex-related locus in this Encephalitozoon species sample. According to sequence-based phylogenetic analyses, the TPT and RNA helicase genes flanking the HMG genes are paralogous rather than orthologous between zygomycetes and microsporidians. CONCLUSION/SIGNIFICANCE: The unique genomic hallmarks between microsporidia and fungi are independent of sequence based phylogenetic comparisons and further contribute to define the borders of the fungal kingdom and support the classification of microsporidia as unusual derived fungi. And the sex/sex-related loci appear to have been subject to frequent gene conversion and translocations in microsporidia and zygomycetes.

Authors
Lee, SC; Corradi, N; Doan, S; Dietrich, FS; Keeling, PJ; Heitman, J
MLA Citation
Lee, SC, Corradi, N, Doan, S, Dietrich, FS, Keeling, PJ, and Heitman, J. "Evolution of the sex-related locus and genomic features shared in microsporidia and fungi. (Published online)" PLoS One 5.5 (May 7, 2010): e10539-.
Website
http://hdl.handle.net/10161/4539
PMID
20479876
Source
pubmed
Published In
PloS one
Volume
5
Issue
5
Publish Date
2010
Start Page
e10539
DOI
10.1371/journal.pone.0010539

Emergence and pathogenicity of highly virulent Cryptococcus gattii genotypes in the northwest United States.

Cryptococcus gattii causes life-threatening disease in otherwise healthy hosts and to a lesser extent in immunocompromised hosts. The highest incidence for this disease is on Vancouver Island, Canada, where an outbreak is expanding into neighboring regions including mainland British Columbia and the United States. This outbreak is caused predominantly by C. gattii molecular type VGII, specifically VGIIa/major. In addition, a novel genotype, VGIIc, has emerged in Oregon and is now a major source of illness in the region. Through molecular epidemiology and population analysis of MLST and VNTR markers, we show that the VGIIc group is clonal and hypothesize it arose recently. The VGIIa/IIc outbreak lineages are sexually fertile and studies support ongoing recombination in the global VGII population. This illustrates two hallmarks of emerging outbreaks: high clonality and the emergence of novel genotypes via recombination. In macrophage and murine infections, the novel VGIIc genotype and VGIIa/major isolates from the United States are highly virulent compared to similar non-outbreak VGIIa/major-related isolates. Combined MLST-VNTR analysis distinguishes clonal expansion of the VGIIa/major outbreak genotype from related but distinguishable less-virulent genotypes isolated from other geographic regions. Our evidence documents emerging hypervirulent genotypes in the United States that may expand further and provides insight into the possible molecular and geographic origins of the outbreak.

Authors
Byrnes, EJ; Li, W; Lewit, Y; Ma, H; Voelz, K; Ren, P; Carter, DA; Chaturvedi, V; Bildfell, RJ; May, RC; Heitman, J
MLA Citation
Byrnes, EJ, Li, W, Lewit, Y, Ma, H, Voelz, K, Ren, P, Carter, DA, Chaturvedi, V, Bildfell, RJ, May, RC, and Heitman, J. "Emergence and pathogenicity of highly virulent Cryptococcus gattii genotypes in the northwest United States. (Published online)" PLoS Pathog 6.4 (April 22, 2010): e1000850-.
Website
http://hdl.handle.net/10161/4598
PMID
20421942
Source
pubmed
Published In
PLoS pathogens
Volume
6
Issue
4
Publish Date
2010
Start Page
e1000850
DOI
10.1371/journal.ppat.1000850

Morphological and genomic characterization of Filobasidiella depauperata: a homothallic sibling species of the pathogenic cryptococcus species complex.

The fungal species Cryptococcus neoformans and Cryptococcus gattii cause respiratory and neurological disease in animals and humans following inhalation of basidiospores or desiccated yeast cells from the environment. Sexual reproduction in C. neoformans and C. gattii is controlled by a bipolar system in which a single mating type locus (MAT) specifies compatibility. These two species are dimorphic, growing as yeast in the asexual stage, and producing hyphae, basidia, and basidiospores during the sexual stage. In contrast, Filobasidiella depauperata, one of the closest related species, grows exclusively as hyphae and it is found in association with decaying insects. Examination of two available strains of F. depauperata showed that the life cycle of this fungal species shares features associated with the unisexual or same-sex mating cycle in C. neoformans. Therefore, F. depauperata may represent a homothallic and possibly an obligately sexual fungal species. RAPD genotyping of 39 randomly isolated progeny from isolate CBS7855 revealed a new genotype pattern in one of the isolated basidiospores progeny, therefore suggesting that the homothallic cycle in F. depauperata could lead to the emergence of new genotypes. Phylogenetic analyses of genes linked to MAT in C. neoformans indicated that two of these genes in F. depauperata, MYO2 and STE20, appear to form a monophyletic clade with the MATa alleles of C. neoformans and C. gattii, and thus these genes may have been recruited to the MAT locus before F. depauperata diverged. Furthermore, the ancestral MATa locus may have undergone accelerated evolution prior to the divergence of the pathogenic Cryptococcus species since several of the genes linked to the MATa locus appear to have a higher number of changes and substitutions than their MATalpha counterparts. Synteny analyses between C. neoformans and F. depauperata showed that genomic regions on other chromosomes displayed conserved gene order. In contrast, the genes linked to the MAT locus of C. neoformans showed a higher number of chromosomal translocations in the genome of F. depauperata. We therefore propose that chromosomal rearrangements appear to be a major force driving speciation and sexual divergence in these closely related pathogenic and saprobic species.

Authors
Rodriguez-Carres, M; Findley, K; Sun, S; Dietrich, FS; Heitman, J
MLA Citation
Rodriguez-Carres, M, Findley, K, Sun, S, Dietrich, FS, and Heitman, J. "Morphological and genomic characterization of Filobasidiella depauperata: a homothallic sibling species of the pathogenic cryptococcus species complex. (Published online)" PLoS One 5.3 (March 10, 2010): e9620-.
Website
http://hdl.handle.net/10161/4531
PMID
20224779
Source
pubmed
Published In
PloS one
Volume
5
Issue
3
Publish Date
2010
Start Page
e9620
DOI
10.1371/journal.pone.0009620

Organization and evolutionary trajectory of the mating type (MAT) locus in dermatophyte and dimorphic fungal pathogens.

Sexual reproduction in fungi is governed by a specialized genomic region, the mating type (MAT) locus, whose gene identity, organization, and complexity are diverse. We identified the MAT locus of five dermatophyte fungal pathogens (Microsporum gypseum, Microsporum canis, Trichophyton equinum, Trichophyton rubrum, and Trichophyton tonsurans) and a dimorphic fungus, Paracoccidioides brasiliensis, and performed phylogenetic analyses. The identified MAT locus idiomorphs of M. gypseum control cell type identity in mating assays, and recombinant progeny were produced. Virulence tests in Galleria mellonella larvae suggest the two mating types of M. gypseum may have equivalent virulence. Synteny analysis revealed common features of the MAT locus shared among these five dermatophytes: namely, a small size ( approximately 3 kb) and a novel gene arrangement. The SLA2, COX13, and APN2 genes, which flank the MAT locus in other Ascomycota are instead linked on one side of the dermatophyte MAT locus. In addition, the transcriptional orientations of the APN2 and COX13 genes are reversed compared to the dimorphic fungi Histoplasma capsulatum, Coccidioides immitis, and Coccidioides posadasii. A putative transposable element, pogo, was found to have inserted in the MAT1-2 idiomorph of one P. brasiliensis strain but not others. In conclusion, the evolution of the MAT locus of the dermatophytes and dimorphic fungi from the last common ancestor has been punctuated by both gene acquisition and expansion, and asymmetric gene loss. These studies further support a foundation to develop molecular and genetic tools for dermatophyte and dimorphic human fungal pathogens.

Authors
Li, W; Metin, B; White, TC; Heitman, J
MLA Citation
Li, W, Metin, B, White, TC, and Heitman, J. "Organization and evolutionary trajectory of the mating type (MAT) locus in dermatophyte and dimorphic fungal pathogens." Eukaryot Cell 9.1 (January 2010): 46-58.
PMID
19880755
Source
pubmed
Published In
Eukaryotic cell
Volume
9
Issue
1
Publish Date
2010
Start Page
46
End Page
58
DOI
10.1128/EC.00259-09

Structure, function, and phylogeny of the mating locus in the Rhizopus oryzae complex

The Rhizopus oryzae species complex is a group of zygomycete fungi that are common, cosmopolitan saprotrophs. Some strains are used beneficially for production of Asian fermented foods but they can also act as opportunistic human pathogens. Although R. oryzae reportedly has a heterothallic (+/2) mating system, most strains have not been observed to undergo sexual reproduction and the genetic structure of its mating locus has not been characterized. Here we report on the mating behavior and genetic structure of the mating locus for 54 isolates of the R. oryzae complex. All 54 strains have a mating locus similar in overall organization to Phycomyces blakesleeanus and Mucor circinelloides (Mucoromycotina, Zygomycota). In all of these fungi, the minus (2) allele features the SexM high mobility group (HMG) gene flanked by an RNA helicase gene and a TP transporter gene (TPT). Within the R. oryzae complex, the plus (+) mating allele includes an inserted region that codes for a BTB/POZ domain gene and the SexP HMG gene. Phylogenetic analyses of multiple genes, including the mating loci (HMG, TPT, RNA helicase), ITS1-5.8S-ITS2 rDNA, RPB2, and LDH genes, identified two distinct groups of strains. These correspond to previously described sibling species R. oryzae sensu stricto and R. delemar. Within each species, discordant gene phylogenies among multiple loci suggest an outcrossing population structure. The hypothesis of random-mating is also supported by a 50:50 ratio of plus and minus mating types in both cryptic species. When crossed with tester strains of the opposite mating type, most isolates of R. delemar failed to produce zygospores, while isolates of R. oryzae produced sterile zygospores. In spite of the reluctance of most strains to mate in vitro, the conserved sex locus structure and evidence for outcrossing suggest that a normal sexual cycle occurs in both species. © 2010 Gryganskyi et al.

Authors
Gryganskyi, AP; Lee, SC; Litvintseva, AP; Smith, ME; Bonito, G; Porter, TM; Anishchenko, IM; Heitman, J; Vilgalys, R
MLA Citation
Gryganskyi, AP, Lee, SC, Litvintseva, AP, Smith, ME, Bonito, G, Porter, TM, Anishchenko, IM, Heitman, J, and Vilgalys, R. "Structure, function, and phylogeny of the mating locus in the Rhizopus oryzae complex." PLoS ONE 5.12 (2010).
Website
http://hdl.handle.net/10161/4588
PMID
21151560
Source
scival
Published In
PloS one
Volume
5
Issue
12
Publish Date
2010
DOI
10.1371/journal.pone.0015273

On the roles of calcineurin in fungal growth and pathogenesis

Calcineurin is a calcium-activated phosphatase that controls morphogenesis and stress responses in eukaryotes. Fungal pathogens have adopted the calcineurin pathway to survive and effectively propagate within the host. The difficulty in treating fungal infections stems from similarities between pathogen and host eukaryotic cells. Using calcineurin inhibitors such as cyclosporin A or tacrolimus (FK506) in combination with antifungal drugs, including azoles or echinocandins, renders these drugs fungicidal, even towards drug-resistant species or strains, making calcineurin a promising drug target. This article summarizes the current understanding of the calcineurin pathway and its roles in governing the growth and virulence of pathogenic fungi, and compares and contrasts the roles of calcineurin in fungal pathogens that infect humans (Candida albicans and Cryptococcus neoformans) or plants (Magnaporthe oryzae and Ustilago maydis). Further investigation of calcineurin biology will advance opportunities to develop novel antifungal therapeutic approaches and provide insight into the evolution of virulence. © 2010 Springer Science+Business Media, LLC.

Authors
Chen, Y-L; Kozubowski, L; Cardenas, ME; Heitman, J
MLA Citation
Chen, Y-L, Kozubowski, L, Cardenas, ME, and Heitman, J. "On the roles of calcineurin in fungal growth and pathogenesis." Current Fungal Infection Reports 4.4 (2010): 244-255.
Source
scival
Published In
Current Fungal Infection Reports
Volume
4
Issue
4
Publish Date
2010
Start Page
244
End Page
255
DOI
10.1007/s12281-010-0027-5

Characterizing the role of RNA silencing components in Cryptococcus neoformans

The RNA interference (RNAi) mediated by homology-dependent degradation of the target mRNA with small RNA molecules plays a key role in controlling transcription and translation processes in a number of eukaryotic organisms. The RNAi machinery is also evolutionarily conserved in a wide variety of fungal species, including pathogenic fungi. To elucidate the physiological functions of the RNAi pathway in Cryptococcus neoformans that causes fungal meningitis, here we performed genetic analyses for genes encoding Argonaute (AGO1 and AGO2), RNA-dependent RNA polymerase (RDP1), and Dicers (DCR1 and DCR2) in both serotype A and D C. neoformans. The present study shows that Ago1, Rdp1, and Dcr2 are the major components of the RNAi process occurring in C. neoformans. However, the RNAi machinery is not involved in regulation of production of two virulence factors (capsule and melanin), sexual differentiation, and diverse stress response. Comparative transcriptome analysis of the serotype A and D RNAi mutants revealed that only modest changes occur in the genome-wide transcriptome profiles when the RNAi process was perturbed. Notably, the serotype D rdp1Δ mutants showed an increase in transcript abundance of active retrotransposons and transposons, such as T2 and T3, the latter of which is a novel serotype D-specific transposon of C. neoformans. In a wild type background both T2 and T3 were found to be weakly active mobile elements, although we found no evidence of Cnl1 retrotransposon mobility. In contrast, all three transposable elements exhibited enhanced mobility in the rdp1Δ mutant strain. In conclusion, the RNAi pathway plays an important role in controlling transposon activity and genome integrity of C. neoformans. © 2010 Elsevier Inc.

Authors
Janbon, G; Maeng, S; Yang, D-H; Ko, Y-J; Jung, K-W; Moyrand, F; Floyd, A; Heitman, J; Bahn, Y-S
MLA Citation
Janbon, G, Maeng, S, Yang, D-H, Ko, Y-J, Jung, K-W, Moyrand, F, Floyd, A, Heitman, J, and Bahn, Y-S. "Characterizing the role of RNA silencing components in Cryptococcus neoformans." Fungal Genetics and Biology 47.12 (2010): 1070-1080.
PMID
21067947
Source
scival
Published In
Fungal Genetics and Biology
Volume
47
Issue
12
Publish Date
2010
Start Page
1070
End Page
1080
DOI
10.1016/j.fgb.2010.10.005

Comparative transcriptome analysis of the CO2 sensing pathway via differential expression of carbonic anhydrase in Cryptococcus neoformans

Carbon dioxide (CO2) sensing and metabolism via carbonic anhydrases (CAs) play pivotal roles in survival and proliferation of pathogenic fungi infecting human hosts from natural environments due to the drastic difference in CO2 levels. In Cryptococcus neoformans, which causes fatal fungal meningoencephalitis, the Can2 CA plays essential roles during both cellular growth in air and sexual differentiation of the pathogen. However the signaling networks downstream of Can2 are largely unknown. To address this question, the present study employed comparative transcriptome DNA microarray analysis of a C. neoformans strain in which CAN2 expression is artificially controlled by the CTR4 (copper transporter) promoter. The PCTR4::CAN2 strain showed growth defects in a CO2-dependent manner when CAN2 was repressed but resumed normal growth when CAN2 was overexpressed. The Can2-dependent genes identified by the transcriptome analysis include FAS1 (fatty acid synthase 1) and GPB1 (G-protein β subunit), supporting the roles of Can2 in fatty acid biosynthesis and sexual differentiation. Cas3, a capsular structure designer protein, was also discovered to be Can2-dependent and yet was not involved in CO2-mediated capsule induction. Most notably, a majority of Can2-dependent genes were environmental stress-regulated (ESR) genes. Supporting this, the CAN2 overexpression strain was hypersensitive to oxidative and genotoxic stress as well as antifungal drugs, such as polyene and azole drugs, potentially due to defective membrane integrity. Finally, an oxidative stress-responsive Atf1 transcription factor was also found to be Can2-dependent. Atf1 not only plays an important role in diverse stress responses, including thermotolerance and antifungal drug resistance, but also represses melanin and capsule production in C. neoformans. In conclusion, this study provides insights into the comprehensive signaling networks orchestrated by CA/CO2-sensing pathways in pathogenic fungi. Copyright © 2010 by the Genetics Society of America.

Authors
Kim, MS; Ko, Y-J; Maeng, S; Floyd, A; Heitman, J; Bahn, Y-S
MLA Citation
Kim, MS, Ko, Y-J, Maeng, S, Floyd, A, Heitman, J, and Bahn, Y-S. "Comparative transcriptome analysis of the CO2 sensing pathway via differential expression of carbonic anhydrase in Cryptococcus neoformans." Genetics 185.4 (2010): 1207-1219.
PMID
20516494
Source
scival
Published In
Genetics
Volume
185
Issue
4
Publish Date
2010
Start Page
1207
End Page
1219
DOI
10.1534/genetics.110.118315

Ferrochelatase is a conserved downstream target of the blue light-sensing White collar complex in fungi

Light is a universal signal perceived by organisms, including fungi, in which light regulates common and unique biological processes depending on the species. Previous research has established that conserved proteins, originally called White collar 1 and 2 from the ascomycete Neurospora crassa, regulate UV/blue light sensing. Homologous proteins function in distant relatives of N. crassa, including the basidiomycetes and zygomycetes, which diverged as long as a billion years ago. Here we conducted microarray experiments on the basidiomycete fungus Cryptococcus neoformans to identify light-regulated genes. Surprisingly, only a single gene was induced by light above the commonly used twofold threshold. This gene, HEM15, is predicted to encode a ferrochelatase that catalyses the final step in haem biosynthesis from highly photoreactive porphyrins. The C. neoformans gene complements a Saccharomyces cerevisiae hem15DΔstrain and is essential for viability, and the Hem15 protein localizes to mitochondria, three lines of evidence that the gene encodes ferrochelatase. Regulation of HEM15 by light suggests a mechanism by which bwc1/bwc2 mutants are photosensitive and exhibit reduced virulence. We show that ferrochelatase is also light-regulated in a white collar-dependent fashion in N. crassa and the zygomycete Phycomyces blakesleeanus, indicating that ferrochelatase is an ancient target of photoregulation in the fungal kingdom. © 2010 SGM.

Authors
Idnurm, A; Heitman, J
MLA Citation
Idnurm, A, and Heitman, J. "Ferrochelatase is a conserved downstream target of the blue light-sensing White collar complex in fungi." Microbiology 156.8 (2010): 2393-2407.
Website
http://hdl.handle.net/10161/4166
PMID
20488877
Source
scival
Published In
Microbiology (Reading, England)
Volume
156
Issue
8
Publish Date
2010
Start Page
2393
End Page
2407
DOI
10.1099/mic.0.039222-0

Evolution of eukaryotic microbial pathogens via covert sexual reproduction

Sexual reproduction enables eukaryotic organisms to reassort genetic diversity and purge deleterious mutations, producing better-fit progeny. Sex arose early and pervades eukaryotes. Fungal and parasite pathogens once thought asexual have maintained cryptic sexual cycles, including unisexual or parasexual reproduction. As pathogens become niche and host adapted, sex appears to specialize to promote inbreeding and clonality yet maintain outcrossing potential. During self-fertile sexual modes, sex itself may generate genetic diversity de novo. Mating-type loci govern fungal sexual identity; how parasites establish sexual identity is unknown. Comparing and contrasting fungal and parasite sex promises to reveal howmicrobial pathogens evolved and are evolving. © 2010 Elsevier Inc.

Authors
Heitman, J
MLA Citation
Heitman, J. "Evolution of eukaryotic microbial pathogens via covert sexual reproduction." Cell Host and Microbe 8.1 (2010): 86-99.
PMID
20638645
Source
scival
Published In
Cell Host & Microbe
Volume
8
Issue
1
Publish Date
2010
Start Page
86
End Page
99
DOI
10.1016/j.chom.2010.06.011

The mating type locus (MAT) and sexual reproduction of Cryptococcus heveanensis: Insights into the evolution of sex and sex-determining chromosomal regions in fungi

Mating in basidiomycetous fungi is often controlled by two unlinked, multiallelic loci encoding homeodomain transcription factors or pheromones/pheromone receptors. In contrast to this tetrapolar organization, Cryptococcus neoformans/Cryptococcus gattii have a bipolar mating system, and a single biallelic locus governs sexual reproduction. The C. neoformans MAT locus is unusually large (>100 kb), contains >20 genes, and enhances virulence. Previous comparative genomic studies provided insights into how this unusual MAT locus might have evolved involving gene acquisitions into two unlinked loci and fusion into one contiguous locus, converting an ancestral tetrapolar system to a bipolar one. Here we tested this model by studying Cryptococcus heveanensis, a sister species to the pathogenic Cryptococcus species complex. An extant sexual cycle was discovered; co-incubating fertile isolates results in the teleomorph (Kwoniella heveanensis) with dikaryotic hyphae, clamp connections, septate basidia, and basidiospores. To characterize the C. heveanensis MAT locus, a fosmid library was screened with C. neoformans/C. gattii MAT genes. Positive fosmids were sequenced and assembled to generate two large probably unlinked MAT gene clusters: one corresponding to the homeodomain locus and the other to the pheromone/receptor locus. Strikingly, two divergent homeodomain genes (SXI1, SXI2) are present, similar to the bE/bW Ustilago maydis paradigm, suggesting one or the other homeodomain gene was recently lost in C. neoformans/C. gattii. Sequencing MAT genes from other C. heveanensis isolates revealed a multiallelic homeodomain locus and at least a biallelic pheromone/receptor locus, similar to known tetrapolar species. Taken together, these studies reveal an extant C. heveanensis sexual cycle, define the structure of its MAT locus consistent with tetrapolar mating, and support the proposed evolutionary model for the bipolar Cryptococcus MAT locus revealing transitions in sexuality concomitant with emergence of a pathogenic clade. These studies provide insight into convergent processes that independently punctuated evolution of sex-determining loci and sex chromosomes in fungi, plants, and animals. © 2010 Metin et al.

Authors
Metin, B; Findley, K; Heitman, J
MLA Citation
Metin, B, Findley, K, and Heitman, J. "The mating type locus (MAT) and sexual reproduction of Cryptococcus heveanensis: Insights into the evolution of sex and sex-determining chromosomal regions in fungi." PLoS Genetics 6.5 (2010): 14--.
PMID
20502678
Source
scival
Published In
PLoS genetics
Volume
6
Issue
5
Publish Date
2010
Start Page
14-
DOI
10.1371/journal.pgen.1000961

Cryptococcal cell morphology affects host cell interactions and pathogenicity

Cryptococcus neoformans is a common life-threatening human fungal pathogen. The size of cryptococcal cells is typically 5 to 10 fim. Cell enlargement was observed in vivo, producing cells up to 100 fim. These morphological changes in cell size affected pathogenicity via reducing phagocytosis by host mononuclear cells, increasing resistance to oxidative and nitrosative stress, and correlated with reduced penetration of the central nervous system. Cell enlargement was stimulated by coinfection with strains of opposite mating type, and ste3aD pheromone receptor mutant strains had reduced cell enlargement. Finally, analysis of DNA content in this novel cell type revealed that these enlarged cells were polyploid, uninucleate, and produced daughter cells in vivo. These results describe a novel mechanism by which C. neoformans evades host phagocytosis to allow survival of a subset of the population at early stages of infection. Thus, morphological changes play unique and specialized roles during infection. © 2010 Okagaki et al.

Authors
Okagaki, LH; Strain, AK; Nielsen, JN; Charlier, C; Baltes, NJ; Chrétien, F; Heitman, JH; Dromer, F; Nielsen, KN
MLA Citation
Okagaki, LH, Strain, AK, Nielsen, JN, Charlier, C, Baltes, NJ, Chrétien, F, Heitman, JH, Dromer, F, and Nielsen, KN. "Cryptococcal cell morphology affects host cell interactions and pathogenicity." PLoS Pathogens 6.6 (2010).
Website
http://hdl.handle.net/10161/4602
PMID
20585559
Source
scival
Published In
PLoS pathogens
Volume
6
Issue
6
Publish Date
2010
DOI
10.1371/journal.ppat.1000953

Transcription factors Mat2 and Znf2 operate cellular circuits orchestrating opposite- and same-sex mating in Cryptococcus neoformans

Cryptococcus neoformans is a human fungal pathogen that undergoes a dimorphic transition from a unicellular yeast to multicellular hyphae during opposite sex (mating) and unisexual reproduction (same-sex mating). Opposite- and same-sex mating are induced by similar environmental conditions and involve many shared components, including the conserved pheromone sensing Cpk1 MAPK signal transduction cascade that governs the dimorphic switch in C. neoformans. However, the homeodomain cell identity proteins Sxi1α/Sxi2a encoded by the mating type locus that are essential for completion of sexual reproduction following cell-cell fusion during opposite-sex mating are dispensable for same-sex mating. Therefore, identification of downstream targets of the Cpk1 MAPK pathway holds the key to understanding molecular mechanisms governing the two distinct developmental fates. Thus far, homology-based approaches failed to identify downstream transcription factors which may therefore be species-specific. Here, we applied insertional mutagenesis via Agrobacteriummediated transformation and transcription analysis using whole genome microarrays to identify factors involved in C. neoformans differentiation. Two transcription factors, Mat2 and Znf2, were identified as key regulators of hyphal growth during same- and opposite-sex mating. Mat2 is an HMG domain factor, and Znf2 is a zinc finger protein; neither is encoded by the mating type locus. Genetic, phenotypic, and transcriptional analyses of Mat2 and Znf2 provide evidence that Mat2 is a downstream transcription factor of the Cpk1 MAPK pathway whereas Znf2 functions as a more terminal hyphal morphogenesis determinant. Although the components of the MAPK pathway including Mat2 are not required for virulence in animal models, Znf2, as a hyphal morphology determinant, is a negative regulator of virulence. Further characterization of these elements and their target circuits will reveal genes controlling biological processes central to fungal development and virulence. © 2010 Lin et al.

Authors
Lin, X; Jackson, JC; Feretzaki, M; Xue, C; Heitman, J
MLA Citation
Lin, X, Jackson, JC, Feretzaki, M, Xue, C, and Heitman, J. "Transcription factors Mat2 and Znf2 operate cellular circuits orchestrating opposite- and same-sex mating in Cryptococcus neoformans." PLoS Genetics 6.5 (2010): 30--.
PMID
20485569
Source
scival
Published In
PLoS genetics
Volume
6
Issue
5
Publish Date
2010
Start Page
30-
DOI
10.1371/journal.pgen.1000953

Emergence and pathogenicity of highly virulent Cryptococcus gattii genotypes in the Northwest United States

Cryptococcus gattii causes life-threatening disease in otherwise healthy hosts and to a lesser extent in immunocompromised hosts. The highest incidence for this disease is on Vancouver Island, Canada, where an outbreak is expanding into neighboring regions including mainland British Columbia and the United States. This outbreak is caused predominantly by C. gattii molecular type VGII, specifically VGIIa/major. In addition, a novel genotype, VGIIc, has emerged in Oregon and is now a major source of illness in the region. Through molecular epidemiology and population analysis of MLST and VNTR markers, we show that the VGIIc group is clonal and hypothesize it arose recently. The VGIIa/IIc outbreak lineages are sexually fertile and studies support ongoing recombination in the global VGII population. This illustrates two hallmarks of emerging outbreaks: high clonality and the emergence of novel genotypes via recombination. In macrophage and murine infections, the novel VGIIc genotype and VGIIa/major isolates from the United States are highly virulent compared to similar non-outbreak VGIIa/major-related isolates. Combined MLST-VNTR analysis distinguishes clonal expansion of the VGIIa/major outbreak genotype from related but distinguishable less-virulent genotypes isolated from other geographic regions. Our evidence documents emerging hypervirulent genotypes in the United States that may expand further and provides insight into the possible molecular and geographic origins of the outbreak. © 2010 Byrnes et al.

Authors
III, EJB; Li, W; Lewit, Y; Ma, H; Voelz, K; Ren, P; Carter, DA; Chaturvedi, V; Bildfell, RJ; May, RC; Heitman, J
MLA Citation
III, EJB, Li, W, Lewit, Y, Ma, H, Voelz, K, Ren, P, Carter, DA, Chaturvedi, V, Bildfell, RJ, May, RC, and Heitman, J. "Emergence and pathogenicity of highly virulent Cryptococcus gattii genotypes in the Northwest United States." PLoS Pathogens 6.4 (2010): 1-16.
Source
scival
Published In
PLoS pathogens
Volume
6
Issue
4
Publish Date
2010
Start Page
1
End Page
16
DOI
10.1371/journal.ppat.1000850

Comparative transcriptome analysis reveals novel roles of the ras and cyclic AMP signaling pathways in environmental stress response and antifungal drug sensitivity in Cryptococcus neoformans

The cyclic AMP (cAMP) pathway plays a central role in the growth, differentiation, and virulence of pathogenic fungi, including Cryptococcus neoformans. Three upstream signaling regulators of adenylyl cyclase (Cac1), Ras, Aca1, and Gpa1, have been demonstrated to control the cAMP pathway in C. neoformans, but their functional relationship remains elusive. We performed a genome-wide transcriptome analysis with a DNA microarray using the ras1δ, gpa1δ, cac1δ, aca1δ, and pka1δ pka2δ mutants. The aca1δ, gpa1δ, cac1δ, and pka1δ pka2δ mutants displayed similar transcriptome patterns, whereas the ras1δ mutant exhibited transcriptome patterns distinct from those of the wild type and the cAMP mutants. Interestingly, a number of environmental stress response genes are modulated differentially in the ras1δ and cAMP mutants. In fact, the Ras signaling pathway was found to be involved in osmotic and genotoxic stress responses and the maintenance of cell wall integrity via the Cdc24-dependent signaling pathway. Notably, the Ras and cAMP mutants exhibited hypersensitivity to a polyene drug, amphotericin B, without showing effects on ergosterol biosynthesis, which suggested a novel method of antifungal combination therapy. Among the cAMP-dependent gene products that we characterized, two small heat shock proteins, Hsp12 and Hsp122, were found to be involved in the polyene antifungal drug susceptibility of C. neoformans. © 2010, American Society for Microbiology. All Rights Reserved.

Authors
Maeng, S; Ko, Y-J; Kim, G-B; Jung, K-W; Floyd, A; Heitman, J; Bahn, Y-S
MLA Citation
Maeng, S, Ko, Y-J, Kim, G-B, Jung, K-W, Floyd, A, Heitman, J, and Bahn, Y-S. "Comparative transcriptome analysis reveals novel roles of the ras and cyclic AMP signaling pathways in environmental stress response and antifungal drug sensitivity in Cryptococcus neoformans." Eukaryotic Cell 9.3 (2010): 360-378.
PMID
20097740
Source
scival
Published In
Eukaryotic cell
Volume
9
Issue
3
Publish Date
2010
Start Page
360
End Page
378
DOI
10.1128/EC.00309-09

Elucidating the Candida albicans calcineurin signaling cascade controlling stress response and virulence

The protein phosphatase calcineurin is a key mediator of virulence and antifungal susceptibility of multiple fungal pathogens including Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus, and has clinical potential as a therapeutic target to increase the efficacy of the current antifungal armamentarium. Despite the importance of this signaling pathway, few components of the calcineurin-signaling pathway are known in C. albicans. Here we identified and analyzed additional components of the C. albicans calcineurin cascade, including the RCN1 (regulator of calcineurin1), MID1, and CCH1 genes, which mediate calcineurin functions in other species. When heterologously expressed in Saccharomyces cerevisiae, C. albicans Rcn1 inhibited calcineurin function. Although rcn1/rcn1, mid1/mid1, and cch1/cch1 mutant strains share some phenotypes with calcineurin mutants, they do not completely recapitulate the phenotypes of a calcineurin mutant strain. These studies extend our understanding of the C. albicans calcineurin signaling cascade and its host-niche specific role in virulence. © 2009 Elsevier Inc. All rights reserved.

Authors
Reedy, JL; Filler, SG; Heitman, J
MLA Citation
Reedy, JL, Filler, SG, and Heitman, J. "Elucidating the Candida albicans calcineurin signaling cascade controlling stress response and virulence." Fungal Genetics and Biology 47.2 (2010): 107-116.
PMID
19755168
Source
scival
Published In
Fungal Genetics and Biology
Volume
47
Issue
2
Publish Date
2010
Start Page
107
End Page
116
DOI
10.1016/j.fgb.2009.09.002

Septins enforce morphogenetic events during sexual reproduction and contribute to virulence of Cryptococcus neoformans

Septins are conserved, cytoskeletal GTPases that contribute to cytokinesis, exocytosis, cell surface organization and vesicle fusion by mechanisms that are poorly understood. Roles of septins in morphogenesis and virulence of a human pathogen and basidiomycetous yeast Cryptococcus neoformans were investigated. In contrast to a well-established paradigm in S. cerevisiae, Cdc3 and Cdc12 septin homologues are dispensable for growth in C. neoformans yeast cells at 24°C but are essential at 37°C. In a bilateral cross between septin mutants, cells fuse but the resulting hyphae exhibit morphological abnormalities, including lack of properly fused specialized clamp cells and failure to produce spores. Interestingly, post-mating hyphae of the septin mutants have a defect in nuclear distribution. Thus, septins are essential for the development of spores, clamp cell fusion and also play a specific role in nuclear dynamics in hyphae. In the post-mating hyphae the septins localize to discrete sites in clamp connections, to the septa and the bases of the initial emerging spores. Strains lacking CDC3 or CDC12 exhibit significantly reduced virulence in a Galleria mellonella model of infection. Thus, C. neoformans septins are vital to morphology of the hyphae and contribute to virulence. © 2010 Blackwell Publishing Ltd.

Authors
Kozubowski, L; Heitman, J
MLA Citation
Kozubowski, L, and Heitman, J. "Septins enforce morphogenetic events during sexual reproduction and contribute to virulence of Cryptococcus neoformans." Molecular Microbiology 75.3 (2010): 658-675.
PMID
19943902
Source
scival
Published In
Molecular Microbiology
Volume
75
Issue
3
Publish Date
2010
Start Page
658
End Page
675
DOI
10.1111/j.1365-2958.2009.06983.x

Transcription factors Mat2 and Znf2 operate cellular circuits orchestrating opposite- and same-sex mating in Cryptococcus neoformans.

Cryptococcus neoformans is a human fungal pathogen that undergoes a dimorphic transition from a unicellular yeast to multicellular hyphae during opposite sex (mating) and unisexual reproduction (same-sex mating). Opposite- and same-sex mating are induced by similar environmental conditions and involve many shared components, including the conserved pheromone sensing Cpk1 MAPK signal transduction cascade that governs the dimorphic switch in C. neoformans. However, the homeodomain cell identity proteins Sxi1alpha/Sxi2a encoded by the mating type locus that are essential for completion of sexual reproduction following cell-cell fusion during opposite-sex mating are dispensable for same-sex mating. Therefore, identification of downstream targets of the Cpk1 MAPK pathway holds the key to understanding molecular mechanisms governing the two distinct developmental fates. Thus far, homology-based approaches failed to identify downstream transcription factors which may therefore be species-specific. Here, we applied insertional mutagenesis via Agrobacterium-mediated transformation and transcription analysis using whole genome microarrays to identify factors involved in C. neoformans differentiation. Two transcription factors, Mat2 and Znf2, were identified as key regulators of hyphal growth during same- and opposite-sex mating. Mat2 is an HMG domain factor, and Znf2 is a zinc finger protein; neither is encoded by the mating type locus. Genetic, phenotypic, and transcriptional analyses of Mat2 and Znf2 provide evidence that Mat2 is a downstream transcription factor of the Cpk1 MAPK pathway whereas Znf2 functions as a more terminal hyphal morphogenesis determinant. Although the components of the MAPK pathway including Mat2 are not required for virulence in animal models, Znf2, as a hyphal morphology determinant, is a negative regulator of virulence. Further characterization of these elements and their target circuits will reveal genes controlling biological processes central to fungal development and virulence.

Authors
Lin, X; Jackson, JC; Feretzaki, M; Xue, C; Heitman, J
MLA Citation
Lin, X, Jackson, JC, Feretzaki, M, Xue, C, and Heitman, J. "Transcription factors Mat2 and Znf2 operate cellular circuits orchestrating opposite- and same-sex mating in Cryptococcus neoformans." PLoS genetics 6.5 (2010): e1000953-.
Website
http://hdl.handle.net/10161/4468
Source
scival
Published In
PLoS genetics
Volume
6
Issue
5
Publish Date
2010
Start Page
e1000953
DOI
10.1371/journal.pgen.1000953

The mating type locus (MAT) and sexual reproduction of Cryptococcus heveanensis: insights into the evolution of sex and sex-determining chromosomal regions in fungi.

Mating in basidiomycetous fungi is often controlled by two unlinked, multiallelic loci encoding homeodomain transcription factors or pheromones/pheromone receptors. In contrast to this tetrapolar organization, Cryptococcus neoformans/Cryptococcus gattii have a bipolar mating system, and a single biallelic locus governs sexual reproduction. The C. neoformans MAT locus is unusually large (>100 kb), contains >20 genes, and enhances virulence. Previous comparative genomic studies provided insights into how this unusual MAT locus might have evolved involving gene acquisitions into two unlinked loci and fusion into one contiguous locus, converting an ancestral tetrapolar system to a bipolar one. Here we tested this model by studying Cryptococcus heveanensis, a sister species to the pathogenic Cryptococcus species complex. An extant sexual cycle was discovered; co-incubating fertile isolates results in the teleomorph (Kwoniella heveanensis) with dikaryotic hyphae, clamp connections, septate basidia, and basidiospores. To characterize the C. heveanensis MAT locus, a fosmid library was screened with C. neoformans/C. gattii MAT genes. Positive fosmids were sequenced and assembled to generate two large probably unlinked MAT gene clusters: one corresponding to the homeodomain locus and the other to the pheromone/receptor locus. Strikingly, two divergent homeodomain genes (SXI1, SXI2) are present, similar to the bE/bW Ustilago maydis paradigm, suggesting one or the other homeodomain gene was recently lost in C. neoformans/C. gattii. Sequencing MAT genes from other C. heveanensis isolates revealed a multiallelic homeodomain locus and at least a biallelic pheromone/receptor locus, similar to known tetrapolar species. Taken together, these studies reveal an extant C. heveanensis sexual cycle, define the structure of its MAT locus consistent with tetrapolar mating, and support the proposed evolutionary model for the bipolar Cryptococcus MAT locus revealing transitions in sexuality concomitant with emergence of a pathogenic clade. These studies provide insight into convergent processes that independently punctuated evolution of sex-determining loci and sex chromosomes in fungi, plants, and animals.

Authors
Metin, B; Findley, K; Heitman, J
MLA Citation
Metin, B, Findley, K, and Heitman, J. "The mating type locus (MAT) and sexual reproduction of Cryptococcus heveanensis: insights into the evolution of sex and sex-determining chromosomal regions in fungi." PLoS genetics 6.5 (2010): e1000961-.
Website
http://hdl.handle.net/10161/4469
Source
scival
Published In
PLoS genetics
Volume
6
Issue
5
Publish Date
2010
Start Page
e1000961
DOI
10.1371/journal.pgen.1000961

Surfactant protein D increases phagocytosis of hypocapsular Cryptococcus neoformans by murine macrophages and enhances fungal survival.

Cryptococcus neoformans is a facultative intracellular opportunistic pathogen and the leading cause of fungal meningitis in humans. In the absence of a protective cellular immune response, the inhalation of C. neoformans cells or spores results in pulmonary infection. C. neoformans cells produce a polysaccharide capsule composed predominantly of glucuronoxylomannan, which constitutes approximately 90% of the capsular material. In the lungs, surfactant protein A (SP-A) and SP-D contribute to immune defense by facilitating the aggregation, uptake, and killing of many microorganisms by phagocytic cells. We hypothesized that SP-D plays a role in C. neoformans pathogenesis by binding to and enhancing the phagocytosis of the yeast. Here, the abilities of SP-D to bind to and facilitate the phagocytosis and survival of the wild-type encapsulated strain H99 and the cap59Delta mutant hypocapsular strain are assessed. SP-D binding to cap59Delta mutant cells was approximately sixfold greater than binding to wild-type cells. SP-D enhanced the phagocytosis of cap59Delta cells by approximately fourfold in vitro. To investigate SP-D binding in vivo, SP-D(-/-) mice were intranasally inoculated with Alexa Fluor 488-labeled cap59Delta or H99 cells. By confocal microscopy, a greater number of phagocytosed C. neoformans cells in wild-type mice than in SP-D(-/-) mice was observed, consistent with in vitro data. Interestingly, SP-D protected C. neoformans cells against macrophage-mediated defense mechanisms in vitro, as demonstrated by an analysis of fungal viability using a CFU assay. These findings provide evidence that C. neoformans subverts host defense mechanisms involving surfactant, establishing a novel virulence paradigm that may be targeted for therapy.

Authors
Geunes-Boyer, S; Oliver, TN; Janbon, G; Lodge, JK; Heitman, J; Perfect, JR; Wright, JR
MLA Citation
Geunes-Boyer, S, Oliver, TN, Janbon, G, Lodge, JK, Heitman, J, Perfect, JR, and Wright, JR. "Surfactant protein D increases phagocytosis of hypocapsular Cryptococcus neoformans by murine macrophages and enhances fungal survival." Infect Immun 77.7 (July 2009): 2783-2794.
PMID
19451250
Source
pubmed
Published In
Infection and immunity
Volume
77
Issue
7
Publish Date
2009
Start Page
2783
End Page
2794
DOI
10.1128/IAI.00088-09

First reported case of Cryptococcus gattii in the Southeastern USA: implications for travel-associated acquisition of an emerging pathogen.

In 2007, the first confirmed case of Cryptococcus gattii was reported in the state of North Carolina, USA. An otherwise healthy HIV negative male patient presented with a large upper thigh cryptococcoma in February, which was surgically removed and the patient was started on long-term high-dose fluconazole treatment. In May of 2007, the patient presented to the Duke University hospital emergency room with seizures. Magnetic resonance imaging revealed two large CNS lesions found to be cryptococcomas based on brain biopsy. Prior chest CT imaging had revealed small lung nodules indicating that C. gattii spores or desiccated yeast were likely inhaled into the lungs and dissemination occurred to both the leg and CNS. The patient's travel history included a visit throughout the San Francisco, CA region in September through October of 2006, consistent with acquisition during this time period. Cultures from both the leg and brain biopsies were subjected to analysis. Based on phenotypic and molecular methods, both isolates were C. gattii, VGI molecular type, and distinct from the Vancouver Island outbreak isolates. Based on multilocus sequence typing of coding and noncoding regions and virulence in a heterologous host model, the leg and brain isolates are identical, but the two differed in mating fertility. Two clinical isolates, one from a transplant recipient in San Francisco and the other from Australia, were identical to the North Carolina clinical isolate at all markers tested. Closely related isolates that differ at only one or a few noncoding markers are present in the Australian environment. Taken together, these findings support a model in which C. gattii VGI was transferred from Australia to California, possibly though an association with its common host plant E. camaldulensis, and the patient was exposed in San Francisco and returned to present with disease in North Carolina.

Authors
Byrnes, EJ; Li, W; Lewit, Y; Perfect, JR; Carter, DA; Cox, GM; Heitman, J
MLA Citation
Byrnes, EJ, Li, W, Lewit, Y, Perfect, JR, Carter, DA, Cox, GM, and Heitman, J. "First reported case of Cryptococcus gattii in the Southeastern USA: implications for travel-associated acquisition of an emerging pathogen. (Published online)" PLoS One 4.6 (June 10, 2009): e5851-.
PMID
19516904
Source
pubmed
Published In
PloS one
Volume
4
Issue
6
Publish Date
2009
Start Page
e5851
DOI
10.1371/journal.pone.0005851

Hsp90 orchestrates temperature-dependent Candida albicans morphogenesis via Ras1-PKA signaling.

BACKGROUND: Hsp90 is an environmentally contingent molecular chaperone that influences the form and function of diverse regulators of cellular signaling. Hsp90 potentiates the evolution of fungal drug resistance by enabling crucial cellular stress responses. Here we demonstrate that in the leading fungal pathogen of humans, Candida albicans, Hsp90 governs cellular circuitry required not only for drug resistance but also for the key morphogenetic transition from yeast to filamentous growth that is crucial for virulence. This transition is normally regulated by environmental cues, such as exposure to serum, that are contingent upon elevated temperature to induce morphogenesis. The basis for this temperature dependence has remained enigmatic. RESULTS: We show that compromising Hsp90 function pharmacologically or genetically induces a transition from yeast to filamentous growth in the absence of external cues. Elevated temperature relieves Hsp90-mediated repression of the morphogenetic program. Hsp90 regulates morphogenetic circuitry by repressing Ras1-PKA signaling. Modest Hsp90 compromise enhances the phenotypic effects of activated Ras1 signaling whereas deletion of positive regulators of the Ras1-PKA cascade blocks the morphogenetic response to Hsp90 inhibition. Consistent with the requirement for morphogenetic flexibility for virulence, depletion of C. albicans Hsp90 attenuates virulence in a murine model of systemic disease. CONCLUSIONS: Hsp90 governs the integration of environmental cues with cellular signaling to orchestrate fungal morphogenesis and virulence, suggesting new therapeutic strategies for life-threatening infectious disease. Hsp90's capacity to govern a key developmental program in response to temperature change provides a new mechanism that complements the elegant repertoire that organisms utilize to sense temperature.

Authors
Shapiro, RS; Uppuluri, P; Zaas, AK; Collins, C; Senn, H; Perfect, JR; Heitman, J; Cowen, LE
MLA Citation
Shapiro, RS, Uppuluri, P, Zaas, AK, Collins, C, Senn, H, Perfect, JR, Heitman, J, and Cowen, LE. "Hsp90 orchestrates temperature-dependent Candida albicans morphogenesis via Ras1-PKA signaling." Curr Biol 19.8 (April 28, 2009): 621-629.
PMID
19327993
Source
pubmed
Published In
Current Biology
Volume
19
Issue
8
Publish Date
2009
Start Page
621
End Page
629
DOI
10.1016/j.cub.2009.03.017

Molecular evidence that the range of the Vancouver Island outbreak of Cryptococcus gattii infection has expanded into the Pacific Northwest in the United States.

Cryptococcus neoformans frequently causes fungal meningitis in immunocompromised patients, whereas the related species C. gattii is restricted to tropical and subtropical regions,where it usually infects immunocompetent individuals.An outbreak of C. gattii infection that began in 1999 on Vancouver Island has resulted in endemic C. gattii infection and caused numerous human and veterinary infections; the outbreak's range has spread to mainland British Columbia. The outbreak-related isolates have been molecular type VGIIa, the major genotype, or VGIIb, the minor genotype. Since 2006, human and veterinary cases of C. gattii infection have emerged in Washington and Oregon. Multilocus sequence typing demonstrates the spread of C. gattii VGIIa and VGIIb from Vancouver Island to the Pacific Northwest. Clinical strains recovered in Oregon represent a unique VGIIc genotype.

Authors
Byrnes, EJ; Bildfell, RJ; Frank, SA; Mitchell, TG; Marr, KA; Heitman, J
MLA Citation
Byrnes, EJ, Bildfell, RJ, Frank, SA, Mitchell, TG, Marr, KA, and Heitman, J. "Molecular evidence that the range of the Vancouver Island outbreak of Cryptococcus gattii infection has expanded into the Pacific Northwest in the United States." J Infect Dis 199.7 (April 1, 2009): 1081-1086.
PMID
19220140
Source
pubmed
Published In
Journal of Infectious Diseases
Volume
199
Issue
7
Publish Date
2009
Start Page
1081
End Page
1086
DOI
10.1086/597306

Aspergillus fumigatus calcipressin CbpA is involved in hyphal growth and calcium homeostasis.

Calcineurin is a conserved protein phosphatase that plays a critical role in Ca(2+) signaling and stress responses. Previously, a new class of conserved calcineurin-binding proteins, the calcipressins, was identified. However, the role of these proteins remains controversial, and both inhibitory and stimulatory effects on calcineurin were observed. In this study, we investigate the role of CbpA, the Aspergillus fumigatus member of the calcipressin family, and report that deletion of the cbpA gene resulted in reduced hyphal growth and limited attenuated virulence. Interestingly, under high-calcium-level conditions, the DeltacbpA strain displayed improved Ca(2+) tolerance compared to the wild-type strain and revealed increased expression of vcxA, chsA, and cnaA, which encode the vacuolar Ca(2+)/H(+) exchanger VcxA, chitin synthase A, and the calcineurin catalytic subunit CnaA, respectively. The increased transcript levels of these three genes were reversed in the presence of the calcineurin inhibitor FK506, indicating a calcineurin-dependent mechanism. Overexpression of cbpA resulted in decreased transcription of vcxA, chsA, and cnaA, associated with wild-type sensitivity to Ca(2+). Taken together, our study highlights the importance of CbpA in the regulation of hyphal growth and calcium adaptation of A. fumigatus and provides evidence that CbpA may serve as a feedback inhibitor in some aspects of calcineurin functions.

Authors
Pinchai, N; Perfect, BZ; Juvvadi, PR; Fortwendel, JR; Cramer, RA; Asfaw, YG; Heitman, J; Perfect, JR; Steinbach, WJ
MLA Citation
Pinchai, N, Perfect, BZ, Juvvadi, PR, Fortwendel, JR, Cramer, RA, Asfaw, YG, Heitman, J, Perfect, JR, and Steinbach, WJ. "Aspergillus fumigatus calcipressin CbpA is involved in hyphal growth and calcium homeostasis." Eukaryot Cell 8.4 (April 2009): 511-519.
PMID
19252123
Source
pubmed
Published In
Eukaryotic cell
Volume
8
Issue
4
Publish Date
2009
Start Page
511
End Page
519
DOI
10.1128/EC.00336-08

Diploids in the Cryptococcus neoformans serotype A population homozygous for the alpha mating type originate via unisexual mating.

The ubiquitous environmental human pathogen Cryptococcus neoformans is traditionally considered a haploid fungus with a bipolar mating system. In nature, the alpha mating type is overwhelmingly predominant over a. How genetic diversity is generated and maintained by this heterothallic fungus in a largely unisexual alpha population is unclear. Recently it was discovered that C. neoformans can undergo same-sex mating under laboratory conditions generating both diploid intermediates and haploid recombinant progeny. Same-sex mating (alpha-alpha) also occurs in nature as evidenced by the existence of natural diploid alphaADalpha hybrids that arose by fusion between two alpha cells of different serotypes (A and D). How significantly this novel sexual style contributes to genetic diversity of the Cryptococcus population was unknown. In this study, approximately 500 natural C. neoformans isolates were tested for ploidy and close to 8% were found to be diploid by fluorescence flow cytometry analysis. The majority of these diploids were serotype A isolates with two copies of the alpha MAT locus allele. Among those, several are intra-varietal allodiploid hybrids produced by fusion of two genetically distinct alpha cells through same-sex mating. The majority, however, are autodiploids that harbor two seemingly identical copies of the genome and arose via either endoreplication or clonal mating. The diploids identified were isolated from different geographic locations and varied genotypically and phenotypically, indicating independent non-clonal origins. The present study demonstrates that unisexual mating produces diploid isolates of C. neoformans in nature, giving rise to populations of hybrids and mixed ploidy. Our findings underscore the importance of same-sex mating in shaping the current population structure of this important human pathogenic fungus, with implications for mechanisms of selfing and inbreeding in other microbial pathogens.

Authors
Lin, X; Patel, S; Litvintseva, AP; Floyd, A; Mitchell, TG; Heitman, J
MLA Citation
Lin, X, Patel, S, Litvintseva, AP, Floyd, A, Mitchell, TG, and Heitman, J. "Diploids in the Cryptococcus neoformans serotype A population homozygous for the alpha mating type originate via unisexual mating." PLoS Pathog 5.1 (January 2009): e1000283-.
PMID
19180236
Source
pubmed
Published In
PLoS pathogens
Volume
5
Issue
1
Publish Date
2009
Start Page
e1000283
DOI
10.1371/journal.ppat.1000283

Phylogeny and phenotypic characterization of pathogenic Cryptococcus species and closely related saprobic taxa in the tremellales

The basidiomycetous yeasts Cryptococcus neoformans and Cryptococcus gattii are closely related sibling species that cause respiratory and neurological disease in humans and animals. Within these two recognized species, phylogenetic analysis reveals at least six cryptic species defined as molecular types (VNI/II/B, VNIV, VGI, VGII, VGIII, and VGIV) that comprise the pathogenic Cryptococcus species complex. These pathogenic species are clustered in the Filobasidiella clade within the order Tremellales. Previous studies have shown that the Filobasidiella clade also includes several saprobic fungi isolated from insect frass, but information evaluating the relatedness of the saprobes and pathogens within this cluster is limited. Here, the phylogeny encompassing a subset of species in the Tremellales lineage that clusters closely with the pathogenic Cryptococcus species complex was resolved by employing a multilocus sequencing approach for phylogenetic analysis. Six highly conserved genomic loci from 15 related basidiomycete species were sequenced, and the alignments from the concatenated gene sequences were evaluated with different tree-building criteria. Furthermore, these 15 species were subjected to virulence and phenotype assays to evaluate their pathogenic potential. These studies revealed that Cryptococcus amylolentus and Tsuchiyaea wingfieldii, two nonpathogenic sibling species, are the taxa most closely related to the pathogens C. neoformans and C. gattii and together with Filobasidiella depauperata form a Cryptococcus sensu stricto group. Five other saprobic yeast species form the Kwoniella clade, which appears to be a part of a more distantly related sensu lato group. This study establishes a foundation for future comparative genomic approaches that will provide insight into the structure, function, and evolution of the mating type locus, the transitions in modes of sexual reproduction, and the emergence of human pathogenic species from related or ancestral saprobic species. Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Authors
Findley, K; Rodriguez-Carres, M; Metin, B; Kroiss, J; Fonseca, Á; Vilgalys, R; Heitman, J
MLA Citation
Findley, K, Rodriguez-Carres, M, Metin, B, Kroiss, J, Fonseca, Á, Vilgalys, R, and Heitman, J. "Phylogeny and phenotypic characterization of pathogenic Cryptococcus species and closely related saprobic taxa in the tremellales." Eukaryotic Cell 8.3 (2009): 353-361.
PMID
19151324
Source
scival
Published In
Eukaryotic cell
Volume
8
Issue
3
Publish Date
2009
Start Page
353
End Page
361
DOI
10.1128/EC.00373-08

The protein kinase Tor1 regulates adhesin gene expression in Candida albicans

Eukaryotic cell growth is coordinated in response to nutrient availability, growth factors, and environmental stimuli, enabling cell-cell interactions that promote survival. The rapamycin-sensitive Tor1 protein kinase, which is conserved from yeasts to humans, participates in a signaling pathway central to cellular nutrient responses. To gain insight into Tor-mediated processes in human fungal pathogens, we have characterized Tor signaling in Candida albicans. Global transcriptional profiling revealed evolutionarily conserved roles for Tor1 in regulating the expression of genes involved in nitrogen starvation responses and ribosome biogenesis. Interestingly, we found that in C. albicans Tor1 plays a novel role in regulating the expression of several cell wall and hyphal specific genes, including adhesins and their transcriptional repressors Nrg1 and Tup1. In accord with this transcriptional profile, rapamycin induced extensive cellular aggregation in an adhesin-dependent fashion. Moreover, adhesin gene induction and cellular aggregation of rapamycin-treated cells were strongly dependent on the transactivators Bcr1 and Efg1. These findings support models in which Tor1 negatively controls cellular adhesion by governing the activities of Bcr1 and Efg1. Taken together, these results provide evidence that Tor1-mediated cellular adhesion might be broadly conserved among eukaryotic organisms. © 2009 Bastidas et al.

Authors
Bastidas, RJ; Heitman, J; Cardenas, ME
MLA Citation
Bastidas, RJ, Heitman, J, and Cardenas, ME. "The protein kinase Tor1 regulates adhesin gene expression in Candida albicans." PLoS Pathogens 5.2 (2009).
PMID
19197361
Source
scival
Published In
PLoS pathogens
Volume
5
Issue
2
Publish Date
2009
DOI
10.1371/journal.ppat.1000294

Microbial genetics: Love the one you're with

Candida albicans is notorious as an opportunistic microbe that causes thrush and serious systemic disease. For geneticists, however, it offers continuing revelations into the wondrously varied sex lives of fungi. ©2009 Macmillan Publishers Limited. All rights reserved.

Authors
Heitman, J
MLA Citation
Heitman, J. "Microbial genetics: Love the one you're with." Nature 460.7257 (2009): 807-808.
PMID
19675638
Source
scival
Published In
Nature
Volume
460
Issue
7257
Publish Date
2009
Start Page
807
End Page
808
DOI
10.1038/460807a

Spores as infectious propagules of Cryptococcus neoformans

Cryptococcus neoformans and Cryptococcus gattii are closely related pathogenic fungi that cause pneumonia and meningitis in both immunocompromised and immunocompetent hosts and are a significant global infectious disease risk. Both species are found in the environment and are acquired via inhalation, leading to an initial pulmonary infection. The infectious propagule is unknown but is hypothesized to be small desiccated yeast cells or spores produced by sexual reproduction (opposite- or same-sex mating). Here we characterize the morphology, germination properties, and virulence of spores. A comparative morphological analysis of hyphae and spores produced by opposite-sex mating, same-sex mating, and self-fertile diploid strains was conducted by scanning electron microscopy, yielding insight into hyphal/basidial morphology and spore size, structure, and surface properties. Spores isolated by microdissection were found to readily germinate even on water agarose medium. Thus, nutritional signals do not appear to be required to stimulate spore germination, and as-yet-unknown environmental factors may normally constrain germination in nature. As few as 500 CFU of a spore-enriched infectious inoculum (∼95% spores) of serotype A C. neoformans var. grubii were fully virulent (100% lethal infection) in both a murine inhalation virulence model and the invertebrate model host Galleria mellonella. In contrast to a previous report on C. neoformans var. neoformans, spores of C. neoformans var. grubii were not more infectious than yeast cells. Molecular analysis of isolates recovered from tissues of infected mice (lung, spleen, and brain) provides evidence for infection and dissemination by recombinant spore products. These studies provide a detailed morphological and physiological analysis of the spore and document that spores can serve as infectious propagules. Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Authors
Velagapudi, R; Hsueh, Y-P; Geunes-Boyer, S; Wright, JR; Heitman, J
MLA Citation
Velagapudi, R, Hsueh, Y-P, Geunes-Boyer, S, Wright, JR, and Heitman, J. "Spores as infectious propagules of Cryptococcus neoformans." Infection and Immunity 77.10 (2009): 4345-4355.
PMID
19620339
Source
scival
Published In
Infection and immunity
Volume
77
Issue
10
Publish Date
2009
Start Page
4345
End Page
4355
DOI
10.1128/IAI.00542-09

Remodeling of global transcription patterns of Cryptococcus neoformans genes mediated by the stress-activated HOG signaling pathways

The ability to sense and adapt to a hostile host environment is a crucial element for virulence of pathogenic fungi, including Cryptococcus neoformans. These cellular responses are evoked by diverse signaling cascades, including the stress-activated HOG pathway. Despite previous analysis of central components of the HOG pathway, its downstream signaling network is poorly characterized in C. neoformans. Here we performed comparative transcriptome analysis with HOG signaling mutants to explore stress-regulated genes and their correlation with the HOG pathway in C. neoformans. In this study, we not only provide important insights into remodeling patterns of global gene expression for counteracting external stresses but also elucidate novel characteristics of the HOG pathway in C. neoformans. First, inhibition of the HOG pathway increases expression of ergosterol biosynthesis genes and cellular ergosterol content, conferring a striking synergistic antifungal activity with amphotericin B and providing an excellent opportunity to develop a novel therapeutic method for treatment of cryptococcosis. Second, a number of cadmium-sensitive genes are differentially regulated by the HOG pathway, and their mutation causes resistance to cadmium. Finally, we have discovered novel stress defense and HOG-dependent genes, which encode a sodium/potassium efflux pump, protein kinase, multidrug transporter system, and elements of the ubiquitin-dependent system. Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Authors
Ko, Y-J; Yu, YM; Kim, G-B; Lee, G-W; Maeng, PJ; Kim, S; Floyd, A; Heitman, J; Bahn, Y-S
MLA Citation
Ko, Y-J, Yu, YM, Kim, G-B, Lee, G-W, Maeng, PJ, Kim, S, Floyd, A, Heitman, J, and Bahn, Y-S. "Remodeling of global transcription patterns of Cryptococcus neoformans genes mediated by the stress-activated HOG signaling pathways." Eukaryotic Cell 8.8 (2009): 1197-1217.
PMID
19542307
Source
scival
Published In
Eukaryotic cell
Volume
8
Issue
8
Publish Date
2009
Start Page
1197
End Page
1217
DOI
10.1128/EC.00120-09

Spread of Cryptococcus gattii into Pacific Northwest Region of the United States

Cryptococcus gattii has emerged as a human and animal pathogen in the Pacific Northwest. First recognized on Vancouver Island, British Columbia, Canada, it now involves mainland British Columbia, and Washington and Oregon in the United States. In Canada, the incidence of disease has been one of the highest worldwide. In the United States, lack of cryptococcal species identification and case surveillance limit our knowledge of C. gattii epidemiology. Infections in the Pacific Northwest are caused by multiple genotypes, but the major strain is genetically novel and may have emerged recently in association with unique mating or environmental changes. C. gattii disease affects immunocompromised and immunocompetent persons, causing substantial illness and death. Successful management requires an aggressive medical and surgical approach and consideration of potentially variable antifungal drug susceptibilities. We summarize the study results of a group of investigators and review current knowledge with the goal of increasing awareness and highlighting areas where further knowledge is required.

Authors
Datta, K; Bartlett, KH; Baer, R; Byrnes, E; Galanis, E; Heitman, J; Hoang, L; Leslie, MJ; MacDougall, L; Magill, SS; Morshed, MG; Marr, KA
MLA Citation
Datta, K, Bartlett, KH, Baer, R, Byrnes, E, Galanis, E, Heitman, J, Hoang, L, Leslie, MJ, MacDougall, L, Magill, SS, Morshed, MG, and Marr, KA. "Spread of Cryptococcus gattii into Pacific Northwest Region of the United States." Emerging Infectious Diseases 15.8 (2009): 1185-1191.
PMID
19757550
Source
scival
Published In
Emerging infectious diseases
Volume
15
Issue
8
Publish Date
2009
Start Page
1185
End Page
1191
DOI
10.3201/eid1508.081384

Evolution of pathogenicity and sexual reproduction in eight Candida genomes

Candida species are the most common cause of opportunistic fungal infection worldwide. Here we report the genome sequences of six Candida species and compare these and related pathogens and non-pathogens. There are significant expansions of cell wall, secreted and transporter gene families in pathogenic species, suggesting adaptations associated with virulence. Large genomic tracts are homozygous in three diploid species, possibly resulting from recent recombination events. Surprisingly, key components of the mating and meiosis pathways are missing from several species. These include major differences at the mating-type loci (MTL); Lodderomyces elongisporus lacks MTL, and components of the a1/α2 cell identity determinant were lost in other species, raising questions about how mating and cell types are controlled. Analysis of the CUG leucine-to-serine genetic-code change reveals that 99% of ancestral CUG codons were erased and new ones arose elsewhere. Lastly, we revise the Candida albicans gene catalogue, identifying many new genes. © 2009 Macmillan Publishers Limited. All rights reserved.

Authors
Butler, G; Rasmussen, MD; Lin, MF; Santos, MAS; Sakthikumar, S; Munro, CA; Rheinbay, E; Grabherr, M; Forche, A; Reedy, JL; Agrafioti, I; Arnaud, MB; Bates, S; Brown, AJP; Brunke, S; Costanzo, MC; Fitzpatrick, DA; Groot, PWJD; Harris, D; Hoyer, LL; Hube, B; Klis, FM; Kodira, C; Lennard, N; Logue, ME; Martin, R; Neiman, AM; Nikolaou, E; Quail, MA; Quinn, J; Santos, MC; Schmitzberger, FF; Sherlock, G; Shah, P; Silverstein, KAT; Skrzypek, MS; Soll, D; Staggs, R; Stansfield, I; Stumpf, MPH et al.
MLA Citation
Butler, G, Rasmussen, MD, Lin, MF, Santos, MAS, Sakthikumar, S, Munro, CA, Rheinbay, E, Grabherr, M, Forche, A, Reedy, JL, Agrafioti, I, Arnaud, MB, Bates, S, Brown, AJP, Brunke, S, Costanzo, MC, Fitzpatrick, DA, Groot, PWJD, Harris, D, Hoyer, LL, Hube, B, Klis, FM, Kodira, C, Lennard, N, Logue, ME, Martin, R, Neiman, AM, Nikolaou, E, Quail, MA, Quinn, J, Santos, MC, Schmitzberger, FF, Sherlock, G, Shah, P, Silverstein, KAT, Skrzypek, MS, Soll, D, Staggs, R, Stansfield, I, and Stumpf, MPH et al. "Evolution of pathogenicity and sexual reproduction in eight Candida genomes." Nature 459.7247 (2009): 657-662.
PMID
19465905
Source
scival
Published In
Nature
Volume
459
Issue
7247
Publish Date
2009
Start Page
657
End Page
662
DOI
10.1038/nature08064

Mechanistic Plasticity of Sexual Reproduction and Meiosis in the Candida Pathogenic Species Complex

Background: Candida species are microbial pathogens originally thought to be asexual, but several are now recognized as sexual or parasexual. Candida albicans, the most common fungus infecting humans, is an obligate diploid with a parasexual cycle involving mating, recombination, and genome reduction but no recognized meiosis. Others (C. lusitaniae, C. guilliermondii) are haploid, and their mating produces spores, suggestive of complete meiotic sexual cycles. However, comparative genomic analysis reveals that these species lack key meiotic components, including the recombinase Dmc1 and cofactors (Mei5/Sae3), synaptonemal-complex proteins (Zip1-Zip4/Hop1), and the crossover interference pathway (Msh4/5). Results: Here we elucidate the structure and functions of the mating-type (MAT) locus and establish that C. lusitaniae undergoes meiosis during its sexual cycle. The MAT-encoded a2 (high-mobility group) and α1 (α domain) factors specify a and α cell identity, whereas the a1 homeodomain protein drives meiosis and sporulation and functions without its canonical heterodimeric partner, α2. Despite the apparent loss of meiotic genes, C. lusitaniae undergoes meiosis during sexual reproduction involving diploid intermediates, frequent SPO11-dependent recombination, and whole-genome reduction generating haploid progeny. The majority of meiotic progeny are euploid, but approximately one-third are diploid/aneuploid. Conclusions: The cell identity and meiotic pathways have been substantially rewired, and meiotic generation of both recombinant and aneuploid progeny may expand genetic diversity. These findings inform our understanding of sexual reproduction in pathogenic microbes and the evolutionary plasticity of the meiotic machinery, with implications for the sexual nature of C. albicans and the generation and consequences of aneuploidy in biology and medicine. © 2009 Elsevier Ltd. All rights reserved.

Authors
Reedy, JL; Floyd, AM; Heitman, J
MLA Citation
Reedy, JL, Floyd, AM, and Heitman, J. "Mechanistic Plasticity of Sexual Reproduction and Meiosis in the Candida Pathogenic Species Complex." Current Biology 19.11 (2009): 891-899.
PMID
19446455
Source
scival
Published In
Current Biology
Volume
19
Issue
11
Publish Date
2009
Start Page
891
End Page
899
DOI
10.1016/j.cub.2009.04.058

A constitutively active GPCR governs morphogenic transitions in Cryptococcus neoformans

Sex in fungi is driven by peptide pheromones sensed through seven-transmembrane pheromone receptors. In Cryptococcus neoformans, sexual reproduction occurs through an outcrossing/heterothallic a- sexual cycle or an inbreeding/homothallic - unisexual mating process. Pheromone receptors encoded by the mating-type locus (MAT) mediate reciprocal pheromone sensing during opposite-sex mating and contribute to but are not essential for unisexual mating. A pheromone receptor-like gene, CPR2, was discovered that is not encoded by MAT and whose expression is induced during a- mating. cpr2 mutants are fertile but have a fusion defect and produce abnormal hyphal structures, whereas CPR2 overexpression elicits unisexual reproduction. When heterologously expressed in Saccharomyces cerevisiae, Cpr2 activates pheromone responses in the absence of any ligand. This constitutive activity results from an unconventional residue, Leu222, in place of a conserved proline in transmembrane domain six; a Cpr2L222P mutant is no longer constitutively active. Cpr2 engages the same G-protein activated signalling cascade as the Ste3a/α pheromone receptors, and thereby competes for pathway activation. This study established a new paradigm in which a naturally occurring constitutively active G protein-coupled receptor governs morphogenesis in fungi.

Authors
Hsueh, Y-P; Xue, C; Heitman, J
MLA Citation
Hsueh, Y-P, Xue, C, and Heitman, J. "A constitutively active GPCR governs morphogenic transitions in Cryptococcus neoformans." EMBO Journal 28.9 (2009): 1220-1233.
PMID
19322200
Source
scival
Published In
EMBO Journal
Volume
28
Issue
9
Publish Date
2009
Start Page
1220
End Page
1233
DOI
10.1038/emboj.2009.68

Phycomyces MADB interacts with MADA to form the primary photoreceptor complex for fungal phototropism

The fungus Phycomyces blakesleeanus reacts to environmental signals, including light, gravity, touch, and the presence of nearby objects, by changing the speed and direction of growth of its fruiting body (sporangiophore). Phototropism, growth toward light, shares many features in fungi and plants but the molecular mechanisms remain to be fully elucidated. Phycomyces mutants with altered phototropism were isolated ≈40 years ago and found to have mutations in the mad genes. All of the responses to light in Phycomyces require the products of the madA and madB genes. We showed that madA encodes a protein similar to the Neurospora blue-light photoreceptor, zinc-finger protein WC-1. We show here that madB encodes a protein similar to the Neurospora zinc-finger protein WC-2. MADA and MADBinteracttoformacomplex in yeast 2-hybrid assays and when coexpressed in E. coli, providing evidence that phototropism and other responses to light are mediated by a photoresponsive transcription factor complex. The Phycomyces genome contains 3 genes similar to wc-1, and 4 genes similar to wc-2, many of which are regulated by light in a madA or madB dependent manner. We did not detect any interactions between additional WC proteins in yeast 2-hybrid assays, which suggest that MADA and MADB form the major photoreceptor complex in Phycomyces. However, the presence of multiple wc genes in Phycomyces may enable perception across a broad range of light intensities, and may provide specialized photoreceptors for distinct photoresponses.

Authors
Sanz, C; Rodríguez-Romero, J; Idnurm, A; Christie, JM; Heitman, J; Corrochano, LM; Eslava, AP
MLA Citation
Sanz, C, Rodríguez-Romero, J, Idnurm, A, Christie, JM, Heitman, J, Corrochano, LM, and Eslava, AP. "Phycomyces MADB interacts with MADA to form the primary photoreceptor complex for fungal phototropism." Proceedings of the National Academy of Sciences of the United States of America 106.17 (2009): 7095-7100.
PMID
19380729
Source
scival
Published In
Proceedings of the National Academy of Sciences of USA
Volume
106
Issue
17
Publish Date
2009
Start Page
7095
End Page
7100
DOI
10.1073/pnas.0900879106

Dynamic duo takes down fungal villains

Authors
Semighini, CP; Heitman, J
MLA Citation
Semighini, CP, and Heitman, J. "Dynamic duo takes down fungal villains." Proceedings of the National Academy of Sciences of the United States of America 106.9 (2009): 2971-2972.
PMID
19251661
Source
scival
Published In
Proceedings of the National Academy of Sciences of USA
Volume
106
Issue
9
Publish Date
2009
Start Page
2971
End Page
2972
DOI
10.1073/pnas.0900801106

Identification of ENA1 as a virulence gene of the human pathogenic fungus Cryptococcus neoformans through signature-tagged insertional mutagenesis

A library of more than 4,500 signature-tagged insertion mutants of the human pathogenic fungus Cryptococcus neoformans was generated, and a subset was screened in a murine inhalation model to identify genes required for virulence. New genes that regulate aspects of C. neoformans virulence were also identified by screening the entire library for in vitro phenotypes related to the ability to cause disease, including melanin production, growth at high temperature, and growth under conditions of nutrient limitation. A screen of 10% of the strain collection in mice identified an avirulent mutant strain with an insertion in the ENA1 gene, which is predicted to encode a fungus-specific sodium or potassium P-type ATPase. The results of the deletion of the gene and complementation experiments confirmed its key role in mammalian virulence. ena1 mutant strains exhibited no change in sensitivity to high salt concentrations but were sensitive to alkaline pH conditions, providing evidence that the fungus may have to survive at elevated pH during infection of the mammalian host. The mutation of the well-characterized virulence factor calcineurin (CNA1) also rendered C. neoformans strains sensitive to elevated pH. ENA1 transcripts in wild-type and cna1 mutant strains were upregulated in response to high pH, and cna1 ena1 double mutant strains exhibited increased sensitivity to elevated pH, indicating that at least two pathways in the fungus mediate survival under alkaline conditions. Signature-tagged mutagenesis is an effective strategy for the discovery of new virulence genes in fungal pathogens of animals. Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Authors
Idnurm, A; Walton, FJ; Floyd, A; Reedy, JL; Heitman, J
MLA Citation
Idnurm, A, Walton, FJ, Floyd, A, Reedy, JL, and Heitman, J. "Identification of ENA1 as a virulence gene of the human pathogenic fungus Cryptococcus neoformans through signature-tagged insertional mutagenesis." Eukaryotic Cell 8.3 (2009): 315-326.
PMID
19151325
Source
scival
Published In
Eukaryotic cell
Volume
8
Issue
3
Publish Date
2009
Start Page
315
End Page
326
DOI
10.1128/EC.00375-08

Trimorphic stepping stones pave the way to fungal virulence

Authors
Bastidas, RJ; Heitman, J
MLA Citation
Bastidas, RJ, and Heitman, J. "Trimorphic stepping stones pave the way to fungal virulence." Proceedings of the National Academy of Sciences of the United States of America 106.2 (2009): 351-352.
PMID
19129500
Source
scival
Published In
Proceedings of the National Academy of Sciences of USA
Volume
106
Issue
2
Publish Date
2009
Start Page
351
End Page
352
DOI
10.1073/pnas.0811994106

Generation of genetic diversity in microsporidia via sexual reproduction and horizontal gene transfer

Microsporidia are obligate intracellular pathogens mainly infecting both vertebrate and invertebrate hosts. The group comprises approximately 150 genera with 1,200 species. Due to sequence divergence phylogenic reconstructions that are solely based on DNA sequence have been unprecise for these pathogens. Our previous study identified that three microsporidian genomes contained a putative sex-related locus similar to that of zygomycetes. In a comparison of genome architecture of the microsporidia to other fungi, Rhizopus oryzae, a zygomycete fungus, shared more common gene clusters with Encephalitozoon cuniculi, a microsporidian. This provides evidence supporting the hypothesis that microsporidia and zygomycete fungi may share a more recent common ancestor than other fungal lineages. Genetic recombination is an important outcome of sexual development. We describe genetic markers which will enable tests of whether sex occurs within E. cuniculi populations by analyzing tandem repeat DNA regions in three different isolates. Taken together, the phylogenetic relationship of microsporidia to fungi and the presence of a sex-related locus in their genomes suggest the microsporidia may have an extant sexual cycle. In addition, we describe recently reported evidence of horizontal gene transfer from Chlamydia to the E. cuniculi genome and show that these two obligate intracellular pathogens can infect the same host cells. © 2009 Landes Bioscience.

Authors
Lee, SC; Weiss, LM; Heitman, J
MLA Citation
Lee, SC, Weiss, LM, and Heitman, J. "Generation of genetic diversity in microsporidia via sexual reproduction and horizontal gene transfer." Communicative and Integrative Biology 2.5 (2009): 414-417.
PMID
19907704
Source
scival
Published In
Communicative & integrative biology
Volume
2
Issue
5
Publish Date
2009
Start Page
414
End Page
417
DOI
10.4161/cib.2.5.8846

Signalling pathways in the pathogenesis of Cryptococcus

Efficient communication with the environment is critical for all living organisms. Fungi utilize complex signalling systems to sense their environments and control proliferation, development and in some cases virulence. Well-studied signalling pathways include the protein kinase A/cyclic AMP (cAMP), protein kinase C (PKC)/mitogen-activated protein kinase (MAPK), lipid signalling cascades, and the calcium-calcineurin signalling pathway. The human pathogenic basidiomycetous fungus Cryptococcus neoformans deploys sensitive signalling systems to survive in the human host, leading to life-threatening meningoencephalitis. Known virulence traits of this fungus, including the antioxidant melanin production, the antiphagocytic polysaccharide capsule and the ability to grow at 37°C, are orchestrated by complex signalling networks, whose understanding is crucial to better treat, diagnose and prevent cryptococcosis. © 2009 Blackwell Publishing Ltd.

Authors
Kozubowski, L; Lee, SC; Heitman, J
MLA Citation
Kozubowski, L, Lee, SC, and Heitman, J. "Signalling pathways in the pathogenesis of Cryptococcus." Cellular Microbiology 11.3 (2009): 370-380.
PMID
19170685
Source
scival
Published In
Cellular Microbiology
Volume
11
Issue
3
Publish Date
2009
Start Page
370
End Page
380
DOI
10.1111/j.1462-5822.2008.01273.x

Loss of allergen 1 confers a hypervirulent phenotype that resembles mucoid switch variants of Cryptococcus neoformans

Microbial survival in a host is usually dependent on the ability of a pathogen to undergo changes that promote escape from host defense mechanisms. The human-pathogenic fungus Cryptococcus neoformans undergoes phenotypic switching in vivo that promotes persistence in tissue. By microarray and real-time PCR analyses, the allergen 1 gene (ALL1) was found to be downregulated in the hypervirulent mucoid switch variant, both during logarithmic growth and during intracellular growth in macrophages. The ALL1 gene encodes a small cytoplasmic protein that is involved in capsule formation. Growth of an all1Δ gene deletion mutant was normal. Similar to cells of the mucoid switch variant, all1Δ cells produced a larger polysaccharide capsule than cells of the smooth parent and the complemented strain produced, and the enlarged capsule inhibited macrophage phagocytosis. The mutant exhibited a modest defect in capsule induction compared to all of the other variants. In animal models the phenotype of the all1Δ mutant mimicked the hypervirulent phenotype of the mucoid switch variant, which is characterized by decreased host survival and elevated intracranial pressure. Decreased survival is likely the result of both an ineffective cell-mediated immune response and impaired phagocytosis by macrophages. Consequently, we concluded that, unlike loss of most virulence-associated genes, where loss of gene function results in attenuated virulence, loss of the ALL1 gene enhances virulence by altering the host-pathogen interaction and thereby impairing clearance. Our data identified the first cryptococcal gene associated with elevated intracranial pressure and support the hypothesis that an environmental opportunistic pathogen has modified its virulence in vivo by epigenetic downregulation of gene function. Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Authors
Jain, N; Li, L; Hsueh, Y-P; Guerrero, A; Heitman, J; Goldman, DL; Fries, BC
MLA Citation
Jain, N, Li, L, Hsueh, Y-P, Guerrero, A, Heitman, J, Goldman, DL, and Fries, BC. "Loss of allergen 1 confers a hypervirulent phenotype that resembles mucoid switch variants of Cryptococcus neoformans." Infection and Immunity 77.1 (2009): 128-140.
PMID
18955480
Source
scival
Published In
Infection and immunity
Volume
77
Issue
1
Publish Date
2009
Start Page
128
End Page
140
DOI
10.1128/IAI.01079-08

Cryptococcus gattii with bimorphic colony types in a dog in western Oregon: Additional evidence for expansion of the Vancouver Island outbreak

Cryptococcus gattii was isolated from a 1.5-year-old dog with systemic cryptococcosis in Oregon. The dog had no link to Vancouver Island or British Columbia, Canada. Samples from a nasal swab and from a granulomatous mass within the cranial cavity were pooled for culture. Colonies on Sabouraud dextrose agar were mucoid and exhibited bimorphic morphology, melanin-pigmented and unpigmented. Pigmented colonies were encapsulated budding spherical yeast, whereas unpigmented colonies were of unencapsulated ovoid budding yeast. In addition to defective melanin production, the unpigmented colony type exhibited defective mating. Genetic analysis by high-resolution multilocus sequence typing revealed that the 2 isolates are genetically identical at 8 unlinked loci tested and that the 2 isolates are both the VGIIa Vancouver Island major genotype. Findings are consistent with expansion of the Vancouver Island outbreak onto the mainland Pacific Northwest region of the United States.

Authors
III, EJB; Bildfell, RJ; Dearing, PL; Valentine, BA; Heitman, J
MLA Citation
III, EJB, Bildfell, RJ, Dearing, PL, Valentine, BA, and Heitman, J. "Cryptococcus gattii with bimorphic colony types in a dog in western Oregon: Additional evidence for expansion of the Vancouver Island outbreak." Journal of Veterinary Diagnostic Investigation 21.1 (2009): 133-136.
PMID
19139515
Source
scival
Published In
Journal of veterinary diagnostic investigation : official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc
Volume
21
Issue
1
Publish Date
2009
Start Page
133
End Page
136

Microsporidia evolved from ancestral sexual fungi.

Microsporidia are obligate, intracellular eukaryotic pathogens that infect animal cells, including humans [1]. Previous studies suggested microsporidia share a common ancestor with fungi [2-7]. However, the exact nature of this phylogenetic relationship is unclear because of unusual features of microsporidial genomes, which are compact with fewer and highly divergent genes [8]. As a consequence, it is unclear whether microsporidia evolved from a specific fungal lineage, or whether microsporidia are a sister group to all fungi. Here, we present evidence addressing this controversial question that is independent of sequence-based phylogenetic reconstruction, but rather based on genome structure. In the zygomycete basal fungal lineage, the sex locus is a syntenic gene cluster governing sexual reproduction in which a high mobility group (HMG) transcription-factor gene is flanked by triose-phosphate transporter (TPT) and RNA helicase genes [9]. Strikingly, microsporidian genomes harbor a sex-related locus with the same genes in the same order. Genome-wide synteny analysis reveals multiple other loci conserved between microsporidia and zygomycetes to the exclusion of all other fungal lineages with sequenced genomes. These findings support the hypothesis that microsporidia are true fungi that descended from a zygomycete ancestor and suggest microsporidia may have an extant sexual cycle.

Authors
Lee, SC; Corradi, N; Byrnes, EJ; Torres-Martinez, S; Dietrich, FS; Keeling, PJ; Heitman, J
MLA Citation
Lee, SC, Corradi, N, Byrnes, EJ, Torres-Martinez, S, Dietrich, FS, Keeling, PJ, and Heitman, J. "Microsporidia evolved from ancestral sexual fungi." Curr Biol 18.21 (November 11, 2008): 1675-1679.
PMID
18976912
Source
pubmed
Published In
Current Biology
Volume
18
Issue
21
Publish Date
2008
Start Page
1675
End Page
1679
DOI
10.1016/j.cub.2008.09.030

Central nervous system cryptococcosis in solid organ transplant recipients: clinical relevance of abnormal neuroimaging findings.

BACKGROUND: Prognostic implications of cryptococcal antigen and outcomes associated with central nervous system (CNS) cryptococcal lesions in solid organ transplant recipients have not been fully defined. METHODS: Patients were derived form a cohort of 122 solid organ transplant recipients with cryptococcosis in a multicenter study from 1999 to 2006. RESULTS: Central nervous system cryptococcosis was documented in 61 patients. Serum or cerebral spinal fluid antigen titers did not correlate with mortality at 90 days or cerebral spinal fluid sterilization at 2 weeks. Central nervous system lesions were identified in 16 patients and included leptomeningeal lesions in eight, parenchymal lesions in six, and hydrocephalus in two. Overall, 13/16 CNS lesions were present at the time of diagnosis. One parenchymal and two hydrocephalus lesions, however, developed after diagnosis and fulfilled the criteria for immune reconstitution syndrome. Cerebral spinal fluid antigen titers were higher with meningeal versus parenchymal lesions, and hydrocephalus (P=0.015). Mortality was 50% (3/6) for patients with parenchymal, 12.5% (1/8) for those with leptomeningeal, and 0/3 for patients with hydrocephalus. Mortality was 31% (4/13) for patients with CNS lesions at baseline and 0/3 in those with new onset lesions. CONCLUSIONS: Despite a higher antigen titer with meningeal lesions, outcomes tended to be worse with parenchymal compared with meningeal lesions or hydrocephalus. New onset CNS lesions may represent immune reconstitution syndrome and seemed to be associated with better outcome.

Authors
Singh, N; Lortholary, O; Dromer, F; Alexander, BD; Gupta, KL; John, GT; del Busto, R; Klintmalm, GB; Somani, J; Lyon, GM; Pursell, K; Stosor, V; Munoz, P; Limaye, AP; Kalil, AC; Pruett, TL; Garcia-Diaz, J; Humar, A; Houston, S; House, AA; Wray, D; Orloff, S; Dowdy, LA; Fisher, RA; Heitman, J; Wagener, MM; Husain, S; Cryptococcal Collaborative Transplant Study Group,
MLA Citation
Singh, N, Lortholary, O, Dromer, F, Alexander, BD, Gupta, KL, John, GT, del Busto, R, Klintmalm, GB, Somani, J, Lyon, GM, Pursell, K, Stosor, V, Munoz, P, Limaye, AP, Kalil, AC, Pruett, TL, Garcia-Diaz, J, Humar, A, Houston, S, House, AA, Wray, D, Orloff, S, Dowdy, LA, Fisher, RA, Heitman, J, Wagener, MM, Husain, S, and Cryptococcal Collaborative Transplant Study Group, . "Central nervous system cryptococcosis in solid organ transplant recipients: clinical relevance of abnormal neuroimaging findings." Transplantation 86.5 (September 15, 2008): 647-651.
PMID
18791444
Source
pubmed
Published In
Transplantation
Volume
86
Issue
5
Publish Date
2008
Start Page
647
End Page
651
DOI
10.1097/TP.0b013e3181814e76

Calcineurin localizes to the hyphal septum in Aspergillus fumigatus: implications for septum formation and conidiophore development.

A functional calcineurin A fusion to enhanced green fluorescent protein (EGFP), CnaA-EGFP, was expressed in the Aspergillus fumigatus DeltacnaA mutant. CnaA-EGFP localized in actively growing hyphal tips, at the septa, and at junctions between the vesicle and phialides in an actin-dependent manner. This is the first study to implicate calcineurin in septum formation and conidiophore development of a filamentous fungus.

Authors
Juvvadi, PR; Fortwendel, JR; Pinchai, N; Perfect, BZ; Heitman, J; Steinbach, WJ
MLA Citation
Juvvadi, PR, Fortwendel, JR, Pinchai, N, Perfect, BZ, Heitman, J, and Steinbach, WJ. "Calcineurin localizes to the hyphal septum in Aspergillus fumigatus: implications for septum formation and conidiophore development." Eukaryot Cell 7.9 (September 2008): 1606-1610.
PMID
18606829
Source
pubmed
Published In
Eukaryotic cell
Volume
7
Issue
9
Publish Date
2008
Start Page
1606
End Page
1610
DOI
10.1128/EC.00200-08

Calcineurin target CrzA regulates conidial germination, hyphal growth, and pathogenesis of Aspergillus fumigatus.

The calcineurin pathway is a critical signal transduction pathway in fungi that mediates growth, morphology, stress responses, and pathogenicity. The importance of the calcineurin pathway in fungal physiology creates an opportunity for the development of new antifungal therapies that target this critical signaling pathway. In this study, we examined the role of the zinc finger transcription factor Crz1 homolog (CrzA) in the physiology and pathogenicity of the opportunistic human fungal pathogen Aspergillus fumigatus. Genetic replacement of the crzA locus in A. fumigatus resulted in a strain with significant defects in conidial germination, polarized hyphal growth, cell wall structure, and asexual development that are similar to but with differences from defects seen in the A. fumigatus DeltacnaA (calcineurin A) strain. Like the DeltacnaA strain, the DeltacrzA strain was incapable of causing disease in an experimental persistently neutropenic inhalational murine model of invasive pulmonary aspergillosis. Our results suggest that CrzA is an important downstream effector of calcineurin that controls morphology in A. fumigatus, but additional downstream effectors that mediate calcineurin signal transduction are likely present in this opportunistic fungal pathogen. In addition, the importance of CrzA to the production of disease is critical, and thus CrzA is an attractive fungus-specific antifungal target for the treatment of invasive aspergillosis.

Authors
Cramer, RA; Perfect, BZ; Pinchai, N; Park, S; Perlin, DS; Asfaw, YG; Heitman, J; Perfect, JR; Steinbach, WJ
MLA Citation
Cramer, RA, Perfect, BZ, Pinchai, N, Park, S, Perlin, DS, Asfaw, YG, Heitman, J, Perfect, JR, and Steinbach, WJ. "Calcineurin target CrzA regulates conidial germination, hyphal growth, and pathogenesis of Aspergillus fumigatus." Eukaryot Cell 7.7 (July 2008): 1085-1097.
PMID
18456861
Source
pubmed
Published In
Eukaryotic cell
Volume
7
Issue
7
Publish Date
2008
Start Page
1085
End Page
1097
DOI
10.1128/EC.00086-08

Impact of mating type, serotype, and ploidy on the virulence of Cryptococcus neoformans.

Hybridization with polyploidization is a significant biological force driving evolution. The effect of combining two distinct genomes in one organism on the virulence potential of pathogenic fungi is not clear. Cryptococcus neoformans, the most common cause of fungal infection of the central nervous system, has a bipolar mating system with a and alpha mating types and occurs as A (haploid), D (haploid), and AD hybrid (mostly diploid) serotypes. Diploid AD hybrids are derived either from a-alpha mating or from unisexual mating between haploid cells. The precise contributions of increased ploidy, the effect of hybridization between serotypes A and D, and the combination of mating types to the virulence potential of AD hybrids have remained elusive. By using in vitro and in vivo characterization of laboratory-constructed isogenic diploids and AD hybrids with all possible mating type combinations in defined genetic backgrounds, we found that higher ploidy has a minor negative effect on virulence in a murine inhalation model of cryptococcosis. The presence of both mating types a and alpha in AD hybrids did not affect the virulence potential, irrespective of the serotype origin. Interestingly, AD hybrids with only one mating type behaved differently, with the virulence of alphaADalpha strains similar to that of other hybrids, while aADa hybrids displayed significantly lower virulence due to negative epistatic interactions between the Aa and Da alleles of the mating type locus. This study provides insights into the impact of ploidy, mating type, and serotype on virulence and the impact of hybridization on the fitness and virulence of a eukaryotic microbial pathogen.

Authors
Lin, X; Nielsen, K; Patel, S; Heitman, J
MLA Citation
Lin, X, Nielsen, K, Patel, S, and Heitman, J. "Impact of mating type, serotype, and ploidy on the virulence of Cryptococcus neoformans." Infect Immun 76.7 (July 2008): 2923-2938.
PMID
18426889
Source
pubmed
Published In
Infection and immunity
Volume
76
Issue
7
Publish Date
2008
Start Page
2923
End Page
2938
DOI
10.1128/IAI.00168-08

Surfactant Protein D enhances phagocytosis of Cryptococcus neoformans hypocapsular strain cap59 Delta by murine macrophages

Authors
Geunes-Boyer, SG; Oliver, TN; Heitman, J; Perfect, JR; Wright, JR
MLA Citation
Geunes-Boyer, SG, Oliver, TN, Heitman, J, Perfect, JR, and Wright, JR. "Surfactant Protein D enhances phagocytosis of Cryptococcus neoformans hypocapsular strain cap59 Delta by murine macrophages." FASEB JOURNAL 22 (April 2008).
Source
wos-lite
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
22
Publish Date
2008

Calcineurin inhibitor agents interact synergistically with antifungal agents in vitro against Cryptococcus neoformans isolates: correlation with outcome in solid organ transplant recipients with cryptococcosis.

Synergistic interactions were observed between CIs and antifungal agents against 53 (90%) of 59 Cryptococcus neoformans isolates from solid organ transplant recipients with cryptococcosis and may account for better outcomes in patients with cryptococcosis receiving these immunosuppressive agents.

Authors
Kontoyiannis, DP; Lewis, RE; Alexander, BD; Lortholary, O; Dromer, F; Gupta, KL; John, GT; Del Busto, R; Klintmalm, GB; Somani, J; Lyon, GM; Pursell, K; Stosor, V; Munoz, P; Limaye, AP; Kalil, AC; Pruett, TL; Garcia-Diaz, J; Humar, A; Houston, S; House, AA; Wray, D; Orloff, S; Dowdy, LA; Fisher, RA; Heitman, J; Albert, ND; Wagener, MM; Singh, N
MLA Citation
Kontoyiannis, DP, Lewis, RE, Alexander, BD, Lortholary, O, Dromer, F, Gupta, KL, John, GT, Del Busto, R, Klintmalm, GB, Somani, J, Lyon, GM, Pursell, K, Stosor, V, Munoz, P, Limaye, AP, Kalil, AC, Pruett, TL, Garcia-Diaz, J, Humar, A, Houston, S, House, AA, Wray, D, Orloff, S, Dowdy, LA, Fisher, RA, Heitman, J, Albert, ND, Wagener, MM, and Singh, N. "Calcineurin inhibitor agents interact synergistically with antifungal agents in vitro against Cryptococcus neoformans isolates: correlation with outcome in solid organ transplant recipients with cryptococcosis." Antimicrob Agents Chemother 52.2 (February 2008): 735-738.
PMID
18070977
Source
pubmed
Published In
Antimicrobial agents and chemotherapy
Volume
52
Issue
2
Publish Date
2008
Start Page
735
End Page
738
DOI
10.1128/AAC.00990-07

Pulmonary cryptococcosis in solid organ transplant recipients: clinical relevance of serum cryptococcal antigen.

BACKGROUND: The role of serum cryptococcal antigen in the diagnosis and determinants of antigen positivity in solid organ transplant (SOT) recipients with pulmonary cryptococcosis has not been fully defined. METHODS: We conducted a prospective, multicenter study of SOT recipients with pulmonary cryptococcosis during 1999-2006. RESULTS: Forty (83%) of 48 patients with pulmonary cryptococcosis tested positive for cryptococcal antigen. Patients with concomitant extrapulmonary disease were more likely to have a positive antigen test result (P=.018), and antigen titers were higher in patients with extrapulmonary disease (P=.003) or fungemia (P=.045). Patients with single nodules were less likely to have a positive antigen test result than were those with all other radiographic presentations (P=.053). Among patients with isolated pulmonary cryptococcosis, lung transplant recipients were less likely to have positive cryptococcal antigen test results than were recipients of other types of SOT (P=.003). In all, 38% of the patients were asymptomatic or had pulmonary cryptococcosis detected as an incidental finding. Nodular densities or mass lesions were more likely to present as asymptomatic or incidentally detected pulmonary cryptococcosis than as pleural effusions and infiltrates (P=.008). CONCLUSIONS: A positive serum cryptococcal antigen test result in SOT recipients with pulmonary cryptococcosis appears to reflect extrapulmonary or more advanced radiographic disease.

Authors
Singh, N; Alexander, BD; Lortholary, O; Dromer, F; Gupta, KL; John, GT; del Busto, R; Klintmalm, GB; Somani, J; Lyon, GM; Pursell, K; Stosor, V; Muñoz, P; Limaye, AP; Kalil, AC; Pruett, TL; Garcia-Diaz, J; Humar, A; Houston, S; House, AA; Wray, D; Orloff, S; Dowdy, LA; Fisher, RA; Heitman, J; Wagener, MM; Husain, S
MLA Citation
Singh, N, Alexander, BD, Lortholary, O, Dromer, F, Gupta, KL, John, GT, del Busto, R, Klintmalm, GB, Somani, J, Lyon, GM, Pursell, K, Stosor, V, Muñoz, P, Limaye, AP, Kalil, AC, Pruett, TL, Garcia-Diaz, J, Humar, A, Houston, S, House, AA, Wray, D, Orloff, S, Dowdy, LA, Fisher, RA, Heitman, J, Wagener, MM, and Husain, S. "Pulmonary cryptococcosis in solid organ transplant recipients: clinical relevance of serum cryptococcal antigen." Clin Infect Dis 46.2 (January 15, 2008): e12-e18.
PMID
18171241
Source
pubmed
Published In
Clinical Infectious Diseases
Volume
46
Issue
2
Publish Date
2008
Start Page
e12
End Page
e18
DOI
10.1086/524738

A Mep2-dependent transcriptional profile links permease function to gene expression during pseudohyphal growth in saccharomyces cerevisiae

The ammonium permease Mep2 is required for the induction of pseudohyphal growth, a process in Saccharomyces cerevisiae that occurs in response to nutrient limitation. Mep2 has both a transport and a regulatory function, supporting models in which Mep2 acts as a sensor of ammonium availability. Potentially similar ammonium permease-dependent regulatory cascades operate in other fungi, and they may also function in animals via the homologous Rh proteins; however, little is known about the molecular mechanisms that mediate ammonium sensing. We show that Mep2 is localized to the cell surface during pseudohyphal growth, and it is required for both filamentous and invasive growth. Analysis of site-directed Mep2 mutants in residues lining the ammonia-conducting channel reveal separation of function alleles (transport and signaling defective; transport-proficient/signaling defective), indicating transport is necessary but not sufficient to sense ammonia. Furthermore, Mep2 overexpression enhances differentiation under normally repressive conditions and induces a transcriptional profile that is consistent with activation of the mitogen-activated protein (MAP) kinase pathway. This finding is supported by epistasis analysis establishing that the known role of the MAP kinase pathway in pseudohyphal growth is linked to Mep2 function. Together, these data strengthen the model that Mep2-like proteins are nutrient sensing transceptors that govern cellular differentiation. © 2008 by The American Society for Cell Biology.

Authors
Rutherford, JC; Chua, G; Hughes, T; Cardenas, ME; Heitman, J
MLA Citation
Rutherford, JC, Chua, G, Hughes, T, Cardenas, ME, and Heitman, J. "A Mep2-dependent transcriptional profile links permease function to gene expression during pseudohyphal growth in saccharomyces cerevisiae." Molecular Biology of the Cell 19.7 (2008): 3028-3039.
PMID
18434596
Source
scival
Published In
Molecular Biology of the Cell
Volume
19
Issue
7
Publish Date
2008
Start Page
3028
End Page
3039
DOI
10.1091/mbc.E08-01-0033

Signaling cascades as drug targets in model and pathogenic fungi

Microbes evolved to produce natural products that inhibit growth of competing soil microorganisms. In many cases these compounds act on fungi, which are eukaryotes with conserved gene sequences closely related to metazoans, including humans. The calcineurin inhibitors cyclosporin A and FK-506, the Tor inhibitor rapamycin, and the Hsp90 inhibitor geldanamycin, all act via targets conserved from yeast to humans. This allows the use of genetically tractable fungi as models to elucidate how these drugs and their targets function in yeast and human cells. These inhibitors also enable studies aimed at harnessing their intrinsic antimicrobial activities to develop novel antifungal therapies.Extensive studies have revealed a globally conserved role for the Tor protein in regulating growth and proliferation in response to nutrients, and targeting its essential functions results in robust antifungal action. Similarly, a conserved and essential role for calcineurin in fungal virulence has been established and could be targeted by inhibitors for therapeutic uses in a variety of clinical settings. Finally, the discovery that inhibitors of calcineurin or Hsp90 result in dramatic synergism with either azoles or glucan synthase inhibitors (candins) providesanother therapeutic vantage point. Taken together, these fungal targets and their inhibitors provide a robust platform from which to develop novel antimicrobial therapies. © The Thomson Corporation.

Authors
Bastidas, RJ; Reedy, JL; Morales-Johansson, H; Heitman, J; Cardenas, ME
MLA Citation
Bastidas, RJ, Reedy, JL, Morales-Johansson, H, Heitman, J, and Cardenas, ME. "Signaling cascades as drug targets in model and pathogenic fungi." Current Opinion in Investigational Drugs 9.8 (2008): 856-864.
PMID
18666033
Source
scival
Published In
Current Opinion in Investigational Drugs
Volume
9
Issue
8
Publish Date
2008
Start Page
856
End Page
864

Orchestration of sexual reproduction and virulence by the fungal mating-type locus

The mating-type locus (MAT) orchestrates sexual reproduction in fungi. Sexual reproduction is related not only to fitness of an organism, but also correlated with virulence in certain pathogens. In the dandruff-associated fungus Malassesia globosa, although the sexual cycle remains to be discovered, whole genome analysis has led to the hypothesis that mating may occur on host skin. Furthermore, the MAT locus of M. globosa and U. hordei provides evidence that transitions between tetrapolar and bipolar systems have independently occurred. These results, together with studies recapitulating the ancestral tetrapolar mating system in Cryptococcus and the structure of MAT in related smut fungi, have furthered understanding on transitions between different mating systems and the evolution of MAT in the Basidiomycota. © 2008 Elsevier Ltd. All rights reserved.

Authors
Hsueh, Y-P; Heitman, J
MLA Citation
Hsueh, Y-P, and Heitman, J. "Orchestration of sexual reproduction and virulence by the fungal mating-type locus." Current Opinion in Microbiology 11.6 (2008): 517-524.
PMID
18935978
Source
scival
Published In
Current Opinion in Microbiology
Volume
11
Issue
6
Publish Date
2008
Start Page
517
End Page
524
DOI
10.1016/j.mib.2008.09.014

Magnificent seven: Roles of G protein-coupled receptors in extracellular sensing in fungi

G protein-coupled receptors (GPCRs) represent the largest family of transmembrane receptors and are responsible for transducing extracellular signals into intracellular responses that involve complex intracellular- signaling networks. This review highlights recent research advances in fungal GPCRs, including classification, extracellular sensing, and G protein-signaling regulation. The involvement of GPCRs in pheromone and nutrient sensing has been studied extensively over the past decade. Following recent advances in fungal genome sequencing projects, a panoply of GPCR candidates has been revealed and some have been documented to play key roles sensing diverse extracellular signals, such as pheromones, sugars, amino acids, nitrogen sources, and even photons. Identification and deorphanization of additional putative GPCRs may require the development of new research tools. Here, we compare research on GPCRs in fungi with information derived from mammalian systems to provide a useful road map on how to better understand ligand-GPCR-G protein interactions in general. We also emphasize the utility of yeast as a discovery tool for systemic studies of GPCRs from other organisms.

Authors
Xue, C; Hsueh, Y-P; Heitman, J
MLA Citation
Xue, C, Hsueh, Y-P, and Heitman, J. "Magnificent seven: Roles of G protein-coupled receptors in extracellular sensing in fungi." FEMS Microbiology Reviews 32.6 (2008): 1010-1032.
PMID
18811658
Source
scival
Published In
Fems Microbiology Reviews
Volume
32
Issue
6
Publish Date
2008
Start Page
1010
End Page
1032
DOI
10.1111/j.1574-6976.2008.00131.x

Transitions in sexuality: Recapitulation of an ancestral tri- and tetrapolar mating system in Cryptococcus neoformans

Sex is orchestrated by the mating-type locus (MAT) in fungi and by sex chromosomes in plants and animals. In fungi, two patterns of sexuality occur: bipolar with a single, typically biallelic sex determinant that promotes inbreeding, and tetrapolar with two unlinked, often multiallelic sex determinants that restrict inbreeding. Multiallelism in either bipolar or tetrapolar mating systems promotes outcrossing. Cryptococcus neoformans is a pathogenic bipolar yeast with two unusually large MAT alleles (a/α) spanning >100 kb, ∼100-fold larger than many other fungal MAT loci. Based on comparative genomic analysis, this unusual MAT locus is hypothesized to have evolved from an ancestral tetrapolar system. In this model, the unlinked homeodomain (HD) transcription factor and pheromone/receptor tetrapolar loci acquired additional sex-related genes and then fused via chromosomal translocation, forming an intermediate transitional mating system (which we term tripolar), which then underwent recombination and gene conversion to fashion the extant bipolar MAT alleles. To experimentally validate this model, C. neoformans was engineered to have a tetrapolar mating system by relocating the MAT SXI1α and SXI2a HD genes to an unlinked genomic locale. Genetic and molecular analyses revealed that this modified organism could complete a tetrapolar sexual cycle. Analysis of progeny generated from bipolar, tripolar, and tetrapolar crosses provides direct experimental evidence that the tripolar state confers decreased fertility and therefore may represent an unstable evolutionary intermediate. These findings illustrate how transitions between outcrossing and inbreeding preference occur by involving sex determinant linkage and collapse from multiallelic to biallelic sex determination, providing insights into both fungal sex evolution and early steps in sex chromosome evolution. Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Authors
Hsueh, Y-P; Fraser, JA; Heitman, J
MLA Citation
Hsueh, Y-P, Fraser, JA, and Heitman, J. "Transitions in sexuality: Recapitulation of an ancestral tri- and tetrapolar mating system in Cryptococcus neoformans." Eukaryotic Cell 7.10 (2008): 1847-1855.
PMID
18723606
Source
scival
Published In
Eukaryotic cell
Volume
7
Issue
10
Publish Date
2008
Start Page
1847
End Page
1855
DOI
10.1128/EC.00271-08

Isolates of Cryptococcus neoformans from infected animals reveal genetic exchange in unisexual, α mating type populations

Sexual reproduction and genetic exchange are important for the evolution of fungal pathogens and for producing potentially infective spores. Studies to determine whether sex occurs in the pathogenic yeast Cryptococcus neoformans var. grubii have produced enigmatic results, however: basidiospores are the most likely infective propagules, and clinical isolates are fertile and genetically diverse, consistent with a sexual species, but almost all populations examined consist of a single mating type and have little evidence for genetic recombination. The choice of population is critical when looking for recombination, particularly when significant asexual propagation is likely and when latency may complicate assessing the origin of an isolate. We therefore selected isolates from infected animals living in the region of Sydney, Australia, with the assumption that the relatively short life spans and limited travels of the animal hosts would provide a very defined population. All isolates were mating type α and were of molecular genotype VNI or VNII. A lack of linkage disequilibrium among loci suggested that genetic exchange occurred within both genotype groups. Four diploid VNII isolates that produced filaments and basidium-like structures when cultured in proximity to an a mating type strain were found. Recent studies suggest that compatible α-α unions can occur in C. neoformans var. neoformans populations and in populations of the sibling species Cryptococcus gattii. As a mating type strains of C. neoformans var. grubii have never been found in Australia, or in the VNII molecular type globally, the potential for α-α unions is evidence that α-α unisexual mating maintains sexual recombination and diversity in this pathogen and may produce infectious propagules. Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Authors
Bui, T; Lin, X; Malik, R; Heitman, J; Carter, D
MLA Citation
Bui, T, Lin, X, Malik, R, Heitman, J, and Carter, D. "Isolates of Cryptococcus neoformans from infected animals reveal genetic exchange in unisexual, α mating type populations." Eukaryotic Cell 7.10 (2008): 1771-1780.
PMID
18552280
Source
scival
Published In
Eukaryotic cell
Volume
7
Issue
10
Publish Date
2008
Start Page
1771
End Page
1780
DOI
10.1128/EC.00097-08

The RGS protein Crg2 regulates both pheromone and cAMP signalling in Cryptococcus neoformans

G proteins orchestrate critical cellular functions by transducing extracellular signals into internal signals and controlling cellular responses to environmental cues. G proteins typically function as switches that are activated by G protein-coupled receptors (GPCRs) and negatively controlled by regulator of G protein signalling (RGS) proteins. In the human fungal pathogen Cryptococcus neoformans, three G protein α subunits (Gpa1, Gpa2 and Gpa3) have been identified. In a previous study, we identified the RGS protein Crg2 involved in regulating the pheromone response pathway through Gpa2 and Gpa3. In this study, a role for Crg2 was established in the Gpa1-cAMP signalling pathway that governs mating and virulence. We show that Crg2 physically interacts with Gpa1 and crg2 mutations increase cAMP production. crg2 mutations also enhance mating filament hyphae production, but reduce cell-cell fusion and sporulation efficiency during mating. Although crg2 mutations and the Gpa1 dominant active allele GPA1Q284L enhanced melanin production under normally repressive conditions, virulence was attenuated in a murine model. We conclude that Crg2 participates in controlling both Gpa1-cAMP-virulence and pheromone-mating signalling cascades and hypothesize it may serve as a molecular interface between these two central signalling conduits.

Authors
Xue, C; Hsueh, Y-P; Chen, L; Heitman, J
MLA Citation
Xue, C, Hsueh, Y-P, Chen, L, and Heitman, J. "The RGS protein Crg2 regulates both pheromone and cAMP signalling in Cryptococcus neoformans." Molecular Microbiology 70.2 (2008): 379-395.
PMID
18761692
Source
scival
Published In
Molecular Microbiology
Volume
70
Issue
2
Publish Date
2008
Start Page
379
End Page
395
DOI
10.1111/j.1365-2958.2008.06417.x

Synergistic effect of calcineurin inhibitors and fluconazole against Candida albicans biofilms

Calcineurin is a Ca2+-calmodulin-activated serine/threonine- specific protein phosphatase that governs multiple aspects of fungal physiology, including cation homeostasis, morphogenesis, antifungal drug susceptibility, and virulence. Growth of Candida albicans planktonic cells is sensitive to the calcineurin inhibitors FK506 and cyclosporine A (CsA) in combination with the azole antifungal fluconazole. This drug synergism is attributable to two effects: first, calcineurin inhibitors render fluconazole fungicidal rather than simply fungistatic, and second, membrane perturbation by azole inhibition of ergosterol biosynthesis increases intracellular calcineurin inhibitor concentrations. C. albicans cells in biofilms are up to 1,000-fold more resistant to fluconazole than planktonic cells. In both in vitro experiments and in an in vivo rat catheter model, C. albicans cells in biofilms were resistant to individually delivered fluconazole or calcineurin inhibitors but exquisitely sensitive to the combination of FK506-fluconazole or CsA-fluconazole. C. albicans strains lacking FKBP12 or expressing a dominant FK506-resistant calcineurin mutant subunit (Cnb1-1) formed biofilms that were resistant to FK506-fluconazole but susceptible to CsA-fluconazole, demonstrating that drug synergism is mediated via direct calcineurin inhibition. These findings reveal that calcineurin contributes to fluconazole resistance of biofilms and provide evidence that synergistic drug combinations may prove efficacious as novel therapeutic interventions to treat or prevent biofilms. Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Authors
Uppuluri, P; Nett, J; Heitman, J; Andes, D
MLA Citation
Uppuluri, P, Nett, J, Heitman, J, and Andes, D. "Synergistic effect of calcineurin inhibitors and fluconazole against Candida albicans biofilms." Antimicrobial Agents and Chemotherapy 52.3 (2008): 1127-1132.
PMID
18180354
Source
scival
Published In
Antimicrobial agents and chemotherapy
Volume
52
Issue
3
Publish Date
2008
Start Page
1127
End Page
1132
DOI
10.1128/AAC.01397-07

Fungal genome sequencing and bioenergy

To date, the number of ongoing filamentous fungal genome sequencing projects is almost tenfold fewer than those of bacterial and archaeal genome projects. The fungi chosen for sequencing represent narrow kingdom diversity; most are pathogens or models. We advocate an ambitious, forward-looking phylogenetic-based genome sequencing program, designed to capture metabolic diversity within the fungal kingdom, thereby enhancing research into alternative bioenergy sources, bioremediation, and fungal-environment interactions.

Authors
Baker, SE; Thykaer, J; Adney, WS; Brettin, TS; Brockman, FJ; D'haeseleer, P; Martinez, AD; Miller, RM; Rokhsar, DS; Schadt, CW; Torok, T; Tuskan, G; Bennett, J; Berka, RM; Briggs, SP; Heitman, J; Taylor, J; Turgeon, BG; Werner-Washburne, M; Himmel, ME
MLA Citation
Baker, SE, Thykaer, J, Adney, WS, Brettin, TS, Brockman, FJ, D'haeseleer, P, Martinez, AD, Miller, RM, Rokhsar, DS, Schadt, CW, Torok, T, Tuskan, G, Bennett, J, Berka, RM, Briggs, SP, Heitman, J, Taylor, J, Turgeon, BG, Werner-Washburne, M, and Himmel, ME. "Fungal genome sequencing and bioenergy." Fungal Biology Reviews 22.1 (2008): 1-5.
Source
scival
Published In
Fungal Biology Reviews
Volume
22
Issue
1
Publish Date
2008
Start Page
1
End Page
5
DOI
10.1016/j.fbr.2008.03.001

Impact of ammonium permeases MepA, MepB, and MepC on nitrogen-regulated secondary metabolism in Fusarium fujikuroi

In Fusarium fujikuroi, the production of gibberellins and bikaverin is repressed by nitrogen sources such as glutamine or ammonium. Sensing and uptake of ammonium by specific permeases play key roles in nitrogen metabolism. Here, we describe the cloning of three ammonium permease genes, mepA, mepB, and mepC, and their participation in ammonium uptake and signal transduction in F. fujikuroi. The expression of all three genes is strictly regulated by the nitrogen regulator AreA. Severe growth defects of ΔmepB mutants on low-ammonium medium and methylamine uptake studies suggest that MepB functions as the main ammonium permease in F. fujikuroi. In ΔmepB mutants, nitrogen-regulated genes such as the gibberellin and bikaverin biosynthetic genes are derepressed in spite of high extracellular ammonium concentrations. mepA mepB and mepC mepB double mutants show a similar phenotype as ΔmepB mutants. All three F. fujikuroi mep genes fully complemented the Saccharomyces cerevisiae mep1 mep2 mep3 triple mutant to restore growth on low-ammonium medium, whereas only MepA and MepC restored pseudohyphal growth in the mep2/mep2 mutant. Overexpression of mepC in the ΔmepB mutants partially suppressed the growth defect but did not prevent derepression of AreA-regulated genes. These studies provide evidence that MepB functions as a regulatory element in a nitrogen sensing system in F. fujikuroi yet does not provide the sensor activity of Mep2 in yeast, indicating differences in the mechanisms by which nitrogen is sensed in S. cerevisiae and F. fujikuroi. Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Authors
Teichert, S; Rutherford, JC; Wottawa, M; Heitman, J; Tudzynski, B
MLA Citation
Teichert, S, Rutherford, JC, Wottawa, M, Heitman, J, and Tudzynski, B. "Impact of ammonium permeases MepA, MepB, and MepC on nitrogen-regulated secondary metabolism in Fusarium fujikuroi." Eukaryotic Cell 7.2 (2008): 187-201.
PMID
18083831
Source
scival
Published In
Eukaryotic cell
Volume
7
Issue
2
Publish Date
2008
Start Page
187
End Page
201
DOI
10.1128/EC.00351-07

Amt2 permease is required to induce ammonium-responsive invasive growth and mating in Cryptococcus neoformans

The conserved AmtB/Mep/Rh family of proteins mediate the transport of ammonium across cellular membranes in a wide range of organisms. Certain fungal members of this group are required to initiate filamentous growth. We have investigated the functions of two members of the AmtB/Mep/Rh family from the pathogenic basidiomycete Cryptococcus neoformans. Amt1 and Amt2 are low- and high-affinity ammonium permeases, respectively, and a mutant lacking both permeases is unable to grow under ammonium-limiting conditions. AMT2 is transcriptionally induced in response to nitrogen limitation, whereas AMT1 is constitutively expressed. Single and double amt mutants exhibit wild-type virulence in two models of cryptococcosis. Consistent with this, the formation of two C. neoformans virulence factors, cell wall melanin and the extracellular polysaccharide capsule, is not impaired in cells lacking either or both of the Amt1 and Amt2 permeases. Amt2 is, however, required for the initiation of invasive growth of haploid cells under low-nitrogen conditions and for the mating of wild-type cells under the same conditions. We propose that Amt2 may be a new fungal ammonium sensor and an element of the signaling cascades that govern the mating of C. neoformans in response to environmental nutritional cues. Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Authors
Rutherford, JC; Lin, X; Nielsen, K; Heitman, J
MLA Citation
Rutherford, JC, Lin, X, Nielsen, K, and Heitman, J. "Amt2 permease is required to induce ammonium-responsive invasive growth and mating in Cryptococcus neoformans." Eukaryotic Cell 7.2 (2008): 237-246.
PMID
18055915
Source
scival
Published In
Eukaryotic cell
Volume
7
Issue
2
Publish Date
2008
Start Page
237
End Page
246
DOI
10.1128/EC.00079-07

Fungal horizons: the Asilomar Fungal Genetics Conference 2007.

This meeting report covers advances presented at the 2007 Asilomar Fungal Genetics Conference that expand our understanding of fungal biology and the myriad ways in which studies of organisms in this ubiquitous and successful kingdom of life advance an understanding of conserved biological principles.

Authors
Heitman, J; Howlett, B
MLA Citation
Heitman, J, and Howlett, B. "Fungal horizons: the Asilomar Fungal Genetics Conference 2007." Fungal genetics and biology : FG & B 45.2 (2008): 77-83.
PMID
17977033
Source
scival
Published In
Fungal Genetics and Biology
Volume
45
Issue
2
Publish Date
2008
Start Page
77
End Page
83
DOI
10.1016/j.fgb.2007.09.009

Identification of the sex genes in an early diverged fungus

Sex determination in fungi is controlled by a small, specialized region of the genome in contrast to the large sex-specific chromosomes of animals and some plants. Different gene combinations reside at these mating-type (MAT) loci and confer sexual identity; invariably they encode homeodomain, α-box, or high mobility group (HMG)-domain transcription factors. So far, MAT loci have been characterized from a single monophyletic clade of fungi, the Dikarya (the ascomycetes and basidiomycetes), and the ancestral state and evolutionary history of these loci have remained a mystery. Mating in the basal members of the kingdom has been less well studied, and even their precise taxonomic inter-relationships are still obscure. Here we apply bioinformatic and genetic mapping to identify the sex-determining (sex) region in Phycomyces blakesleeanus (Zygomycota), which represents an early branch within the fungi. Each sex allele contains a single gene that encodes an HMG-domain protein, implicating the HMG-domain proteins as an earlier form of fungal MAT loci. Additionally, one allele also contains a copy of a unique, chromosome-specific repetitive element, suggesting a generalized mechanism for the earliest steps in the evolution of sex determination and sex chromosome structure in eukaryotes. ©2007 Nature Publishing Group.

Authors
Idnurm, A; Walton, FJ; Floyd, A; Heitman, J
MLA Citation
Idnurm, A, Walton, FJ, Floyd, A, and Heitman, J. "Identification of the sex genes in an early diverged fungus." Nature 451.7175 (2008): 193-196.
PMID
18185588
Source
scival
Published In
Nature
Volume
451
Issue
7175
Publish Date
2008
Start Page
193
End Page
196
DOI
10.1038/nature06453

Yeast as a tool in cancer research

Yeast is an experimental system that has led to critical discoveries in cell and molecular biology. The wide range of tools available in yeast has also made it an important system in many areas relevant to cancer including anti-cancer drug discovery, mechanisms of cell cycle control, and biological responses to stress. The present volume represents a state-of-the-art description of many areas of cancer research where yeast based systems are proving particularly valuable. This volume is of particular value to cancer researchers who lack extensive experience with yeast, but are interested in current results with this highly relevant experimental system. © 2007 Springer. All rights reserved.

Authors
Nitiss, JL; Heitman, J
MLA Citation
Nitiss, JL, and Heitman, J. "Yeast as a tool in cancer research." Yeast as a Tool in Cancer Research (December 1, 2007): 1-433.
Source
scopus
Published In
Yeast as a Tool in Cancer Research
Publish Date
2007
Start Page
1
End Page
433
DOI
10.1007/978-1-4020-5963-6

alpha AD alpha hybrids of Cryptococcus neoformans: evidence of same-sex mating in nature and hybrid fitness.

Cryptococcus neoformans is a ubiquitous human fungal pathogen that causes meningoencephalitis in predominantly immunocompromised hosts. The fungus is typically haploid, and sexual reproduction involves two individuals with opposite mating types/sexes, alpha and a. However, the overwhelming predominance of mating type (MAT) alpha over a in C. neoformans populations limits alpha-a mating in nature. Recently it was discovered that C. neoformans can undergo same-sex mating under laboratory conditions, especially between alpha isolates. Whether same-sex mating occurs in nature and contributes to the current population structure was unknown. In this study, natural alpha AD alpha hybrids that arose by fusion between two alpha cells of different serotypes (A and D) were identified and characterized, providing definitive evidence that same-sex mating occurs naturally. A novel truncated allele of the mating-type-specific cell identity determinant SXI1 alpha was also identified as a genetic factor likely involved in this process. In addition, laboratory-constructed alpha AD alpha strains exhibited hybrid vigor both in vitro and in vivo, providing a plausible explanation for their relative abundance in nature despite the fact that AD hybrids are inefficient in meiosis/sporulation and are trapped in the diploid state. These findings provide insights on the origins, genetic mechanisms, and fitness impact of unisexual hybridization in the Cryptococcus population.

Authors
Lin, X; Litvintseva, AP; Nielsen, K; Patel, S; Floyd, A; Mitchell, TG; Heitman, J
MLA Citation
Lin, X, Litvintseva, AP, Nielsen, K, Patel, S, Floyd, A, Mitchell, TG, and Heitman, J. "alpha AD alpha hybrids of Cryptococcus neoformans: evidence of same-sex mating in nature and hybrid fitness." PLoS Genet 3.10 (October 2007): 1975-1990.
PMID
17953489
Source
pubmed
Published In
PLoS genetics
Volume
3
Issue
10
Publish Date
2007
Start Page
1975
End Page
1990
DOI
10.1371/journal.pgen.0030186

Targeting the calcineurin pathway enhances ergosterol biosynthesis inhibitors against Trichophyton mentagrophytes in vitro and in a human skin infection model.

Fluconazole-FK506 or fluconazole-cyclosporine drug combinations were tested in an ex vivo Trichophyton mentagrophytes human skin infection model. Conidia colonization was monitored by scanning electron microscopy over a 7-day treatment period. The fluconazole-FK506 combination demonstrated the most obvious advantage over single-drug therapy by clearing conidia and protecting skin from damage at low drug concentrations.

Authors
Onyewu, C; Eads, E; Schell, WA; Perfect, JR; Ullmann, Y; Kaufman, G; Horwitz, BA; Berdicevsky, I; Heitman, J
MLA Citation
Onyewu, C, Eads, E, Schell, WA, Perfect, JR, Ullmann, Y, Kaufman, G, Horwitz, BA, Berdicevsky, I, and Heitman, J. "Targeting the calcineurin pathway enhances ergosterol biosynthesis inhibitors against Trichophyton mentagrophytes in vitro and in a human skin infection model." Antimicrob Agents Chemother 51.10 (October 2007): 3743-3746.
PMID
17664323
Source
pubmed
Published In
Antimicrobial agents and chemotherapy
Volume
51
Issue
10
Publish Date
2007
Start Page
3743
End Page
3746
DOI
10.1128/AAC.00492-07

Many globally isolated AD hybrid strains of Cryptococcus neoformans originated in Africa.

Interspecific and intervarietal hybridization may contribute to the biological diversity of fungal populations. Cryptococcus neoformans is a pathogenic yeast and the most common fungal cause of meningitis in patients with AIDS. Most patients are infected with either of the two varieties of C. neoformans, designated as serotype A (C. neoformans var. grubii) or serotype D (C. neoformans var. neoformans). In addition, serotype AD strains, which are hybrids of these two varieties, are commonly isolated from clinical and environmental samples. While most isolates of serotype A and serotype D are haploid, AD strains are diploid or aneuploid, and contain two sets of chromosomes and two mating type alleles, MATa and MATalpha, one from each of the serotypes. The global population of serotype A is dominated by isolates with the MATalpha mating type (Aalpha); however, about half of the globally analyzed AD strains possess the extremely rare serotype A MATa allele (Aa). We previously described an unusual population of serotype A in Botswana, in which 25% of the strains contain the rare MATa allele. Here we utilized two methods, phylogenetic analysis of three genes and genotyping by scoring amplified fragment length polymorphisms, and discovered that AD hybrid strains possessing the rare serotype A MATa allele (genotype AaDalpha) cluster with isolates of serotype A from Botswana, whereas AD hybrids that possess the MATalpha serotype A allele (AalphaDa and AalphaDalpha) cluster with cosmopolitan isolates of serotype A. We also determined that AD hybrid strains are more resistant to UV irradiation than haploid serotype A strains from Botswana. These findings support two hypotheses: (i) AaDalpha strains originated in sub-Saharan Africa from a cross between strains of serotypes A and D; and (ii) this fusion produced hybrid strains with increased fitness, enabling the Botswanan serotype A MATa genome, which is otherwise geographically restricted, to survive, emigrate, and propagate throughout the world.

Authors
Litvintseva, AP; Lin, X; Templeton, I; Heitman, J; Mitchell, TG
MLA Citation
Litvintseva, AP, Lin, X, Templeton, I, Heitman, J, and Mitchell, TG. "Many globally isolated AD hybrid strains of Cryptococcus neoformans originated in Africa." PLoS Pathog 3.8 (August 17, 2007): e114-.
PMID
17708680
Source
pubmed
Published In
PLoS pathogens
Volume
3
Issue
8
Publish Date
2007
Start Page
e114
DOI
10.1371/journal.ppat.0030114

Calcineurin inhibition or mutation enhances cell wall inhibitors against Aspergillus fumigatus.

Calcineurin mutation or inhibition enhanced the antifungal morphological effect of cell wall inhibitors caspofungin or nikkomycin Z against Aspergillus fumigatus. Quantification of 1,3-beta-d-glucan revealed decreased amounts in the calcineurin A (DeltacnaA) mutant. Calcineurin can be an excellent adjunct therapeutic target in combination with other cell wall inhibitors against A. fumigatus.

Authors
Steinbach, WJ; Cramer, RA; Perfect, BZ; Henn, C; Nielsen, K; Heitman, J; Perfect, JR
MLA Citation
Steinbach, WJ, Cramer, RA, Perfect, BZ, Henn, C, Nielsen, K, Heitman, J, and Perfect, JR. "Calcineurin inhibition or mutation enhances cell wall inhibitors against Aspergillus fumigatus." Antimicrob Agents Chemother 51.8 (August 2007): 2979-2981.
PMID
17502415
Source
pubmed
Published In
Antimicrobial agents and chemotherapy
Volume
51
Issue
8
Publish Date
2007
Start Page
2979
End Page
2981
DOI
10.1128/AAC.01394-06

The human fungal pathogen Cryptococcus can complete its sexual cycle during a pathogenic association with plants.

Cryptococcus is a globally distributed human fungal pathogen that primarily afflicts immunocompromised individuals. How and why this human fungal pathogen associates with plants and how this environmental niche influences its life cycle remains a mystery. We established Cryptococcus-Arabidopsis and Cryptococcus-Eucalyptus systems and discovered that Cryptococcus proliferates and mates on plant surfaces. Mating efficiency of C. gattii was markedly enhanced on plants and myo-inositol and indole acetic acid were specific plant products that stimulated mating. On Arabidopsis, dwarfing and chlorosis were observed following infection with a fungal mixture of two opposite mating-type strains, but not with either mating-type alone. This infection process is countered by the plant jasmonate-mediated defense mechanism. These findings reveal that Cryptococcus can parasitically interact with plants to complete its sexual cycle, which may impact an understanding of the origin and evolution of both plant and animal fungal pathogens in nature.

Authors
Xue, C; Tada, Y; Dong, X; Heitman, J
MLA Citation
Xue, C, Tada, Y, Dong, X, and Heitman, J. "The human fungal pathogen Cryptococcus can complete its sexual cycle during a pathogenic association with plants." Cell Host Microbe 1.4 (June 14, 2007): 263-273.
PMID
18005707
Source
pubmed
Published In
Cell Host and Microbe
Volume
1
Issue
4
Publish Date
2007
Start Page
263
End Page
273
DOI
10.1016/j.chom.2007.05.005

Endosymbiosis: the evil within.

A recent study has revealed a novel feature of the symbiosis between a bacterium and a fungal pathogen. In addition to producing a pathogenic toxin, the endosymbiont of the rice pathogen Rhizopus microsporus controls the ability of the fungus to form sporangia and spores.

Authors
Valdivia, RH; Heitman, J
MLA Citation
Valdivia, RH, and Heitman, J. "Endosymbiosis: the evil within." Curr Biol 17.11 (June 5, 2007): R408-R410.
PMID
17550764
Source
pubmed
Published In
Current Biology
Volume
17
Issue
11
Publish Date
2007
Start Page
R408
End Page
R410
DOI
10.1016/j.cub.2007.04.001

Harnessing calcineurin as a novel anti-infective agent against invasive fungal infections.

The number of immunocompromised patients with invasive fungal infections continues to increase and new antifungal therapies are not keeping pace with the growing incidence of these infections and their associated mortality. Calcineurin inhibition is currently used to exert effective immunosuppression following organ transplantation and in treating various other conditions. However, the calcineurin pathway is also intricately involved in the growth and pathogenesis of the three major fungal pathogens of humans, Cryptococcus neoformans, Candida albicans and Aspergillus fumigatus, and the exploitation of fungal calcineurin pathways holds great promise for the future development of novel antifungal agents. This Review summarizes our current understanding of calcineurin biology in these fungal species, and its exciting potential role in treating invasive fungal infections.

Authors
Steinbach, WJ; Reedy, JL; Cramer, RA; Perfect, JR; Heitman, J
MLA Citation
Steinbach, WJ, Reedy, JL, Cramer, RA, Perfect, JR, and Heitman, J. "Harnessing calcineurin as a novel anti-infective agent against invasive fungal infections." Nat Rev Microbiol 5.6 (June 2007): 418-430. (Review)
PMID
17505522
Source
pubmed
Published In
Nature Reviews Microbiology
Volume
5
Issue
6
Publish Date
2007
Start Page
418
End Page
430
DOI
10.1038/nrmicro1680

Cryptococcus neoformans in organ transplant recipients: impact of calcineurin-inhibitor agents on mortality.

Variables influencing the risk of dissemination and outcome of Cryptococcus neoformans infection were assessed in 111 organ transplant recipients with cryptococcosis in a prospective, multicenter, international study. Sixty-one percent (68/111) of the patients had disseminated infection. The risk of disseminated cryptococcosis was significantly higher for liver transplant recipients (adjusted hazard ratio [HR], 6.65; P=.048). The overall mortality rate at 90 days was 14% (16/111). The mortality rate was higher in patients with abnormal mental status (P=.023), renal failure at baseline (P=.028), fungemia (P=.006), and disseminated infection (P=.035) and was lower in those receiving a calcineurin-inhibitor agent (P=.003). In a multivariable analysis, the receipt of a calcineurin-inhibitor agent was independently associated with a lower mortality (adjusted HR, 0.21; P=.008), and renal failure at baseline with a higher mortality rate (adjusted HR, 3.14; P=.037). Thus, outcome in transplant recipients with cryptococcosis appears to be influenced by the type of immunosuppressive agent employed. Additionally, discerning the basis for transplant type-specific differences in disease severity has implications relevant for yielding further insights into the pathogenesis of C. neoformans infection in transplant recipients.

Authors
Singh, N; Alexander, BD; Lortholary, O; Dromer, F; Gupta, KL; John, GT; del Busto, R; Klintmalm, GB; Somani, J; Lyon, GM; Pursell, K; Stosor, V; Munoz, P; Limaye, AP; Kalil, AC; Pruett, TL; Garcia-Diaz, J; Humar, A; Houston, S; House, AA; Wray, D; Orloff, S; Dowdy, LA; Fisher, RA; Heitman, J; Wagener, MM; Husain, S; Cryptococcal Collaborative Transplant Study Group,
MLA Citation
Singh, N, Alexander, BD, Lortholary, O, Dromer, F, Gupta, KL, John, GT, del Busto, R, Klintmalm, GB, Somani, J, Lyon, GM, Pursell, K, Stosor, V, Munoz, P, Limaye, AP, Kalil, AC, Pruett, TL, Garcia-Diaz, J, Humar, A, Houston, S, House, AA, Wray, D, Orloff, S, Dowdy, LA, Fisher, RA, Heitman, J, Wagener, MM, Husain, S, and Cryptococcal Collaborative Transplant Study Group, . "Cryptococcus neoformans in organ transplant recipients: impact of calcineurin-inhibitor agents on mortality." J Infect Dis 195.5 (March 1, 2007): 756-764.
PMID
17262720
Source
pubmed
Published In
Journal of Infectious Diseases
Volume
195
Issue
5
Publish Date
2007
Start Page
756
End Page
764
DOI
10.1086/511438

Peroxisome function regulates growth on glucose in the basidiomycete fungus Cryptococcus neoformans.

The function of the peroxisomes was examined in the pathogenic basidiomycete Cryptococcus neoformans. Recent studies reveal the glyoxylate pathway is required for virulence of diverse microbial pathogens of plants and animals. One exception is C. neoformans, in which isocitrate lyase (encoded by ICL1) was previously shown not to be required for virulence, and here this was extended to exclude also a role for malate synthase (encoded by MLS1). The role of peroxisomes, in which the glyoxylate pathway enzymes are localized in many organisms, was examined by mutation of two genes (PEX1 and PEX6) encoding AAA (ATPases associated with various cellular activities)-type proteins required for peroxisome formation. The pex1 and pex6 deletion mutants were unable to localize the fluorescent DsRED-SKL protein to peroxisomal punctate structures, in contrast to wild-type cells. pex1 and pex6 single mutants and a pex1 pex6 double mutant exhibit identical phenotypes, including abolished growth on fatty acids but no growth difference on acetate. Because both icl1 and mls1 mutants are unable to grow on acetate as the sole carbon source, these findings demonstrate that the glyoxylate pathway can function efficiently outside the peroxisome in C. neoformans. The pex1 mutant exhibits wild-type virulence in a murine inhalation model and in an insect host, demonstrating that peroxisomes are not required for virulence under these conditions. An unusual phenotype of the pex1 and pex6 mutants was that they grew poorly with glucose as the carbon source, but nearly wild type with galactose, which suggested impaired hexokinase function and that C. neoformans peroxisomes might function analogously to the glycosomes of the trypanosomid parasites. Deletion of the hexokinase HXK2 gene reduced growth in the presence of glucose and suppressed the growth defect of the pex1 mutant on glucose. The hexokinase 2 protein of C. neoformans contains a predicted peroxisome targeting signal (type 2) motif; however, Hxk2 fused to fluorescent proteins was not localized to peroxisomes. Thus, we hypothesize that glucose or glycolytic metabolites are utilized in the peroxisome by an as yet unidentified enzyme or regulate a pathway required by the fungus in the absence of peroxisomes.

Authors
Idnurm, A; Giles, SS; Perfect, JR; Heitman, J
MLA Citation
Idnurm, A, Giles, SS, Perfect, JR, and Heitman, J. "Peroxisome function regulates growth on glucose in the basidiomycete fungus Cryptococcus neoformans." Eukaryot Cell 6.1 (January 2007): 60-72.
PMID
17041184
Source
pubmed
Published In
Eukaryotic cell
Volume
6
Issue
1
Publish Date
2007
Start Page
60
End Page
72
DOI
10.1128/EC.00214-06

Sensing the environment: Lessons from fungi

All living organisms use numerous signal-transduction systems to sense and respond to their environments and thereby survive and proliferate in a range of biological niches. Molecular dissection of these signalling networks has increased our understanding of these communication processes and provides a platform for therapeutic intervention when these pathways malfunction in disease states, including infection. Owing to the expanding availability of sequenced genomes, a wealth of genetic and molecular tools and the conservation of signalling networks, members of the fungal kingdom serve as excellent model systems for more complex, multicellular organisms. Here, we review recent progress in our understanding of how fungal-signalling circuits operate at the molecular level to sense and respond to a plethora of environmental cues.

Authors
Bahn, Y-S; Xue, C; Idnurm, A; Rutherford, JC; Heitman, J; Cardenas, ME
MLA Citation
Bahn, Y-S, Xue, C, Idnurm, A, Rutherford, JC, Heitman, J, and Cardenas, ME. "Sensing the environment: Lessons from fungi." Nature Reviews Microbiology 5.1 (2007): 57-69.
PMID
17170747
Source
scival
Published In
Nature Reviews Microbiology
Volume
5
Issue
1
Publish Date
2007
Start Page
57
End Page
69
DOI
10.1038/nrmicro1578

Ssk2 mitogen-activated protein kinase kinase kinase governs divergent patterns of the stress-activated Hog1 signaling pathway in Cryptococcus neoformans

The stress-activated p38/Hog1 mitogen-activated protein kinase (MAPK) pathway is structurally conserved in many diverse organisms, including fungi and mammals, and modulates myriad cellular functions. The Hog1 pathway is uniquely specialized to control differentiation and virulence factors in a majority of clinical Cryptococcus neoformans serotype A and D strains. Here, we identified and characterized the Ssk2 MAPKKK that functions upstream of the MAPKK Pbs2 and the MAPK Hog1 in C. neoformans. The SSK2 gene was identified as a potential component responsible for the difference in Hog1 phosphorylation between the serotype D f1 sibling strains B-3501 and B-3502 through comparative analysis of meiotic maps showing their meiotic segregation patterns of Hog1-dependent sensitivity to the antifungal drug fludioxonil. Ssk2 is the only component of the Hog1 MAPK cascade that is polymorphic between the two strains, and the B-3501 and B-3502 SSK2 alleles were distinguished by two coding sequence changes. Supporting this finding, SSK2 allele exchange completely interchanged the Hog1-controlled signaling patterns, related phenotypes, and virulence levels of strains B-3501 and JEC21. In the serotype A strain H99, disruption of the SSK2 gene enhanced capsule and melanin biosynthesis and mating efficiency, similar to pbs2 and hog1 mutations. Furthermore, ssk2Δ, pbs2Δ, and hog1Δ mutants were hypersensitive to a variety of stresses and resistant to fludioxonil. In agreement with these results, Hog1 phosphorylation was abolished in the ssk2Δ mutant, similar to what occurred in the pbs2Δ mutant. Taken together, these findings indicate that Ssk2 is a critical interface connecting the two-component system and the Pbs2-Hog1 MAPK pathway in C. neoformans. Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Authors
Bahn, Y-S; Geunes-Boyer, S; Heitman, J
MLA Citation
Bahn, Y-S, Geunes-Boyer, S, and Heitman, J. "Ssk2 mitogen-activated protein kinase kinase kinase governs divergent patterns of the stress-activated Hog1 signaling pathway in Cryptococcus neoformans." Eukaryotic Cell 6.12 (2007): 2278-2289.
PMID
17951522
Source
scival
Published In
Eukaryotic cell
Volume
6
Issue
12
Publish Date
2007
Start Page
2278
End Page
2289
DOI
10.1128/EC.00349-07

Renaming the DSCR1/Adapt78 gene family as RCAN: Regulators of calcineurin

Authors
Davies, KJA; Ermak, G; Rothermel, BA; Pritchard, M; Heitman, J; Ahnn, J; Henrique-Silva, F; Crawford, D; Canaider, S; Strippoli, P; Carinci, P; Min, K-T; Fox, DS; Cunningham, KW; Bassel-Duby, R; Olson, EN; Zhang, Z; Williams, RS; Gerber, H-P; Pérez-Riba, M; Seo, H; Cao, X; Klee, CB; Redondo, JM; Maltais, LJ; Bruford, EA; Povey, S; Molkentin, JD; McKeon, FD; Duh, EJ; Crabtree, GR; Cyert, MS; Luna, SDL; Estivill, X
MLA Citation
Davies, KJA, Ermak, G, Rothermel, BA, Pritchard, M, Heitman, J, Ahnn, J, Henrique-Silva, F, Crawford, D, Canaider, S, Strippoli, P, Carinci, P, Min, K-T, Fox, DS, Cunningham, KW, Bassel-Duby, R, Olson, EN, Zhang, Z, Williams, RS, Gerber, H-P, Pérez-Riba, M, Seo, H, Cao, X, Klee, CB, Redondo, JM, Maltais, LJ, Bruford, EA, Povey, S, Molkentin, JD, McKeon, FD, Duh, EJ, Crabtree, GR, Cyert, MS, Luna, SDL, and Estivill, X. "Renaming the DSCR1/Adapt78 gene family as RCAN: Regulators of calcineurin." FASEB Journal 21.12 (2007): 3023-3028.
PMID
17595344
Source
scival
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
21
Issue
12
Publish Date
2007
Start Page
3023
End Page
3028
DOI
10.1096/fj.06-7246com

First contemporary case of human infection with Cryptococcus gattii in puget sound: Evidence for spread of the Vancouver Island outbreak

We report a case of cryptococcosis due to C. gattii which appears to have been acquired in the Puget Sound region, Washington State. Genotyping confirmed identity to the predominant Vancouver Island genotype. This is the first documented case of human disease by the major Vancouver Island emergence strain acquired within the United States. Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Authors
Upton, A; Fraser, JA; Kidd, SE; Bretz, C; Bartlett, KH; Heitman, J; Marr, KA
MLA Citation
Upton, A, Fraser, JA, Kidd, SE, Bretz, C, Bartlett, KH, Heitman, J, and Marr, KA. "First contemporary case of human infection with Cryptococcus gattii in puget sound: Evidence for spread of the Vancouver Island outbreak." Journal of Clinical Microbiology 45.9 (2007): 3086-3088.
PMID
17596366
Source
scival
Published In
Journal of clinical microbiology
Volume
45
Issue
9
Publish Date
2007
Start Page
3086
End Page
3088
DOI
10.1128/JCM.00593-07

G protein signaling governing cell fate decisions involves opposing Gα subunits in Cryptococcus neoformans

Communication between cells and their environments is often mediated by G protein-coupled receptors and cognate G proteins. In fungi, one such signaling cascade is the mating pathway triggered by pheromone/pheromone receptor recognition. Unlike Saccharomyces cerevisiae, which expresses two Gα subunits, most filamentous ascomycetes and basidiomycetes have three Gα subunits. Previous studies have defined the Gα subunit acting upstream of the cAMP-protein kinase A pathway, but it has been unclear which Gα subunit is coupled to the pheromone receptor and response pathway. Here we report that in the pathogenic basidiomycetous yeast Cryptococcus neoformans, two Gα subunits (Gpa2, Gpa3) sense pheromone and govern mating. gpa2 gpa3 double mutants, but neither gpa2 nor gpa3 single mutants, are sterile in bilateral crosses. By contrast, deletion of GPA3 (but not GPA2) constitutively activates pheromone response and filamentation. Expression of GPA2 and GPA3 is differentially regulated: GPA3 expression is induced by nutrient-limitation, whereas GPA2 is induced during mating. Based on the phenotype of dominant active alleles, Gpa2 and Gpa3 signal in opposition: Gpa2 promotes mating, whereas Gpa3 inhibits. The incorporation of an additional Gα into the regulatory circuit enabled increased signaling complexity and facilitated cell fate decisions involving choice between yeast growth and filamentous asexual/sexual development. © 2007 by The American Society for Cell Biology.

Authors
Hsueh, Y-P; Xue, C; Heitman, J
MLA Citation
Hsueh, Y-P, Xue, C, and Heitman, J. "G protein signaling governing cell fate decisions involves opposing Gα subunits in Cryptococcus neoformans." Molecular Biology of the Cell 18.9 (2007): 3237-3249.
PMID
17581859
Source
scival
Published In
Molecular Biology of the Cell
Volume
18
Issue
9
Publish Date
2007
Start Page
3237
End Page
3249
DOI
10.1091/mbc.E07-02-0133

Many globally isolated AD hybrid strains of Cryptococcus neoformans originated in Africa

Interspecific and intervarietal hybridization may contribute to the biological diversity of fungal populations. Cryptococcus neoformans is a pathogenic yeast and the most common fungal cause of meningitis in patients with AIDS. Most patients are infected with either of the two varieties of C. neoformans, designated as serotype A (C. neoformans var. grubii) or serotype D (C. neoformans var. neoformans). In addition, serotype AD strains, which are hybrids of these two varieties, are commonly isolated from clinical and environmental samples. While most isolates of serotype A and serotype D are haploid, AD strains are diploid or aneuploid, and contain two sets of chromosomes and two mating type alleles, MATa and MATα, one from each of the serotypes. The global population of serotype A is dominated by isolates with the MATα mating type (Aα); however, about half of the globally analyzed AD strains possess the extremely rare serotype A MATa allele (Aa). We previously described an unusual population of serotype A in Botswana, in which 25% of the strains contain the rare MATa allele. Here we utilized two methods, phylogenetic analysis of three genes and genotyping by scoring amplified fragment length polymorphisms, and discovered that AD hybrid strains possessing the rare serotype A MATa allele (genotype AaDα) cluster with isolates of serotype A from Botswana, whereas AD hybrids that possess the MATα serotype A allele (AαDa and AαDα) cluster with cosmopolitan isolates of serotype A. We also determined that AD hybrid strains are more resistant to UV irradiation than haploid serotype A strains from Botswana. These findings support two hypotheses: (i) AaDα strains originated in sub-Saharan Africa from a cross between strains of serotypes A and D; and (ii) this fusion produced hybrid strains with increased fitness, enabling the Botswanan serotype A MATa genome, which is otherwise geographically restricted, to survive, emigrate, and propagate throughout the world. © 2007 Litvintseva et al.

Authors
Litvintseva, AP; Lin, X; Templeton, I; Heitman, J; Mitchell, TG
MLA Citation
Litvintseva, AP, Lin, X, Templeton, I, Heitman, J, and Mitchell, TG. "Many globally isolated AD hybrid strains of Cryptococcus neoformans originated in Africa." PLoS Pathogens 3.8 (2007): 1109-1117.
Source
scival
Published In
PLoS pathogens
Volume
3
Issue
8
Publish Date
2007
Start Page
1109
End Page
1117
DOI
10.1371/journal.ppat.0030114

The Virulence of Human Pathogenic Fungi: Notes from the South of France

The Second FEBS Advanced Lecture Course on Human Fungal Pathogens: Molecular Mechanisms of Host-Pathogen Interactions and Virulence, organized by Christophe d'Enfert (Institut Pasteur, France), Anita Sil (UCSF, USA), and Steffen Rupp (Fraunhofer, IGB, Germany), occurred May 2007 in La Colle sur Loup, France. Here we review the advances presented and the current state of knowledge in key areas of fungal pathogenesis.

Authors
Reedy, JL; Bastidas, RJ; Heitman, J
MLA Citation
Reedy, JL, Bastidas, RJ, and Heitman, J. "The Virulence of Human Pathogenic Fungi: Notes from the South of France." Cell Host and Microbe 2.2 (2007): 77-83.
PMID
18005722
Source
scival
Published In
Cell Host & Microbe
Volume
2
Issue
2
Publish Date
2007
Start Page
77
End Page
83
DOI
10.1016/j.chom.2007.07.004

Eca1, a sarcoplasmic/endoplasmic reticulum Ca2+-ATPase, is involved in stress tolerance and virulence in Cryptococcus neoformans

The basidiomycetous fungal pathogen Cryptococcus neoformans is adapted to survive challenges in the soil and environment and within the unique setting of the mammalian host. A C. neoformans mutant was isolated with enhanced virulence in a soil amoeba model that nevertheless exhibits dramatically reduced growth at mammalian body temperature (37°C). This mutant phenotype results from an insertion in the ECA1 gene, which encodes a sarcoplasmic/endoplasmic reticulum (ER) Ca2+-ATPase (SERCA)-type calcium pump. Infection in murine macrophages, amoebae (Acanthamoeba castellanii), nematodes (Caenorhabditis elegans), and wax moth (Galleria mellonella) larvae revealed that the eca1 mutants are virulent or hypervirulent at permissive growth temperatures but attenuated at 37°C. Deletion mutants lacking the entire ECA1 gene were also hypersensitive to the calcineurin inhibitors cyclosporin and FK506 and to ER and osmotic stresses. An eca1Δ cna1Δ mutant lacking both Eca1 and the calcineurin catalytic subunit was more sensitive to high temperature and ER stresses than the single mutants and exhibited reduced survival in C. elegans and attenuated virulence towards wax moth larvae at temperatures that permit normal growth in vitro. Eca1 is likely involved in maintaining ER function, thus contributing to stress tolerance and virulence acting in parallel with Ca 2+-calcineurin signaling. Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Authors
Fan, W; Idnurm, A; Breger, J; Mylonakis, E; Heitman, J
MLA Citation
Fan, W, Idnurm, A, Breger, J, Mylonakis, E, and Heitman, J. "Eca1, a sarcoplasmic/endoplasmic reticulum Ca2+-ATPase, is involved in stress tolerance and virulence in Cryptococcus neoformans." Infection and Immunity 75.7 (2007): 3394-3405.
PMID
17502401
Source
scival
Published In
Infection and immunity
Volume
75
Issue
7
Publish Date
2007
Start Page
3394
End Page
3405
DOI
10.1128/IAI.01977-06

Cryptococcus neoformans mates on pigeon guano: Implications for the realized ecological niche and globalization

The ecological niche that a species can occupy is determined by its resource requirements and the physical conditions necessary for survival. The niche to which an organism is most highly adapted is the realized niche, whereas the complete range of habitats that an organism can occupy represents the fundamental niche. The growth and development of Cryptococcus neoformans and Cryptococcus gattii on pigeon guano were examined to determine whether these two species occupy the same or different ecological niches. C. neoformans is a cosmopolitan pathogenic yeast that infects predominantly immunocompromised individuals, exists in two varieties (grubii [serotype A] and neoformans [serotype D]), and is commonly isolated from pigeon guano worldwide. By contrast, C. gattii often infects immunocompetent individuals and is associated with geographically restricted environments, most notably, eucalyptus trees. Pigeon guano supported the growth of both species, and a brown pigment related to melanin, a key virulence factor, was produced. C. neoformans exhibited prolific mating on pigeon guano, whereas C. gattii did not. The observations that C. neoformans completes the life cycle on pigeon guano but that C. gattii does not indicates that pigeon guano could represent the realized ecological niche for C. neoformans. Because C. gattii grows on pigeon guano but cannot sexually reproduce, pigeon guano represents a fundamental but not a realized niche for C. gattii. Based on these studies, we hypothesize that an ancestral Cryptococcus strain gained the ability to sexually reproduce in pigeon guano and then swept the globe. Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Authors
Nielsen, K; Obaldia, ALD; Heitman, J
MLA Citation
Nielsen, K, Obaldia, ALD, and Heitman, J. "Cryptococcus neoformans mates on pigeon guano: Implications for the realized ecological niche and globalization." Eukaryotic Cell 6.6 (2007): 949-959.
PMID
17449657
Source
scival
Published In
Eukaryotic cell
Volume
6
Issue
6
Publish Date
2007
Start Page
949
End Page
959
DOI
10.1128/EC.00097-07

An EcoRI-RsrI chimeric restriction endonuclease retains parental sequence specificity

To test their structural and functional similarity, hybrids were constructed between EcoRI and RsrI, two restriction endonucleases recognizing the same DNA sequence and sharing 50% amino acid sequence identity. One of the chimeric proteins (EERE), in which the EcoRI segment His147-Ala206 was replaced with the corresponding RsrI segment, showed EcoRI/RsrI-specific endonuclease activity. EERE purified from inclusion bodies was found to have ∼ 100-fold weaker activity but higher specific DNA binding affinity, than EcoRI. Increased binding is consistent with results of molecular dynamics simulations, which indicate that the number of hydrogen bonds formed with the recognition sequence increased in the chimera as compared to EcoRI. The success of obtaining an EcoRI-RsrI hybrid endonuclease, which differs from EcoRI by 22 RsrI-specific amino acid substitutions and still preserves canonical cleavage specificity, is a sign of structural and functional similarity shared by the parental enzymes. This conclusion is also supported by computational studies, which indicate that construction of the EERE chimera did not induce substantial changes in the structure of EcoRI. Surprisingly, the chimeric endonuclease was more toxic to cells not protected by EcoRI methyltransferase, than the parental EcoRI mutant. Molecular modelling revealed structural alterations, which are likely to impede coupling between substrate recognition and cleavage and suggest a possible explanation for the toxic phenotype. © 2007 Elsevier B.V. All rights reserved.

Authors
Chuluunbaatar, T; Ivanenko-Johnston, T; Fuxreiter, M; Meleshko, R; Raskó, T; Simon, I; Heitman, J; Kiss, A
MLA Citation
Chuluunbaatar, T, Ivanenko-Johnston, T, Fuxreiter, M, Meleshko, R, Raskó, T, Simon, I, Heitman, J, and Kiss, A. "An EcoRI-RsrI chimeric restriction endonuclease retains parental sequence specificity." Biochimica et Biophysica Acta - Proteins and Proteomics 1774.5 (2007): 583-594.
PMID
17442645
Source
scival
Published In
BBA - Proteins and Proteomics
Volume
1774
Issue
5
Publish Date
2007
Start Page
583
End Page
594
DOI
10.1016/j.bbapap.2007.02.011

Evolution of the mating type locus: Insights gained from the dimorphic primary fungal pathogens Histoplasma capsulatum, Coccidioides immitis, and Coccidioides posadasii

Sexual reproduction of fungi is governed by the mating type (MAT) locus, a specialized region of the genome encoding key transcriptional regulators that direct regulatory networks to specify cell identity and fate. Knowledge of MAT locus structure and evolution has been considerably advanced in recent years as a result of genomic analyses that enable the definition of MAT locus sequences in many species as well as provide an understanding of the evolutionary plasticity of this unique region of the genome. Here, we extend this analysis to define the mating type locus of three dimorphic primary human fungal pathogens, Histoplasma capsulatum, Coccidioides immitis, and Coccidioides posadasii, using genomic analysis, direct sequencing, and bioinformatics. These studies provide evidence that all three species possess heterothallic bipolar mating type systems, with isolates encoding either a high-mobility-group (HMG) domain or an α-hox transcriptional regulator. These genes are intact in all loci examined and have not been subject to loss or decay, providing evidence that the loss of fertility upon passage in H. capsulatum is not attributable to mutations at the MAT locus. These findings also suggest that an extant sexual cycle remains to be defined in both Coccidioides species, in accord with population genetic evidence. Based on these MAT sequences, a facile PCR test was developed that allows the mating type to be rapidly ascertained. Finally, these studies highlight the evolutionary forces shaping the MAT locus, revealing examples in which flanking genes have been inverted or subsumed and incorporated into an expanding MAT locus, allowing us to propose an expanded model for the evolution of the MAT locus in the phylum Ascomycota. Copyright © 2007. American Society for Microbiology. All Rights Reserved.

Authors
Fraser, JA; Stajich, JE; Tarcha, EJ; Cole, GT; Inglis, DO; Sil, A; Heitman, J
MLA Citation
Fraser, JA, Stajich, JE, Tarcha, EJ, Cole, GT, Inglis, DO, Sil, A, and Heitman, J. "Evolution of the mating type locus: Insights gained from the dimorphic primary fungal pathogens Histoplasma capsulatum, Coccidioides immitis, and Coccidioides posadasii." Eukaryotic Cell 6.4 (2007): 622-629.
PMID
17337636
Source
scival
Published In
Eukaryotic cell
Volume
6
Issue
4
Publish Date
2007
Start Page
622
End Page
629
DOI
10.1128/EC.00018-07

The mating type-specific homeodomain genes SXI1α and SXI2a coordinately control uniparental mitochondrial inheritance in Cryptococcus neoformans

In the great majority of sexual eukaryotes, mitochondrial genomes are inherited almost exclusively from a single parent. While many hypotheses have been proposed to explain this phenomenon, very little is known about the genetic elements controlling uniparental mitochondria inheritance. In the bipolar, isogamous basidiomycete yeast Cryptococcus neoformans, progeny from crosses between strains of mating type a (MATa) and mating type α (MATα) typically inherit mitochondrial DNA (mtDNA) from the MATa parent. We recently demonstrated that a mating type α (MATα)- specific gene SXI1α, controls mitochondrial inheritance in C. neoformans. Here, we show that another homeodomain gene SXI2a in the alternative mating type MATa is also required for uniparental mtDNA inheritance in this fungus. Disruption of SXI2a resulted in biparental mtDNA inheritance in the zygote population with significant numbers of progeny inheriting mtDNA from the MATa parent, the MATα parent, and both the MATa and the MATα parents. In addition, progeny from same-sex mating between MATα strains showed a biparental mitochondrial inheritance pattern. Our results suggest that SXI1 and SXI2a coordinately control uniparental mitochondrial inheritance in C. neoformans. © Springer-Verlag 2006.

Authors
Yan, Z; Hull, CM; Sun, S; Heitman, J; Xu, J
MLA Citation
Yan, Z, Hull, CM, Sun, S, Heitman, J, and Xu, J. "The mating type-specific homeodomain genes SXI1α and SXI2a coordinately control uniparental mitochondrial inheritance in Cryptococcus neoformans." Current Genetics 51.3 (2007): 187-195.
PMID
17186242
Source
scival
Published In
Current Genetics
Volume
51
Issue
3
Publish Date
2007
Start Page
187
End Page
195
DOI
10.1007/s00294-006-0115-9

Divergence of protein kinase A catalytic subunits in Cryptococcus neoformans and Cryptococcus gattii illustrates evolutionary reconfiguration of a signaling cascade

Gene duplication and divergence via both the loss and gain of gene activities are powerful evolutionary forces underlying the origin off new biological functions. Here a comparative genetics approach was applied to examine the roles of protein kinase A (PKA) catalytic subunits in three closely related varieties or sibling species of the pathogenic fungus genus Cryptococcus. Previous studies revealed that two PKA catalytic subanits, Pka1 and Pka2, control virulence factor production and mating. However, only one of the two plays the predominant physiological role, and this function has been exchanged between Pka1 and Pka2 in strains of the Cryptococcus neoformans var. grubii serotype A lineage compared to divergent C. neoformans var. neoformans serotype D isolates. To understand the basis for this functional plasticity, here the activities of Pka1 and Pka2 were defined in the two varieties and the related sibling species Cryptococcus gattii by gene disruption and characterization, heterologous complementation, and analysis of serotype AD hybrid mutant strains. The findings provide evidence for a shared ancestral role of PKA in governing mating and virulence factor production and indicate that the exchange of catalytic subunit roles is attributable to loss of function. Our studies illustrate the plasticity of signaling networks enabling rapid rewiring during speciation of a clade of common human fungal pathogens. Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Authors
Hicks, JK; Heitman, J
MLA Citation
Hicks, JK, and Heitman, J. "Divergence of protein kinase A catalytic subunits in Cryptococcus neoformans and Cryptococcus gattii illustrates evolutionary reconfiguration of a signaling cascade." Eukaryotic Cell 6.3 (2007): 413-420.
PMID
17189488
Source
scival
Published In
Eukaryotic cell
Volume
6
Issue
3
Publish Date
2007
Start Page
413
End Page
420
DOI
10.1128/EC.00213-06

Fungal Pathogenesis: Gene Clusters Unveiled as Secrets within the Ustilago maydis Code

The genome sequence of a second plant pathogenic fungus is now available, revealing unique gene clusters encoding secretory proteins that are induced during infection and regulate pathogenesis. Gene clusters play important roles in pathogenic fungi, yet their evolution and maintenance remain a mystery. © 2007 Elsevier Ltd. All rights reserved.

Authors
Howlett, BJ; Idnurm, A; Heitman, J
MLA Citation
Howlett, BJ, Idnurm, A, and Heitman, J. "Fungal Pathogenesis: Gene Clusters Unveiled as Secrets within the Ustilago maydis Code." Current Biology 17.3 (2007): R87-R90.
PMID
17276906
Source
scival
Published In
Current Biology
Volume
17
Issue
3
Publish Date
2007
Start Page
R87
End Page
R90
DOI
10.1016/j.cub.2006.11.047

Sex and Virulence of Human Pathogenic Fungi

Over the past decade, opportunistic fungal infectious diseases have increased in prevalence as the population of immunocompromised individuals escalated due to HIV/AIDS and immunosuppression associated with organ transplantation and cancer therapies. In the three predominant human pathogenic fungi (Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus), a unifying feature is that all three retained the machinery needed for sex, and yet all limit their access to sexual reproduction. While less well characterized, many of the other human pathogenic fungi also appear to have the ability to undergo sexual reproduction. Recent studies with engineered pairs of diploid strains of the model yeast Saccharomyces cerevisiae, one that is sexual and the other an obligate asexual, provide direct experimental validation of the benefits of both sexual and asexual reproduction. The obligate asexual strain had an advantage in response to constant environmental conditions whereas the sexual strain had a competitive edge under stressful conditions (Goddard et al., 2005; Grimberg and Zeyl, 2005). The human pathogenic fungi have gone to great lengths to maintain all of the machinery required for sex, including the mating-type locus and the pheromone response and cell fusion pathways. Yet these pathogens limit their access to sexual or parasexual reproduction in unique and specialized ways. Our hypothesis is that this has enabled the pathogenic fungi to proliferate in their environmental niche, but to also undergo genetic exchange via sexual reproduction in response to stressful conditions such as new environments, different host organisms, or changes in the human host such as antimicrobial therapy. Further study of the sexual nature of the human pathogenic fungi will illuminate how these unique microbes have evolved into successful pathogens in humans. © 2006 Elsevier Inc. All rights reserved.

Authors
Nielsen, K; Heitman, J
MLA Citation
Nielsen, K, and Heitman, J. "Sex and Virulence of Human Pathogenic Fungi." Advances in Genetics 57 (2007): 143-173.
PMID
17352904
Source
scival
Published In
Advances in genetics
Volume
57
Publish Date
2007
Start Page
143
End Page
173
DOI
10.1016/S0065-2660(06)57004-X

Unique applications of novel antifungal drug combinations

Candida albicans is a commensal fungal organism that can over-proliferate and cause disease in the appropriate host setting. C. albicans can cause irritating superficial skin and mucocutaneous infections such as diaper rash and vaginal yeast infections, respectively. In immunocompromised hosts, these infections can progress to disseminated disease in which the organism enters the blood and colonizes multiple organs. Consequently, Candida infections result in a considerable amount of morbidity and mortality every year. Most modern-day antifungal drugs block ergosterol biosynthesis. Several of these agents are fungistatic and do not kill the fungal cell, thus facilitating the emergence of drug-resistant species, which further complicate therapy. Alternatively, some of the most effective antifungal drugs are too toxic for continuous use or can only be administered intravenously. The ideal antifungal drug would be non-toxic, fungicidal, and amenable to self-administration. Previous studies have demonstrated that specific commercially available drugs from two unrelated drug classes (calcineurin inhibitors and ergosterol biosynthesis inhibitors) act synergistically to kill Candida by targeting distinct molecular pathways in the organism. Calcineurin inhibitors are immunosuppressive agents, so systemic administration of these drugs would be counter-intuitive for treatment of already immunocompromised individuals. However, this drug combination can be applied to topical antifungal therapies for a variety of cutaneous and mucocutaneous fungal infections that afflict a diverse population, including immunocompromised patients. © 2007 Bentham Science Publishers Ltd.

Authors
Onyewu, C; Heitman, J
MLA Citation
Onyewu, C, and Heitman, J. "Unique applications of novel antifungal drug combinations." Anti-Infective Agents in Medicinal Chemistry 6.1 (2007): 3-15.
Source
scival
Published In
Anti-Infective Agents in Medicinal Chemistry
Volume
6
Issue
1
Publish Date
2007
Start Page
3
End Page
15

Virulence attributes and hyphal growth of C. neoformans are quantitative traits and the MATalpha allele enhances filamentation.

Cryptococcus neoformans is a fungal human pathogen with a bipolar mating system. It undergoes a dimorphic transition from a unicellular yeast to hyphal filamentous growth during mating and monokaryotic fruiting. The traditional sexual cycle that leads to the production of infectious basidiospores involves cells of both alpha and a mating type. Monokaryotic fruiting is a modified form of sexual reproduction that involves cells of the same mating type, most commonly alpha, which is the predominant mating type in both the environment and clinical isolates. However, some a isolates can also undergo monokaryotic fruiting. To determine whether mating type and other genetic loci contribute to the differences in fruiting observed between alpha and a cells, we applied quantitative trait loci (QTL) mapping to an inbred population of F2 progeny. We discovered that variation in hyphal length produced during fruiting is a quantitative trait resulting from the combined effects of multiple genetic loci, including the mating type (MAT) locus. Importantly, the alpha allele of the MAT locus enhanced hyphal growth compared with the a allele. Other virulence traits, including melanization and growth at 39 degrees C, also are quantitative traits that share a common QTL with hyphal growth. The Mac1 transcription factor, encoded in this common QTL, regulates copper homeostasis. MAC1 allelic differences contribute to phenotypic variation, and mac1Delta mutants exhibit defects in filamentation, melanin production, and high temperature growth. Further characterization of these QTL regions will reveal additional quantitative trait genes controlling biological processes central to fungal development and pathogenicity.

Authors
Lin, X; Huang, JC; Mitchell, TG; Heitman, J
MLA Citation
Lin, X, Huang, JC, Mitchell, TG, and Heitman, J. "Virulence attributes and hyphal growth of C. neoformans are quantitative traits and the MATalpha allele enhances filamentation." PLoS Genet 2.11 (November 17, 2006): e187-.
PMID
17112316
Source
pubmed
Published In
PLoS genetics
Volume
2
Issue
11
Publish Date
2006
Start Page
e187
DOI
10.1371/journal.pgen.0020187

A unique fungal two-component system regulates stress responses, drug sensitivity, sexual development, and virulence of Cryptococcus neoformans.

The stress-activated mitogen-activated protein kinase (MAPK) pathway is widely used by eukaryotic organisms as a central conduit via which cellular responses to the environment effect growth and differentiation. The basidiomycetous human fungal pathogen Cryptococcus neoformans uniquely uses the stress-activated Pbs2-Hog1 MAPK system to govern a plethora of cellular events, including stress responses, drug sensitivity, sexual reproduction, and virulence. Here, we characterized a fungal "two-component" system that controls these fundamental cellular functions via the Pbs2-Hog1 MAPK cascade. A typical response regulator, Ssk1, modulated all Hog1-dependent phenotypes by controlling Hog1 phosphorylation, indicating that Ssk1 is the major upstream signaling component of the Pbs2-Hog1 pathway. A second response regulator, Skn7, governs sensitivity to Na+ ions and the antifungal agent fludioxonil, negatively controls melanin production, and functions independently of Hog1 regulation. To control these response regulators, C. neoformans uses multiple sensor kinases, including two-component-like (Tco) 1 and Tco2. Tco1 and Tco2 play shared and distinct roles in stress responses and drug sensitivity through the Hog1 MAPK system. Furthermore, each sensor kinase mediates unique cellular functions for virulence and morphological differentiation. Our findings highlight unique adaptations of this global two-component MAPK signaling cascade in a ubiquitous human fungal pathogen.

Authors
Bahn, Y-S; Kojima, K; Cox, GM; Heitman, J
MLA Citation
Bahn, Y-S, Kojima, K, Cox, GM, and Heitman, J. "A unique fungal two-component system regulates stress responses, drug sensitivity, sexual development, and virulence of Cryptococcus neoformans." Mol Biol Cell 17.7 (July 2006): 3122-3135.
PMID
16672377
Source
pubmed
Published In
Molecular Biology of the Cell
Volume
17
Issue
7
Publish Date
2006
Start Page
3122
End Page
3135
DOI
10.1091/mbc.E06-02-0113

Calcineurin controls growth, morphology, and pathogenicity in Aspergillus fumigatus.

Calcineurin is implicated in a myriad of human diseases as well as homeostasis and virulence in several major human pathogenic microorganisms. The fungus Aspergillus fumigatus is a leading cause of infectious death in the rapidly expanding immunocompromised patient population. Current antifungal treatments for invasive aspergillosis are often ineffective, and novel therapeutic approaches are urgently needed. We demonstrate that a mutant of A. fumigatus lacking the calcineurin A (cnaA) catalytic subunit exhibited defective hyphal morphology related to apical extension and polarized growth, which resulted in drastically decreased filamentation. The delta cnaA mutant lacked the extensive lattice of invading hyphae seen with the wild-type and complemented strains. Sporulation was also affected in the delta cnaA mutant, including morphological conidial defects with the absence of surface rodlets and the added presence of disjunctors creating long conidial chains. Infection with the delta cnaA mutant in several distinct animal models with different types of immunosuppression and inoculum delivery led to a profound attenuation of pathogenicity compared to infection with the wild-type and complemented strains. Lung tissue from animals infected with the delta cnaA mutant showed a complete absence of hyphae, in contrast to tissue from animals infected with the wild-type and complemented strains. Quantitative fungal burden and pulmonary infarct scoring confirmed these findings. Our results support the clinical observation that substantially decreasing fungal growth can prevent disease establishment and decrease mortality. Our findings reveal that calcineurin appears to play a globally conserved role in the virulence of several pathogenic fungi and yet plays specialized roles in each and can be an excellent target for therapeutic intervention.

Authors
Steinbach, WJ; Cramer, RA; Perfect, BZ; Asfaw, YG; Sauer, TC; Najvar, LK; Kirkpatrick, WR; Patterson, TF; Benjamin, DK; Heitman, J; Perfect, JR
MLA Citation
Steinbach, WJ, Cramer, RA, Perfect, BZ, Asfaw, YG, Sauer, TC, Najvar, LK, Kirkpatrick, WR, Patterson, TF, Benjamin, DK, Heitman, J, and Perfect, JR. "Calcineurin controls growth, morphology, and pathogenicity in Aspergillus fumigatus." Eukaryot Cell 5.7 (July 2006): 1091-1103.
PMID
16835453
Source
pubmed
Published In
Eukaryotic cell
Volume
5
Issue
7
Publish Date
2006
Start Page
1091
End Page
1103
DOI
10.1128/EC.00139-06

Yeast diversity sampling on the San Juan Islands reveals no evidence for the spread of the Vancouver Island Cryptococcus gattii outbreak to this locale.

Biological diversity has been estimated for various phyla of life, such as insects and mammals, but in the microbe world is has been difficult to determine species richness and abundance. Here we describe a study of species diversity of fungi with a yeast-like colony morphology from the San Juan Islands, a group of islands that lies southeast of Vancouver Island, Canada. Our sampling revealed that the San Juan archipelago biosphere contains a diverse range of such fungi predominantly belonging to the Basidiomycota, particularly of the order Tremellales. One member of this group, Cryptococcus gattii, is the etiological agent of a current and ongoing outbreak of cryptococcosis on nearby Vancouver Island. Our sampling did not, however, reveal this species. While the lack of recovery of C. gattii does not preclude its presence on the San Juan Islands, our results suggest that the Strait of Juan de Fuca may be serving as a geographical barrier to restrict the dispersal of this primary human fungal pathogen into the United States.

Authors
Fraser, JA; Lim, SMC; Diezmann, S; Wenink, EC; Arndt, CG; Cox, GM; Dietrich, FS; Heitman, J
MLA Citation
Fraser, JA, Lim, SMC, Diezmann, S, Wenink, EC, Arndt, CG, Cox, GM, Dietrich, FS, and Heitman, J. "Yeast diversity sampling on the San Juan Islands reveals no evidence for the spread of the Vancouver Island Cryptococcus gattii outbreak to this locale." FEMS Yeast Res 6.4 (June 2006): 620-624.
PMID
16696658
Source
pubmed
Published In
Fems Yeast Research
Volume
6
Issue
4
Publish Date
2006
Start Page
620
End Page
624
DOI
10.1111/j.1567-1364.2006.00075.x

G protein-coupled receptor Gpr4 senses amino acids and activates the cAMP-PKA pathway in Cryptococcus neoformans.

The Galpha protein Gpa1 governs the cAMP-PKA signaling pathway and plays a central role in virulence and differentiation in the human fungal pathogen Cryptococcus neoformans, but the signals and receptors that trigger this pathway were unknown. We identified seven putative proteins that share identity with known G protein-coupled receptors (GPCRs). One protein, Gpr4, shares limited sequence identity with the Dictyostelium discoideum cAMP receptor cAR1 and the Aspergillus nidulans GPCR protein GprH and also shares structural similarity with the Saccharomyces cerevisiae receptor Gpr1. gpr4 mutants exhibited reduced capsule production and mating defects, similar to gpa1 mutants, and exogenous cAMP suppressed both gpr4 mutant phenotypes. Epistasis analysis provides further evidence that Gpr4 functions upstream of the Galpha subunit Gpa1. Gpr4-Gpr4 homomeric interactions were observed in the yeast two-hybrid assay, and Gpr4 was shown to physically interact with Gpa1 in the split-ubiquitin system. A Gpr4::DsRED fusion protein was localized to the plasma membrane and methionine was found to trigger receptor internalization. The analysis of intracellular cAMP levels showed that gpr4 mutants still respond to glucose but not to certain amino acids, such as methionine. Amino acids might serve as ligands for Gpr4 and could contribute to engage the cAMP-PKA pathway. Activation of the cAMP-PKA pathway by glucose and amino acids represents a nutrient coincidence detection system shared in other pathogenic fungi.

Authors
Xue, C; Bahn, Y-S; Cox, GM; Heitman, J
MLA Citation
Xue, C, Bahn, Y-S, Cox, GM, and Heitman, J. "G protein-coupled receptor Gpr4 senses amino acids and activates the cAMP-PKA pathway in Cryptococcus neoformans." Mol Biol Cell 17.2 (February 2006): 667-679.
PMID
16291861
Source
pubmed
Published In
Molecular Biology of the Cell
Volume
17
Issue
2
Publish Date
2006
Start Page
667
End Page
679
DOI
10.1091/mbc.E05-07-0699

Recombination hotspots flank the Cryptococcus mating-type locus: Implications for the evolution of a fungal sex chromosome

Recombination increases dramatically during meiosis to promote genetic exchange and generate recombinant progeny. Interestingly, meiotic recombination is unevenly distributed throughout genomes, and, as a consequence, genetic and physical map distances do not have a simple linear relationship. Recombination hotspots and coldspots have been described in many organisms and often reflect global features of chromosome structure. In particular, recombination frequencies are often distorted within or outside sex-determining regions of the genome. Here, we report that recombination is elevated adjacent to the mating-type locus (MAT) in the pathogenic basidiomycete Cryptococcus neoformans. Among fungi, C. neoformans has an unusually large MAT locus, and recombination is suppressed between the two >100-kilobase mating-type specific alleles. When genetic markers were introduced at defined physical distances from MAT, we found the meiotic recombination frequency to be ∼20% between MAT and a flanking marker at 5, 10, 50, or 100 kilobases from the right border. As a result, the physical/genetic map ratio in the regions adjacent to MAT is distorted ∼10- to 50-fold compared to the genome-wide average. Moreover, recombination frequently occurred on both sides of MAT and negative interference between crossovers was observed. MAT heterozygosity was not required for enhanced recombination, implying that this process is not due to a physical distortion from the two non-paired alleles and could also occur during same-sex mating. Sequence analysis revealed a correlation between high G + C content and these hotspot regions. We hypothesize that the presence of recombinational activators may have driven several key events during the assembly and reshaping of the MAT locus and may have played similar roles in the origins of both metabolic and biosynthetic gene clusters. Our findings suggest that during meiosis the MAT locus may be exchanged onto different genetic backgrounds and therefore have broad evolutionary implications with respect to mating-type switching in both model and pathogenic yeasts. © 2006 Hsueh et al.

Authors
Hsueh, Y-P; Idnurm, A; Heitman, J
MLA Citation
Hsueh, Y-P, Idnurm, A, and Heitman, J. "Recombination hotspots flank the Cryptococcus mating-type locus: Implications for the evolution of a fungal sex chromosome." PLoS Genetics 2.11 (2006): 1702-1714.
PMID
17083277
Source
scival
Published In
PLoS genetics
Volume
2
Issue
11
Publish Date
2006
Start Page
1702
End Page
1714
DOI
10.1371/journal.pgen.0020184

Recombination hotspots flank the Cryptococcus mating-type locus: implications for the evolution of a fungal sex chromosome.

Recombination increases dramatically during meiosis to promote genetic exchange and generate recombinant progeny. Interestingly, meiotic recombination is unevenly distributed throughout genomes, and, as a consequence, genetic and physical map distances do not have a simple linear relationship. Recombination hotspots and coldspots have been described in many organisms and often reflect global features of chromosome structure. In particular, recombination frequencies are often distorted within or outside sex-determining regions of the genome. Here, we report that recombination is elevated adjacent to the mating-type locus (MAT) in the pathogenic basidiomycete Cryptococcus neoformans. Among fungi, C. neoformans has an unusually large MAT locus, and recombination is suppressed between the two >100-kilobase mating-type specific alleles. When genetic markers were introduced at defined physical distances from MAT, we found the meiotic recombination frequency to be approximately 20% between MAT and a flanking marker at 5, 10, 50, or 100 kilobases from the right border. As a result, the physical/genetic map ratio in the regions adjacent to MAT is distorted approximately 10- to 50-fold compared to the genome-wide average. Moreover, recombination frequently occurred on both sides of MAT and negative interference between crossovers was observed. MAT heterozygosity was not required for enhanced recombination, implying that this process is not due to a physical distortion from the two non-paired alleles and could also occur during same-sex mating. Sequence analysis revealed a correlation between high G + C content and these hotspot regions. We hypothesize that the presence of recombinational activators may have driven several key events during the assembly and reshaping of the MAT locus and may have played similar roles in the origins of both metabolic and biosynthetic gene clusters. Our findings suggest that during meiosis the MAT locus may be exchanged onto different genetic backgrounds and therefore have broad evolutionary implications with respect to mating-type switching in both model and pathogenic yeasts.

Authors
Hsueh, YP; Idnurm, A; Heitman, J
MLA Citation
Hsueh, YP, Idnurm, A, and Heitman, J. "Recombination hotspots flank the Cryptococcus mating-type locus: implications for the evolution of a fungal sex chromosome." PLoS genetics 2.11 (2006): e184-.
Source
scival
Published In
PLoS genetics
Volume
2
Issue
11
Publish Date
2006
Start Page
e184
DOI
10.1371/journal.pgen.0020184

Virulence attributes and hyphal growth of C. neoformans are quantitative traits and the MATα allele enhances filamentation

Cryptococcus neoformans is a fungal human pathogen with a bipolar mating system. It undergoes a dimorphic transition from a unicellular yeast to hyphal filamentous growth during mating and monokaryotic fruiting. The traditional sexual cycle that leads to the production of infectious basidiospores involves cells of both α and a mating type. Monokaryotic fruiting is a modified form of sexual reproduction that involves cells of the same mating type, most commonly α, which is the predominant mating type in both the environment and clinical isolates. However, some a isolates can also undergo monokaryotic fruiting. To determine whether mating type and other genetic loci contribute to the differences in fruiting observed between α and a cells, we applied quantitative trait loci (QTL) mapping to an inbred population of F2 progeny. We discovered that variation in hyphal length produced during fruiting is a quantitative trait resulting from the combined effects of multiple genetic loci, including the mating type (MAT) locus. Importantly, the α allele of the MAT locus enhanced hyphal growth compared with the a allele. Other virulence traits, including melanization and growth at 39°C, also are quantitative traits that share a common QTL with hyphal growth. The Mac1 transcription factor, encoded in this common QTL, regulates copper homeostasis. MAC1 allelic differences contribute to phenotypic variation, and mac1Δ mutants exhibit defects in filamentation, melanin production, and high temperature growth. Further characterization of these QTL regions will reveal additional quantitative trait genes controlling biological processes central to fungal development and pathogenicity. © 2006 Lin et al.

Authors
Lin, X; Huang, JC; Mitchell, TG; Heitman, J
MLA Citation
Lin, X, Huang, JC, Mitchell, TG, and Heitman, J. "Virulence attributes and hyphal growth of C. neoformans are quantitative traits and the MATα allele enhances filamentation." PLoS Genetics 2.11 (2006): 1801-1814.
Source
scival
Published In
PLoS genetics
Volume
2
Issue
11
Publish Date
2006
Start Page
1801
End Page
1814
DOI
10.1371/journal.pgen.0020187

Sexual Reproduction and the Evolution of Microbial Pathogens

Three common systemic human fungal pathogens - Cryptococcus neoformans, Candida albicans and Aspergillus fumigatus - have retained all the machinery to engage in sexual reproduction, and yet their populations are often clonal with limited evidence for recombination. Striking parallels have emerged with four protozoan parasites that infect humans: Toxoplasma gondii, Trypanosoma brucei, Trypanosoma cruzi and Plasmodium falciparum. Limiting sexual reproduction appears to be a common virulence strategy, enabling generation of clonal populations well adapted to host and environmental niches, yet retaining the ability to engage in sexual or parasexual reproduction and respond to selective pressure. Continued investigation of the sexual nature of microbial pathogens should facilitate both laboratory investigation and an understanding of the complex interplay between pathogens, hosts, vectors, and their environments. © 2006 Elsevier Ltd. All rights reserved.

Authors
Heitman, J
MLA Citation
Heitman, J. "Sexual Reproduction and the Evolution of Microbial Pathogens." Current Biology 16.17 (2006): R711-R725.
PMID
16950098
Source
scival
Published In
Current Biology
Volume
16
Issue
17
Publish Date
2006
Start Page
R711
End Page
R725
DOI
10.1016/j.cub.2006.07.064

Conserved elements of the RAM signaling pathway establish cell polarity in the basidiomycete Cryptococcus neoformans in a divergent fashion from other fungi

In eukaryotes the complex processes of development, differentiation, and proliferation require carefully orchestrated changes in cellular morphology. Single-celled eukaryotes provide tractable models for the elucidation of signaling pathways involved in morphogenesis. Here we describe a pathway regulating cell polarization and separation in the human pathogenic fungus Cryptococcus neoformans. An insertional mutagenesis screen identified roles for the ARF1, CAP60, NDH1, KIC1, CBK1, SOG2, and TAO3 genes in establishing normal colony morphology. ARF1 and CAP60 are also required for capsule production, a virulence factor, and ARF1 confers resistance to the antifungal fluconazole. KIC1, CBK1, SOG2, and TAO3 are homologues of genes conserved in other eukaryotes; in Saccharomyces cerevisiae they constitute components of the RAM (regulation of Ace2p activity and cellular morphogenesis) signaling pathway. A targeted deletion of a fifth component of RAM (MOB2) conferred identical phenotypes to kic1, cbk1, sog2, or tao3 mutations. Characterization of these genes in C. neoformans revealed unique features of the RAM pathway in this organism. Loss of any of these genes caused constitutive hyperpolarization instead of the loss of polarity seen in S. cerevisiae. Furthermore, sensitivity to the drugs FK506 and cyclosporin A demonstrates that the RAM pathway acts in parallel with the protein phosphatase calcineurin in C. neoformans but not in S. cerevisiae. These results indicate that conserved signaling pathways serve both similar and divergent cellular roles in morphogenesis in these divergent organisms. © 2006 by The American Society for Cell Biology.

Authors
Walton, FJ; Heitman, J; Idnurm, A
MLA Citation
Walton, FJ, Heitman, J, and Idnurm, A. "Conserved elements of the RAM signaling pathway establish cell polarity in the basidiomycete Cryptococcus neoformans in a divergent fashion from other fungi." Molecular Biology of the Cell 17.9 (2006): 3768-3780.
PMID
16775005
Source
scival
Published In
Molecular Biology of the Cell
Volume
17
Issue
9
Publish Date
2006
Start Page
3768
End Page
3780
DOI
10.1091/mbc.E06-02-0125

The Kelch Proteins Gpb1 and Gpb2 Inhibit Ras Activity via Association with the Yeast RasGAP Neurofibromin Homologs Ira1 and Ira2

The G protein-coupled receptor Gpr1 and associated Gα subunit Gpa2 govern dimorphic transitions in response to extracellular nutrients by signaling coordinately with Ras to activate adenylyl cyclase in the yeast Saccharomyces cerevisiae. Gpa2 forms a protein complex with the kelch Gβ mimic subunits Gpb1/2, and previous studies demonstrate that Gpb1/2 negatively control cAMP-PKA signaling via Gpa2 and an unknown second target. Here, we define these targets of Gpb1/2 as the yeast neurofibromin homologs Ira1 and Ira2, which function as GTPase activating proteins of Ras. Gpb1/2 bind to a conserved C-terminal domain of Ira1/2, and loss of Gpb1/2 results in a destabilization of Ira1 and Ira2, leading to elevated levels of Ras2-GTP and unbridled cAMP-PKA signaling. Because the Gpb1/2 binding domain on Ira1/2 is conserved in the human neurofibromin protein, an analogous signaling network may contribute to the neoplastic development of neurofibromatosis type 1. © 2006 Elsevier Inc. All rights reserved.

Authors
Harashima, T; Anderson, S; III, JRY; Heitman, J
MLA Citation
Harashima, T, Anderson, S, III, JRY, and Heitman, J. "The Kelch Proteins Gpb1 and Gpb2 Inhibit Ras Activity via Association with the Yeast RasGAP Neurofibromin Homologs Ira1 and Ira2." Molecular Cell 22.6 (2006): 819-830.
PMID
16793550
Source
scival
Published In
Molecular Cell
Volume
22
Issue
6
Publish Date
2006
Start Page
819
End Page
830
DOI
10.1016/j.molcel.2006.05.011

Eukaryotes

Authors
Heitman, J
MLA Citation
Heitman, J. "Eukaryotes." Current Opinion in Microbiology 9.6 (2006): 537-539.
Source
scival
Published In
Current Opinion in Microbiology
Volume
9
Issue
6
Publish Date
2006
Start Page
537
End Page
539
DOI
10.1016/j.mib.2006.10.013

Calcineurin promotes infection of the cornea by Candida albicans and can be targeted to enhance fluconazole therapy

In an established Candida albicans murine keratitis model, combination therapy with ophthalmic preparations of fluconazole and cyclosporine A (CsA) demonstrated in vivo drug synergy and effectively resolved wild-type C. albicans infection more rapidly than monotherapy with either drug. Calcineurin, the target of CsA, was also found to contribute to pathogenicity. Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Authors
Onyewu, C; Afshari, NA; Heitman, J
MLA Citation
Onyewu, C, Afshari, NA, and Heitman, J. "Calcineurin promotes infection of the cornea by Candida albicans and can be targeted to enhance fluconazole therapy." Antimicrobial Agents and Chemotherapy 50.11 (2006): 3963-3965.
PMID
16923949
Source
scival
Published In
Antimicrobial agents and chemotherapy
Volume
50
Issue
11
Publish Date
2006
Start Page
3963
End Page
3965
DOI
10.1128/AAC.00393-06

Immunotherapy with tacrolimus (FK506) does not select for resistance to calcineurin inhibitors in Candida albicans isolates from liver transplant patients

In Candida albicans, calcineurin mediates tolerance to azole antifungal drugs, survival in serum, and virulence. In this study, we examined 24 Candida isolates from liver transplant recipients receiving a calcineurin inhibitor as a component of their immunosuppressive therapy. We were unable to detect a difference in susceptibility to calcineurin inhibitors in combination with fluconazole, serum, or calcium in these isolates. Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Authors
Reedy, JL; Husain, S; Ison, M; Pruett, TL; Singh, N; Heitman, J
MLA Citation
Reedy, JL, Husain, S, Ison, M, Pruett, TL, Singh, N, and Heitman, J. "Immunotherapy with tacrolimus (FK506) does not select for resistance to calcineurin inhibitors in Candida albicans isolates from liver transplant patients." Antimicrobial Agents and Chemotherapy 50.4 (2006): 1573-1577.
PMID
16569889
Source
scival
Published In
Antimicrobial agents and chemotherapy
Volume
50
Issue
4
Publish Date
2006
Start Page
1573
End Page
1577
DOI
10.1128/AAC.50.4.1573-1577.2006

The Phycomyces madA gene encodes a blue-light photoreceptor for phototropism and other light responses

Phycomyces blakesleeanus is a filamentous zygomycete fungus that produces striking elongated single cells that extend up to 10 cm into the air, with each such sporangiophore supporting a sphere containing the spores for dispersal. This organism has served as a model for the detection of environmental signals as diverse as light, chemicals, touch, wind, gravity, and adjacent objects. In particular, sporangiophore growth is regulated by light, and it exhibits phototropism by bending toward near-UV and blue wave-lengths and away from far-UV wavelengths in a manner that is physiologically similar to plant phototropic responses. The Phycomyces madA mutants were first isolated more than 40 years ago, and they exhibit reduced sensitivity to light. Here, we identify two (duplicated) homologs in the White Collar 1 family of blue-light photoreceptors in Phycomyces. We describe that the madA mutant strains contain point mutations in one of these genes and that these mutations cosegregate with a defect in phototropism after genetic crosses. Thus, the phototropic responses of fungi through madA and plants through phototropin rely on diverse proteins; however, these proteins share a conserved flavin-binding domain for photon detection. © 2006 by The National Academy of Sciences of the USA.

Authors
Idnurm, A; Rodríguez-Romero, J; Corrochano, LM; Sanz, C; Iturriaga, EA; Eslava, AP; Heitman, J
MLA Citation
Idnurm, A, Rodríguez-Romero, J, Corrochano, LM, Sanz, C, Iturriaga, EA, Eslava, AP, and Heitman, J. "The Phycomyces madA gene encodes a blue-light photoreceptor for phototropism and other light responses." Proceedings of the National Academy of Sciences of the United States of America 103.12 (2006): 4546-4551.
PMID
16537433
Source
scival
Published In
Proceedings of the National Academy of Sciences of USA
Volume
103
Issue
12
Publish Date
2006
Start Page
4546
End Page
4551
DOI
10.1073/pnas.0600633103

Calcineurin, Mpk1 and Hog1 MAPK pathways independently control fludioxonil antifungal sensitivity in Cryptococcus neoformans

Fludioxonil is employed as an agricultural fungicide to control plant-pathogenic fungi such as Botrytis cinerea. Cryptococcus neoformans is a basidiomycetous human fungal pathogen that causes fatal disease in immunocompromised hosts. This paper demonstrates that three different signalling cascades regulate sensitivity of C. neoformans to fludioxonil. Fludioxonil inhibited growth of the serotype A sequence reference strain H99 but not that of the sequenced serotype D strain JEC21. In the drug-sensitive wild-type strain, fludioxonil exposure activated the Hog1 osmosensing pathway, and hog1Δ mutations conferred fludioxonil resistance. Fludioxonil treatment caused cell growth inhibition following cell swelling and cytokinesis defects in the sensitive wild-type but not in a hog1Δ mutant strain, suggesting that Hog1 activation results in morphological cellular defects. Fludioxonil exerted a fungistatic effect on the wild-type strain H99, but exhibited fungicidal activity against calcineurin mutant strains, indicating that the calcineurin pathway contributes to drug resistance in this fungus. Combination of fludioxonil and the calcineurin inhibitor FK506 synergistically inhibited C. neoformans growth. mpk1Δ MAPK mutant strains exhibited fludioxonil hypersensitivity, indicating that this pathway also contributes to drug resistance. These studies provide evidence that the broad-spectrum antifungal drug fludioxonil exerts its action via activation of the Hog1 MAPK pathway and provide insight into novel targets for synergistic antifungal drug combinations. © 2006 SGM.

Authors
Kojima, K; Bahn, Y-S; Heitman, J
MLA Citation
Kojima, K, Bahn, Y-S, and Heitman, J. "Calcineurin, Mpk1 and Hog1 MAPK pathways independently control fludioxonil antifungal sensitivity in Cryptococcus neoformans." Microbiology 152.3 (2006): 591-604.
PMID
16514140
Source
scival
Published In
Microbiology (Reading, England)
Volume
152
Issue
3
Publish Date
2006
Start Page
591
End Page
604
DOI
10.1099/mic.0.28571-0

The biology of the Cryptococcus neoformans species complex

Cryptococcus neoformans is a major cause of fungal meningoencephalitis in immunocompromised patients. Despite recent advances in the genetics and molecular biology of C. neoformans, and improved techniques for molecular epidemiology, aspects of the ecology, population structure, and mode of reproduction of this environmental pathogen remain to be established. Application of recent insights into the life cycle of C. neoformans and its different ways of engaging in sexual reproduction under laboratory conditions has just begun to affect research on the ecology and epidemiology of this human pathogenic fungus. The melding of these disparate disciplines should yield rich dividends in our understanding of the evolution of microbial pathogens, providing insights relevant to diagnosis, treatment, and prevention. Copyright © 2006 by Annual Reviews. All rights reserved.

Authors
Lin, X; Heitman, J
MLA Citation
Lin, X, and Heitman, J. "The biology of the Cryptococcus neoformans species complex." Annual Review of Microbiology 60 (2006): 69-105.
PMID
16704346
Source
scival
Published In
Annual Review of Microbiology
Volume
60
Publish Date
2006
Start Page
69
End Page
105
DOI
10.1146/annurev.micro.60.080805.142102

Carbonic anhydrase and CO2 sensing during Cryptococcus neoformans growth, differentiation, and virulence.

The gas carbon dioxide (CO2) plays a critical role in microbial and mammalian respiration, photosynthesis in algae and plants, chemoreception in insects, and even global warming . However, how CO2 is transported, sensed, and metabolized by microorganisms is largely not understood. For instance, CO2 is known to induce production of polysaccharide capsule virulence determinants in pathogenic bacteria and fungi via unknown mechanisms . Therefore, we studied CO2 actions in growth, differentiation, and virulence of the basidiomycetous human fungal pathogen Cryptococcus neoformans. The CAN2 gene encoding beta-carbonic anhydrase in C. neoformans was found to be essential for growth in environmental ambient conditions but dispensable for in vivo proliferation and virulence at the high CO2 levels in the host. The can2Delta mutant in vitro growth defect is largely attributable to defective fatty acid synthesis. CO2 was found to inhibit cell-cell fusion but not filamentation during sexual reproduction. The can2 mutation restored early mating events in high CO2 but not later steps (fruiting body formation, sporulation), indicating a major role for carbonic anhydrase and CO2/HCO3- in this developmental cascade leading to the production of infectious spores. Our studies illustrate diverse roles of an ancient enzyme class in enabling environmental survival of a ubiquitous human pathogen.

Authors
Bahn, Y-S; Cox, GM; Perfect, JR; Heitman, J
MLA Citation
Bahn, Y-S, Cox, GM, Perfect, JR, and Heitman, J. "Carbonic anhydrase and CO2 sensing during Cryptococcus neoformans growth, differentiation, and virulence." Curr Biol 15.22 (November 22, 2005): 2013-2020.
PMID
16303560
Source
pubmed
Published In
Current Biology
Volume
15
Issue
22
Publish Date
2005
Start Page
2013
End Page
2020
DOI
10.1016/j.cub.2005.09.047

Interaction between genetic background and the mating-type locus in Cryptococcus neoformans virulence potential.

The study of quantitative traits provides a window on the interactions between multiple unlinked genetic loci. The interaction between hosts and pathogenic microbes, such as fungi, involves aspects of quantitative genetics for both partners in this dynamic equilibrium. One important pathogenic fungus is Cryptococcus neoformans, a basidiomycete yeast that can infect the human brain and whose mating system has two mating type alleles, a and alpha. The alpha mating-type allele has previously been linked to increased virulence potential. Here congenic C. neoformans strains were generated in the two well-characterized genetic backgrounds B3501alpha and NIH433a to examine the potential influence of genes outside of the mating-type locus on the virulence potential of mating type. The congenic nature of these new strain pairs was established by karyotyping, amplified fragment length polymorphism genotyping, and whole-genome molecular allele mapping (congenicity mapping). Virulence studies revealed that virulence was equivalent between the B3501 a and alpha congenic strains but the alpha strain was more virulent than its a counterpart in the NIH433 genetic background. These results demonstrate that genomic regions outside the mating type locus contribute to differences in virulence between a and alpha cells. The congenic strains described here provide a foundation upon which to elucidate at genetic and molecular levels how mating-type and other unlinked loci interact to enable microbial pathogenesis.

Authors
Nielsen, K; Marra, RE; Hagen, F; Boekhout, T; Mitchell, TG; Cox, GM; Heitman, J
MLA Citation
Nielsen, K, Marra, RE, Hagen, F, Boekhout, T, Mitchell, TG, Cox, GM, and Heitman, J. "Interaction between genetic background and the mating-type locus in Cryptococcus neoformans virulence potential." Genetics 171.3 (November 2005): 975-983.
PMID
15965241
Source
pubmed
Published In
Genetics
Volume
171
Issue
3
Publish Date
2005
Start Page
975
End Page
983
DOI
10.1534/genetics.105.045039

Antifungal management practices and evolution of infection in organ transplant recipients with cryptococcus neoformans infection.

BACKGROUND: Therapeutic practices for Cryptococcus neoformans infection in transplant recipients vary, particularly with regards to antifungal agent employed, and duration of therapy. The risk of relapse and time to recurrence is not known. We assessed antifungal treatment practices for cryptococcosis in a cohort of prospectively followed organ transplant recipients. METHODS: The patients comprised 83 transplant recipients with cryptococcosis followed for a median of 2.1 and up to 5.2 years. RESULTS: Patients with central nervous system infection (69% vs. 16%, P = 0.00001), disseminated infection (82.7% vs. 20%, P = 0.00001), and fungemia (29% vs. 8%, P = 0.046) were more likely to receive regimens containing amphotericin B than fluconazole as primary therapy. The use of fluconazole, on the other hand, was more likely for infection limited to the lungs (64% vs. 14%, P = 0.00002). Survival at 6 months tended to be lower in patients whose CSF cultures at 2 weeks were positive compared to those whose CSF cultures were negative (50% vs. 91%, P = 0.06). Maintenance therapy was employed in 68% (54/79) of the patients who survived >3 weeks. The median duration of maintenance therapy was 183 days; 55% received maintenance for > or = 6 months and 25% for >1 year. Relapse was documented in 1.3% (1/79) of the patients. CONCLUSIONS: A majority of the organ transplant recipients with cryptococcosis receive maintenance antifungal therapy for 6 months with low risk of relapse. These data can assist in trials to assess the optimal therapeutic approach and duration of therapy for cryptococcosis in transplant recipients.

Authors
Singh, N; Lortholary, O; Alexander, BD; Gupta, KL; John, GT; Pursell, KJ; Muñoz, P; Klintmalm, GB; Stosor, V; Del Busto, R; Limaye, AP; Somani, J; Lyon, M; Houston, S; House, AA; Pruett, TL; Orloff, S; Humar, A; Dowdy, LA; Garcia-Diaz, J; Kalil, AC; Fisher, RA; Heitman, J; Husain, S
MLA Citation
Singh, N, Lortholary, O, Alexander, BD, Gupta, KL, John, GT, Pursell, KJ, Muñoz, P, Klintmalm, GB, Stosor, V, Del Busto, R, Limaye, AP, Somani, J, Lyon, M, Houston, S, House, AA, Pruett, TL, Orloff, S, Humar, A, Dowdy, LA, Garcia-Diaz, J, Kalil, AC, Fisher, RA, Heitman, J, and Husain, S. "Antifungal management practices and evolution of infection in organ transplant recipients with cryptococcus neoformans infection." Transplantation 80.8 (October 27, 2005): 1033-1039.
PMID
16278582
Source
pubmed
Published In
Transplantation
Volume
80
Issue
8
Publish Date
2005
Start Page
1033
End Page
1039

Same-sex mating and the origin of the Vancouver Island Cryptococcus gattii outbreak.

Genealogy can illuminate the evolutionary path of important human pathogens. In some microbes, strict clonal reproduction predominates, as with the worldwide dissemination of Mycobacterium leprae, the cause of leprosy. In other pathogens, sexual reproduction yields clones with novel attributes, for example, enabling the efficient, oral transmission of the parasite Toxoplasma gondii. However, the roles of clonal or sexual propagation in the origins of many other microbial pathogen outbreaks remain unknown, like the recent fungal meningoencephalitis outbreak on Vancouver Island, Canada, caused by Cryptococcus gattii. Here we show that the C. gattii outbreak isolates comprise two distinct genotypes. The majority of isolates are hypervirulent and have an identical genotype that is unique to the Pacific Northwest. A minority of the isolates are significantly less virulent and share an identical genotype with fertile isolates from an Australian recombining population. Genotypic analysis reveals evidence of sexual reproduction, in which the majority genotype is the predicted offspring. However, instead of the classic a-alpha sexual cycle, the majority outbreak clone appears to have descended from two alpha mating-type parents. Analysis of nuclear content revealed a diploid environmental isolate homozygous for the major genotype, an intermediate produced during same-sex mating. These studies demonstrate how cryptic same-sex reproduction can enable expansion of a human pathogen to a new geographical niche and contribute to the ongoing production of infectious spores. This has implications for the emergence of other microbial pathogens and inbreeding in host range expansion in the fungal and other kingdoms.

Authors
Fraser, JA; Giles, SS; Wenink, EC; Geunes-Boyer, SG; Wright, JR; Diezmann, S; Allen, A; Stajich, JE; Dietrich, FS; Perfect, JR; Heitman, J
MLA Citation
Fraser, JA, Giles, SS, Wenink, EC, Geunes-Boyer, SG, Wright, JR, Diezmann, S, Allen, A, Stajich, JE, Dietrich, FS, Perfect, JR, and Heitman, J. "Same-sex mating and the origin of the Vancouver Island Cryptococcus gattii outbreak." Nature 437.7063 (October 27, 2005): 1360-1364.
PMID
16222245
Source
pubmed
Published In
Nature
Volume
437
Issue
7063
Publish Date
2005
Start Page
1360
End Page
1364
DOI
10.1038/nature04220

Isolation of Cryptococcus gattii and Cryptococcus neoformans var. grubii from the flowers and bark of Eucalyptus trees in India.

The association of Cryptococcus gattii with Eucalyptus trees has been well established. Here we report the isolation of both C. gattii and Cryptococcus neoformans var. grubii from the flowers and bark of Eucalyptus trees in India. We investigated a total of 233 samples of Eucalyptus trees: 120 flowers, 81 fragments of bark, and 32 leaves. C. gattii was isolated from two samples of flowers of Eucalyptus terreticornis. C. neoformans var. grubii was recovered twice from the bark of Eucalyptus camaldulensis, initially from one of three samples, and again 2 months later, from one of four samples collected beneath the canopy of the tree. The primary isolation medium was Nigerseed agar, and brown colonies were presumptively identified as C. gattii or C. neoformans. The species identification was confirmed by morphological and biochemical characteristics. Using the Crypto-Check kit (Iatron, Tokyo, Japan), the first two isolates were identified as serotype B (C. gattii) and the other two were serotype A (C. neoformans var. grubii). PCR analysis of the isolates of C. neoformans var. grubii revealed that they possessed the MATalpha mating type allele. Molecular typing by amplified fragment length polymorphism markers indicated that both isolates of C. neoformans var. grubii possessed the same genotype. This study demonstrates that C. neoformans var. grubii, as well as C. gattii, may be associated with Eucalyptus trees.

Authors
Gugnani, HC; Mitchell, TG; Litvintseva, AP; Lengeler, KB; Heitman, J; Kumar, A; Basu, S; Paliwal-Joshi, A
MLA Citation
Gugnani, HC, Mitchell, TG, Litvintseva, AP, Lengeler, KB, Heitman, J, Kumar, A, Basu, S, and Paliwal-Joshi, A. "Isolation of Cryptococcus gattii and Cryptococcus neoformans var. grubii from the flowers and bark of Eucalyptus trees in India." Med Mycol 43.6 (September 2005): 565-569.
PMID
16323312
Source
pubmed
Published In
Medical Mycology (Informa)
Volume
43
Issue
6
Publish Date
2005
Start Page
565
End Page
569

Cryptococcus neoformans {alpha} strains preferentially disseminate to the central nervous system during coinfection.

Cryptococcus neoformans is a fungal pathogen that has evolved over the past 40 million years into three distinct varieties or sibling species (gattii, grubii, and neoformans). Each variety manifests differences in epidemiology and disease, and var. grubii strains are responsible for the vast majority of human disease. In previous studies, alpha strains were more virulent than congenic a strains in var. neoformans, whereas var. grubii congenic a and alpha strains exhibited equivalent levels of virulence. Here the role of mating type in the virulence of var. grubii was further characterized in a panel of model systems. Congenic var. grubii a and alpha strains had equivalent survival rates when cultured with amoebae, nematodes, and macrophages. No difference in virulence was observed between a and alpha congenic strains in multiple inbred-mouse genetic backgrounds, and there was no difference in accumulations in the central nervous system (CNS) late in infection. In contrast, during coinfections, a and alpha strains are equivalent in peripheral tissues but alpha cells have an enhanced predilection to penetrate the CNS. These studies reveal the first virulence difference between congenic a and alpha strains in the most common pathogenic variety and suggest an explanation for the prevalence of alpha strains in clinical isolates.

Authors
Nielsen, K; Cox, GM; Litvintseva, AP; Mylonakis, E; Malliaris, SD; Benjamin, DK; Giles, SS; Mitchell, TG; Casadevall, A; Perfect, JR; Heitman, J
MLA Citation
Nielsen, K, Cox, GM, Litvintseva, AP, Mylonakis, E, Malliaris, SD, Benjamin, DK, Giles, SS, Mitchell, TG, Casadevall, A, Perfect, JR, and Heitman, J. "Cryptococcus neoformans {alpha} strains preferentially disseminate to the central nervous system during coinfection." Infect Immun 73.8 (August 2005): 4922-4933.
PMID
16041006
Source
pubmed
Published In
Infection and immunity
Volume
73
Issue
8
Publish Date
2005
Start Page
4922
End Page
4933
DOI
10.1128/IAI.73.8.4922-4933.2005

Cyclophilin B escorts the hepatitis C virus RNA polymerase: a viral achilles heel?

A recent report by Watashi et al. (2005) in Molecular Cell reveals a role for the host cell prolyl isomerase cyclophilin B (CyPB) in the replication of the hepatitis C viral genome, opening potential avenues for antiviral therapeutic intervention.

Authors
Heitman, J; Cullen, BR
MLA Citation
Heitman, J, and Cullen, BR. "Cyclophilin B escorts the hepatitis C virus RNA polymerase: a viral achilles heel?." Mol Cell 19.2 (July 22, 2005): 145-146. (Review)
PMID
16039584
Source
pubmed
Published In
Molecular Cell
Volume
19
Issue
2
Publish Date
2005
Start Page
145
End Page
146
DOI
10.1016/j.molcel.2005.07.001

Specialization of the HOG pathway and its impact on differentiation and virulence of Cryptococcus neoformans.

The human pathogenic fungus Cryptococcus neoformans has diverged from a common ancestor into three biologically distinct varieties or sibling species over the past 10-40 million years. During evolution of these divergent forms, serotype A C. neoformans var. grubii has emerged as the most virulent and cosmopolitan pathogenic clade. Therefore, understanding how serotype A C. neoformans is distinguished from less successful pathogenic serotypes will provide insights into the evolution of fungal virulence. Here we report that the structurally conserved Pbs2-Hog1 MAP kinase cascade has been specifically recruited as a global regulator to control morphological differentiation and virulence factors in the highly virulent serotype A H99 clinical isolate, but not in the laboratory-generated and less virulent serotype D strain JEC21. The mechanisms of Hog1 regulation are strikingly different between the two strains, and the phosphorylation kinetics and localization pattern of Hog1 are opposite in H99 compared with JEC21 and other yeasts. The unique Hog1 regulatory pattern observed in the H99 clinical isolate is widespread in serotype A strains and is also present in some clinical serotype D isolates. Serotype A hog1delta and pbs2delta mutants are attenuated in virulence, further underscoring the role of the Pbs2-Hog1 MAPK cascade in the pathogenesis of cryptococcosis.

Authors
Bahn, Y-S; Kojima, K; Cox, GM; Heitman, J
MLA Citation
Bahn, Y-S, Kojima, K, Cox, GM, and Heitman, J. "Specialization of the HOG pathway and its impact on differentiation and virulence of Cryptococcus neoformans." Mol Biol Cell 16.5 (May 2005): 2285-2300.
PMID
15728721
Source
pubmed
Published In
Molecular Biology of the Cell
Volume
16
Issue
5
Publish Date
2005
Start Page
2285
End Page
2300
DOI
10.1091/mbc.E04-11-0987

The genome of the basidiomycetous yeast and human pathogen Cryptococcus neoformans.

Cryptococcus neoformans is a basidiomycetous yeast ubiquitous in the environment, a model for fungal pathogenesis, and an opportunistic human pathogen of global importance. We have sequenced its approximately 20-megabase genome, which contains approximately 6500 intron-rich gene structures and encodes a transcriptome abundant in alternatively spliced and antisense messages. The genome is rich in transposons, many of which cluster at candidate centromeric regions. The presence of these transposons may drive karyotype instability and phenotypic variation. C. neoformans encodes unique genes that may contribute to its unusual virulence properties, and comparison of two phenotypically distinct strains reveals variation in gene content in addition to sequence polymorphisms between the genomes.

Authors
Loftus, BJ; Fung, E; Roncaglia, P; Rowley, D; Amedeo, P; Bruno, D; Vamathevan, J; Miranda, M; Anderson, IJ; Fraser, JA; Allen, JE; Bosdet, IE; Brent, MR; Chiu, R; Doering, TL; Donlin, MJ; D'Souza, CA; Fox, DS; Grinberg, V; Fu, J; Fukushima, M; Haas, BJ; Huang, JC; Janbon, G; Jones, SJM; Koo, HL; Krzywinski, MI; Kwon-Chung, JK; Lengeler, KB; Maiti, R; Marra, MA; Marra, RE; Mathewson, CA; Mitchell, TG; Pertea, M; Riggs, FR; Salzberg, SL; Schein, JE; Shvartsbeyn, A; Shin, H; Shumway, M; Specht, CA et al.
MLA Citation
Loftus, BJ, Fung, E, Roncaglia, P, Rowley, D, Amedeo, P, Bruno, D, Vamathevan, J, Miranda, M, Anderson, IJ, Fraser, JA, Allen, JE, Bosdet, IE, Brent, MR, Chiu, R, Doering, TL, Donlin, MJ, D'Souza, CA, Fox, DS, Grinberg, V, Fu, J, Fukushima, M, Haas, BJ, Huang, JC, Janbon, G, Jones, SJM, Koo, HL, Krzywinski, MI, Kwon-Chung, JK, Lengeler, KB, Maiti, R, Marra, MA, Marra, RE, Mathewson, CA, Mitchell, TG, Pertea, M, Riggs, FR, Salzberg, SL, Schein, JE, Shvartsbeyn, A, Shin, H, Shumway, M, and Specht, CA et al. "The genome of the basidiomycetous yeast and human pathogen Cryptococcus neoformans." Science 307.5713 (February 25, 2005): 1321-1324.
PMID
15653466
Source
pubmed
Published In
Science
Volume
307
Issue
5713
Publish Date
2005
Start Page
1321
End Page
1324
DOI
10.1126/science.1103773

Chromosomal translocation and segmental duplication in Cryptococcus neoformans.

Large chromosomal events such as translocations and segmental duplications enable rapid adaptation to new environments. Here we marshal genomic, genetic, meiotic mapping, and physical evidence to demonstrate that a chromosomal translocation and segmental duplication occurred during construction of a congenic strain pair in the fungal human pathogen Cryptococcus neoformans. Two chromosomes underwent telomere-telomere fusion, generating a dicentric chromosome that broke to produce a chromosomal translocation, forming two novel chromosomes sharing a large segmental duplication. The duplication spans 62,872 identical nucleotides and generated a second copy of 22 predicted genes, and we hypothesize that this event may have occurred during meiosis. Gene disruption studies of one embedded gene (SMG1) corroborate that this region is duplicated in an otherwise haploid genome. These findings resolve a genome project assembly anomaly and illustrate an example of rapid genome evolution in a fungal genome rich in repetitive elements.

Authors
Fraser, JA; Huang, JC; Pukkila-Worley, R; Alspaugh, JA; Mitchell, TG; Heitman, J
MLA Citation
Fraser, JA, Huang, JC, Pukkila-Worley, R, Alspaugh, JA, Mitchell, TG, and Heitman, J. "Chromosomal translocation and segmental duplication in Cryptococcus neoformans." Eukaryot Cell 4.2 (February 2005): 401-406.
PMID
15701802
Source
pubmed
Published In
Eukaryotic cell
Volume
4
Issue
2
Publish Date
2005
Start Page
401
End Page
406
DOI
10.1128/EC.4.2.401-406.2005

Transcriptional network of multiple capsule and melanin genes governed by the Cryptococcus neoformans cyclic AMP cascade.

Cryptococcus neoformans is an opportunistic human fungal pathogen that elaborates several virulence attributes, including a polysaccharide capsule and melanin pigments. A conserved Galpha protein/cyclic AMP (cAMP) pathway controls melanin and capsule production. To identify targets of this pathway, we used an expression profiling approach to define genes that are transcriptionally regulated by the Galpha protein Gpa1. This approach revealed that Gpa1 transcriptionally regulates multiple genes involved in capsule assembly and identified two additional genes with a marked dependence on Gpa1 for transcription. The first is the LAC1 gene, encoding the laccase enzyme that catalyzes a rate-limiting step in diphenol oxidation and melanin production. The second gene identified (LAC2) is adjacent to the LAC1 gene and encodes a second laccase that shares 75% nucleotide identity with LAC1. Similar to the LAC1 gene, LAC2 is induced in response to glucose deprivation. However, LAC2 basal transcript levels are much lower than those for LAC1. Accordingly, a lac2 mutation results in only a modest delay in melanin formation. LAC2 overexpression suppresses the melanin defects of gpa1 and lac1 mutants and partially restores virulence of these strains. These studies provide mechanistic insights into the regulation of capsule and melanin production by the C. neoformans cAMP pathway and demonstrate that multiple laccases contribute to C. neoformans melanin production and pathogenesis.

Authors
Pukkila-Worley, R; Gerrald, QD; Kraus, PR; Boily, M-J; Davis, MJ; Giles, SS; Cox, GM; Heitman, J; Alspaugh, JA
MLA Citation
Pukkila-Worley, R, Gerrald, QD, Kraus, PR, Boily, M-J, Davis, MJ, Giles, SS, Cox, GM, Heitman, J, and Alspaugh, JA. "Transcriptional network of multiple capsule and melanin genes governed by the Cryptococcus neoformans cyclic AMP cascade." Eukaryot Cell 4.1 (January 2005): 190-201.
PMID
15643074
Source
pubmed
Published In
Eukaryotic cell
Volume
4
Issue
1
Publish Date
2005
Start Page
190
End Page
201
DOI
10.1128/EC.4.1.190-201.2005

Cryptococcus neoformans isolates from transplant recipients are not selected for resistance to calcineurin inhibitors by current immunosuppressive regimens.

The immunosuppressants tacrolimus (FK506) and cyclosporine A inhibit calcineurin and have potent antifungal activity. In this study, 24% of Cryptococcus neoformans isolates from solid-organ transplant patients exhibited altered sensitivity to these drugs, which may have an impact on the infectious course but does not appear to be the consequence of immunosuppressive therapy.

Authors
Blankenship, JR; Singh, N; Alexander, BD; Heitman, J
MLA Citation
Blankenship, JR, Singh, N, Alexander, BD, and Heitman, J. "Cryptococcus neoformans isolates from transplant recipients are not selected for resistance to calcineurin inhibitors by current immunosuppressive regimens." J Clin Microbiol 43.1 (January 2005): 464-467.
PMID
15635017
Source
pubmed
Published In
Journal of clinical microbiology
Volume
43
Issue
1
Publish Date
2005
Start Page
464
End Page
467
DOI
10.1128/JCM.43.1.464-467.2005

Clinical and environmental isolates of Cryptococcus gattii from Australia that retain sexual fecundity

Cryptococcus gattii is a primary pathogenic yeast that causes disease in both animals and humans. It is closely related to Cryptococcus neoformans and diverged from a common ancestor ∼40 million years ago. While C. gattii has a characterized sexual cycle dependent upon a dimorphic region of the genome known as the MAT locus, mating has rarely been observed in this species. In this study, we identify for the first time clinical (both human and veterinary) and environmental isolates from Australia that retain sexual fecundity. A collection of 120 isolates from a variety of geographic locations was analyzed for molecular type, mating type, and the ability to develop mating structures when cocultured with fertile tester strains. Nine isolates produced dikaryotic filaments with paired nuclei, fused clamp connections, and basidiospores. DNA sequence analysis of three genes (URA5, the MATα-specific SXI1α gene, and the MATa-specific SXI2a gene) revealed little or no variability in URA5 and SXI2a, respectively. However across the 108 MATα strains sequenced, the SXI1α gene was found to exist as 11 different alleles. Phylogenetic analysis found most variation to occur in the more fertile genotypes. Although some lineages of Australian C. gattii have retained the ability to mate, the majority of isolates were sterile, suggesting that asexuality is the dominant mode of propagation in these populations. Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Authors
Campbell, LT; Fraser, JA; Nichols, CB; Dietrich, FS; Carter, D; Heitman, J
MLA Citation
Campbell, LT, Fraser, JA, Nichols, CB, Dietrich, FS, Carter, D, and Heitman, J. "Clinical and environmental isolates of Cryptococcus gattii from Australia that retain sexual fecundity." Eukaryotic Cell 4.8 (2005): 1410-1419.
PMID
16087746
Source
scival
Published In
Eukaryotic cell
Volume
4
Issue
8
Publish Date
2005
Start Page
1410
End Page
1419
DOI
10.1128/EC.4.8.1410-1419.2005

Cryptococcus neoformans gene involved in mammalian pathogenesis identified by a Caenorhabditis elegans progeny-based approach

Caenorhabditis elegans can serve as a substitute host for the study of microbial pathogenesis. We found that mutations in genes of the fungal pathogen Cryptococcus neoformans involved in mammalian virulence allow C. elegans to produce greater numbers of progeny than when exposed to wild-type fungus. We used this property to screen a library of C. neoformans mutants for strains that permit larger C. elegans brood sizes. In this screen, we identified a gene homologous to Saccharomyces cerevisiae ROM2. C. neoformans rom2 mutation resulted in a defect in mating and growth defects at elevated temperature or in the presence of cell wall or hyperosmolar stresses. An effect of the C. neoformans rom2 mutation in virulence was confirmed in a murine inhalation infection model. We propose that a screen for progeny-permissive mutants of microorganisms can serve as a high-throughput method for identifying novel loci related to mammalian pathogenesis. Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Authors
Tang, RJ; Breger, J; Idnurm, A; Gerik, KJ; Lodge, JK; Heitman, J; Calderwood, SB; Mylonakis, E
MLA Citation
Tang, RJ, Breger, J, Idnurm, A, Gerik, KJ, Lodge, JK, Heitman, J, Calderwood, SB, and Mylonakis, E. "Cryptococcus neoformans gene involved in mammalian pathogenesis identified by a Caenorhabditis elegans progeny-based approach." Infection and Immunity 73.12 (2005): 8219-8225.
PMID
16299318
Source
scival
Published In
Infection and immunity
Volume
73
Issue
12
Publish Date
2005
Start Page
8219
End Page
8225
DOI
10.1128/IAI.73.12.8219-8225.2005

Pde1 phosphodiesterase modulates cyclic AMP levels through a protein kinase A-mediated negative feedback loop in Cryptococcus neoformans

The virulence of the human pathogenic fungus Cryptococcus neoformans is regulated by a cyclic AMP (cAMP)-dependent protein kinase A (PKA) signaling cascade that promotes mating and the production of melanin and capsule. In this study, genes encoding homologs of the Saccharomyces cerevisiae low- and high-affinity phosphodiesterases, PDE1 and PDE2, respectively, were deleted in serotype A strains of C. neoformans. The resulting mutants exhibited moderately elevated levels of melanin and capsule production relative to the wild type. Epistasis experiments indicate that Pde1 functions downstream of the Gα subunit Gpa1, which initiates cAMP-dependent signaling in response to an extracellular signal. Previous work has shown that the PKA catalytic subunit Pka1 governs cAMP levels via a negative feedback loop. Here we show that a pde1Δ. pka1Δ mutant strain exhibits cAMP levels that are dramatically increased (∼15-fold) relative to those in a pka1Δ single mutant strain and that a site-directed mutation in a consensus PKA phosphorylation site reduces Pde1 function. These data provide evidence that fluctuations in cAMP levels are modulated by both Pka1-dependent regulation of Pde1 and another target that comprise a robust negative feedback loop to tightly constrain intracellular cAMP levels. Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Authors
Hicks, JK; Bahn, Y-S; Heitman, J
MLA Citation
Hicks, JK, Bahn, Y-S, and Heitman, J. "Pde1 phosphodiesterase modulates cyclic AMP levels through a protein kinase A-mediated negative feedback loop in Cryptococcus neoformans." Eukaryotic Cell 4.12 (2005): 1971-1981.
PMID
16339715
Source
scival
Published In
Eukaryotic cell
Volume
4
Issue
12
Publish Date
2005
Start Page
1971
End Page
1981
DOI
10.1128/EC.4.12.1971-1981.2005

Chromosomal sex-determining regions in animals, plants and fungi

The independent evolution of sex chromosomes in many eukaryotic species raises questions about the evolutionary forces that drive their formation. Recent advances in our understanding of these genomic structures in mammals in parallel with alternate models such as the monotremes, fish, dioecious plants, and fungi support the idea of a remarkable convergence in structure to form large, non-recombining regions with discrete evolutionary strata. The discovery that evolutionary events similar to those that have transpired in humans have also occurred during the formation of sex chromosomes in organisms as divergent as the plant Silene, the fungus Cryptococcus and the fish medaka highlights the importance of future studies in these systems. Such investigation will broaden our knowledge of the evolution and plasticity of these ubiquitous genomic features underlying sexual dimorphism and reproduction. © 2005 Elsevier Ltd. All rights reserved.

Authors
Fraser, JA; Heitman, J
MLA Citation
Fraser, JA, and Heitman, J. "Chromosomal sex-determining regions in animals, plants and fungi." Current Opinion in Genetics and Development 15.6 (2005): 645-651.
PMID
16182521
Source
scival
Published In
Current Opinion in Genetics & Development
Volume
15
Issue
6
Publish Date
2005
Start Page
645
End Page
651
DOI
10.1016/j.gde.2005.09.002

Deciphering the model pathogenic fungus Cryptococcus neoformans

Cryptococcus neoformans is a basidiomycete fungal pathogen of humans that has diverged considerably from other model fungi such as Neurospora crassa, Aspergillus nidulans, Saccharomyces cerevisiae and the common human fungal pathogen Candida albicans. The recent completion of the genome sequences of two related C. neoformans strains and the ongoing genome sequencing of three other divergent Cryptococcus strains with different virulence phenotypes and environmental distributions should improve our understanding of this important pathogen. We discuss the biology of C. neoformans in light of this genomic data, with a special emphasis on the role that evolution and sexual reproduction have in the complex relationships of the fungus with the environment and the host. © 2005 Nature Publishing Group.

Authors
Idnurm, A; Bahn, Y-S; Nielsen, K; Lin, X; Fraser, JA; Heitman, J
MLA Citation
Idnurm, A, Bahn, Y-S, Nielsen, K, Lin, X, Fraser, JA, and Heitman, J. "Deciphering the model pathogenic fungus Cryptococcus neoformans." Nature Reviews Microbiology 3.10 (2005): 753-764.
PMID
16132036
Source
scival
Published In
Nature Reviews Microbiology
Volume
3
Issue
10
Publish Date
2005
Start Page
753
End Page
764
DOI
10.1038/nrmicro1245

Chlamydospore formation during hyphal growth in Cryptococcus neoformans

Cryptococcus neoformans, a basidiomycetous fungal pathogen, infects hosts through inhalation and can cause fatal meningoencephalitis in individuals if untreated. This fungus undergoes a dimorphic transition from yeast to filamentous growth during mating and monokaryotic fruiting, which leads to the production of hyphae and airborne infectious basidiospores. Here we characterized a novel morphological feature associated with the filamentous stages of the life cycle of C. neoformans which resembles resting or survival structures known as chlamydospores in other fungi. The C. neoformans chlamydospore-like structure is rich in glycogen, suggesting that it might have a role as an energy store. However, characterization of mutants with decreased or increased levels of glycogen production showed that glycogen levels have little effect on filamentous growth, sporulation, or chlamydospore formation. These results suggest that the formation of chlamydospores is independent of glycogen accumulation level. We also show that chlamydospore formation does not require successful sporulation and that the presence of chlamydospores is not sufficient for sporulation. Although the biological functions of chlamydospores remain to be established for this pathogenic fungus, their formation appears to be an integral part of the filamentation process, suggesting that they could be necessary to support sexual sporulation under adverse conditions and thereby facilitate the production of infectious basidiospores or long-term survival propagules in harsh environments. Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Authors
Lin, X; Heitman, J
MLA Citation
Lin, X, and Heitman, J. "Chlamydospore formation during hyphal growth in Cryptococcus neoformans." Eukaryotic Cell 4.10 (2005): 1746-1754.
PMID
16215181
Source
scival
Published In
Eukaryotic cell
Volume
4
Issue
10
Publish Date
2005
Start Page
1746
End Page
1754
DOI
10.1128/EC.4.10.1746-1754.2005

Photosensing fungi: Phytochrome in the spotlight

Red light triggers asexual development and represses sexual development in the fungus Aspergillus nidulans. This response has been shown to require a phytochrome red/far-red light photoreceptor, FphA, which is cytoplasmic and binds a tetrapyrrole chromophore. FphA exhibits similarities to both plant and bacterial phytochromes.

Authors
Idnurm, A; Heitman, J
MLA Citation
Idnurm, A, and Heitman, J. "Photosensing fungi: Phytochrome in the spotlight." Current Biology 15.20 (2005): R829-R832.
PMID
16243020
Source
scival
Published In
Current Biology
Volume
15
Issue
20
Publish Date
2005
Start Page
R829
End Page
R832
DOI
10.1016/j.cub.2005.10.001

Gα subunit Gpa2 recruits kelch repeat subunits that inhibit receptor-G protein coupling during cAMP-induced dimorphic transitions in Saccharomyces cerevisiae

All eukaryotic cells sense extracellular stimuli and activate intracellular signaling cascades via G protein-coupled receptors (GPCR) and associated heterotrimeric G proteins. The Saccharomyces cerevisiae GPCR Gpr1 and associated Gα subunit Gpa2 sense extracellular carbon sources (including glucose) to govern filamentous growth. In contrast to conventional Gα subunits, Gpa2 forms an atypical G protein complex with the kelch repeat Gβ mimic proteins Gpb1 and Gpb2. Gpb1/2 negatively regulate cAMP signaling by inhibiting Gpa2 and an as yet unidentified target. Here we show that Gpa2 requires lipid modifications of its N-terminus for membrane localization but association with the Gpr1 receptor or Gpb1/2 subunits is dispensable for membrane targeting. Instead, Gpa2 promotes membrane localization of its associated Gβ mimic subunit Gpb2. We also show that the Gpa2 N-terminus binds both to Gpb2 and to the C-terminal tail of the Gpr1 receptor and that Gpb1/2 binding interferes with Gpr1 receptor coupling to Gpa2. Our studies invoke novel mechanisms involving GPCR-G protein modules that may be conserved in multicellular eukaryotes. © 2005 by The American Society for Cell Biology.

Authors
Harashima, T; Heitman, J
MLA Citation
Harashima, T, and Heitman, J. "Gα subunit Gpa2 recruits kelch repeat subunits that inhibit receptor-G protein coupling during cAMP-induced dimorphic transitions in Saccharomyces cerevisiae." Molecular Biology of the Cell 16.10 (2005): 4557-4571.
PMID
16030250
Source
scival
Published In
Molecular Biology of the Cell
Volume
16
Issue
10
Publish Date
2005
Start Page
4557
End Page
4571
DOI
10.1091/mbc.E05-05-0403

A fungal Achilles' heel

Authors
Heitman, J
MLA Citation
Heitman, J. "A fungal Achilles' heel." Science 309.5744 (2005): 2175-2176.
PMID
16195450
Source
scival
Published In
Science
Volume
309
Issue
5744
Publish Date
2005
Start Page
2175
End Page
2176
DOI
10.1126/science.1119321

Calcineurin-binding protein Cbp1 directs the specificity of calcineurin-dependent hyphal elongation during mating in Cryptococcus neoformans

Mating and virulence of the human fungal pathogen Cryptococcus neoformans are controlled by calcineurin, a serine-threonine-specific calcium-activated phosphatase that is the target of the immunosuppressive drugs cyclosporine A and FK506. In previous studies, a calcineurin binding protein (Cbp1, Rcn1, Dscr1/Csp1-3/ MCIP1-3) that is conserved from yeasts to humans has been identified, but whether this protein functions to regulate calcineurin activity or facilitate calcineurin function as a signaling effector has been unclear. Here we show that, like calcineurin, Cbp1 is required for mating in C. neoformans. By contrast, Cbp1 plays no role in promoting calcineurin-dependent growth at 37°C and is not essential for haploid fruiting. Site-directed mutagenesis studies provide evidence that tandem phosphorylation and dephosphorylation of two serine residues in the conserved SP repeat motif are critical for Cbp1 function. Epistasis analysis supports models in which Cbp1 functions coordinately with calcineurin to direct hyphal elongation during mating. Taken together, these findings provide insights into the roles of Cbp1 as an accessory subunit or effector of calcineurin-specific signaling pathways, which may be features conserved among the calcipressins to govern calcineurin signaling in immune cells, cardiomyocytes, and neurons of multicellular eukaryotes. Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Authors
Fox, DS; Heitman, J
MLA Citation
Fox, DS, and Heitman, J. "Calcineurin-binding protein Cbp1 directs the specificity of calcineurin-dependent hyphal elongation during mating in Cryptococcus neoformans." Eukaryotic Cell 4.9 (2005): 1526-1538.
PMID
16151246
Source
scival
Published In
Eukaryotic Cell
Volume
4
Issue
9
Publish Date
2005
Start Page
1526
End Page
1538
DOI
10.1128/EC.4.9.1526-1538.2005

Clonality and recombination in genetically differentiated subgroups of Cryptococcus gattii

Cryptococcus gattii is a pathogenic yeast that together with Cryptococcus neoformans causes cryptococcosis in humans and animals. High numbers of viable C. gattii propagules can be obtained from certain species of Australian Eucalyptus camaldulensis trees, and an epidemiological link between Eucalyptus colonization and human exposure has been proposed. However, the highest prevalence of C. gattii cryptococcosis occurs in Papua New Guinea and in regions of Australia where the eucalypt species implicated to date are not endemic. This study investigated the population structure of three geographically distinct clinical and veterinary populations of C. gattii from Australia and Papua New Guinea. All populations that consisted of a genotype found frequently in Australia (VGI) were strongly clonal and were highly differentiated from one another. Two populations of the less common VGII genotype from Sydney and the Northern Territory had population structures inferring recombination. In addition, there was some evidence of reduced genetic differentiation between these geographically remote regions. In a companion study presented in this issue, VGII isolates were overwhelmingly more fertile than those of the VGI genotype, giving biological support to the indirect assessment of sexual exchange. It appears that the VGI genotype propagates clonally on eucalypts in Australia and on an unknown substrate in Papua New Guinea, with infection initiated by an unidentified infectious propagule. VGII isolates are completing their life cycles and may be dispersed via sexually produced basidiospores, which are also likely to initiate respiratory infection. Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Authors
Campbell, LT; Currie, BJ; Krockenberger, M; Malik, R; Meyer, W; Heitman, J; Carter, D
MLA Citation
Campbell, LT, Currie, BJ, Krockenberger, M, Malik, R, Meyer, W, Heitman, J, and Carter, D. "Clonality and recombination in genetically differentiated subgroups of Cryptococcus gattii." Eukaryotic Cell 4.8 (2005): 1403-1409.
PMID
16087745
Source
scival
Published In
Eukaryotic cell
Volume
4
Issue
8
Publish Date
2005
Start Page
1403
End Page
1409
DOI
10.1128/EC.4.8.1403-1409.2005

Calcineurin is required for Candida albicans to survive calcium stress in serum

The calcium-activated protein phosphatase calcineurin plays a critical role in the virulence of Candida albicans. Previous studies demonstrated that calcineurin is not required for the yeast-hypha dimorphic transition, host cell adherence, or host cell injury, which are all established virulence attributes of this organism. Calcineurin is, however, essential for survival in serum and disseminated infection. Here we identify the component of serum that is toxic to calcineurin mutant cells. Proteins, peptides, lipids, and other hydrophobic components were all excluded as essential toxic elements. Upon testing of small molecules present in serum, we discovered that calcineurin protects cells from stress caused by the endogenous levels of calcium ions present in serum. These studies illustrate how calcineurin functions in a calcium homeostatic pathway that enables a common human commensal to survive passage through the hostile environment of the bloodstream to establish deep-seated infections and cause disease. Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Authors
Blankenship, JR; Heitman, J
MLA Citation
Blankenship, JR, and Heitman, J. "Calcineurin is required for Candida albicans to survive calcium stress in serum." Infection and Immunity 73.9 (2005): 5767-5774.
PMID
16113294
Source
scival
Published In
Infection and Immunity
Volume
73
Issue
9
Publish Date
2005
Start Page
5767
End Page
5774
DOI
10.1128/IAI.73.9.5767-5774.2005

Novel gene functions required for melanization of the human pathogen Cryptococcus neoformans

The ability to produce melanin is a key virulence factor in many fungal pathogens including the human basidiomycete pathogen Cryptococcus neoformans, a major cause of life-threatening infections among immunocompromised persons. Despite the significance of melanin biosynthesis in virulence of C. neoformans, the cellular and molecular processes involved in this pathway have not yet been fully elucidated. Here, we used Agrobacterium to isolate insertional mutants and screened 12000 mutants to uncover genes involved in melanin production in C. neoformans. Four new mutant alleles of the well-known melanin biosynthesis gene, LAC1, which encodes laccase were identified, and the T-DNA was shown to have a possible predisposition for insertion into the promoters of genes, in particular LAC1. Melanization in C. neoformans is dependent on five additional genes identified in this screen encoding homologues of the copper transporter Ccc2, the copper chaperone Atx1, the chitin synthase Chs3, the transcriptional coactivator Mbf1 and the chromatin-remodelling enzyme Snf5. Illumination of the molecular and genetic components of this virulence pathway reveals potential novel targets for drug development against C. neoformans and provides further insight into the intimate relationship between metal ion homeostasis and melanin biosynthesis. © 2005 Blackwell Publishing Ltd.

Authors
Walton, FJ; Idnurm, A; Heitman, J
MLA Citation
Walton, FJ, Idnurm, A, and Heitman, J. "Novel gene functions required for melanization of the human pathogen Cryptococcus neoformans." Molecular Microbiology 57.5 (2005): 1381-1396.
PMID
16102007
Source
scival
Published In
Molecular Microbiology
Volume
57
Issue
5
Publish Date
2005
Start Page
1381
End Page
1396
DOI
10.1111/j.1365-2958.2005.04779.x

Cryptococcus neoformans gene expression during murine macrophage infection

The fungal pathogen Cryptococcus neoformans survives phagocytosis by macrophages and proliferates within, ultimately establishing latent infection as a facultative intracellular pathogen that can escape macrophage control to cause disseminated disease. This process is hypothesized to be important for C. neoformans pathogenesis; however, it is poorly understood how C. neoformans adapts to and overcomes the hostile intracellular environment of the macrophage. Using DNA microarray technology, we have investigated the transcriptional response of C. neoformans to phagocytosis by murine macrophages. The expression profiles of several genes were verified using quantitative reverse transcription-PCR and a green fluorescent protein reporter strain. Multiple membrane transporters for hexoses, amino acids, and iron were up-regulated, as well as genes involved in responses to oxidative stress. Genes involved in autophagy, peroxisome function, and lipid metabolism were also induced. Interestingly, almost the entire mating type locus displayed increased expression 24 h after internalization, suggesting an intrinsic connection between infection and the MAT locus. Genes in the Gpa1-cyclic AMP-protein kinase A pathway were also up-regulated. Both gpa1 and pka1 mutants were found to be compromised in macrophage infection, confirming the important role of this virulence pathway. A large proportion of the repressed genes are involved in ribosome-related functions, rRNA processing, and translation initiation/elongation, implicating a reduction in translation as a central response to phagocytosis. In summary, this gene expression profile allows us to interpret the adaptation of C. neoformans to the intracellular infection process and informs the search for genes encoding novel virulence attributes. Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Authors
Fan, W; Kraus, PR; Boily, M-J; Heitman, J
MLA Citation
Fan, W, Kraus, PR, Boily, M-J, and Heitman, J. "Cryptococcus neoformans gene expression during murine macrophage infection." Eukaryotic Cell 4.8 (2005): 1420-1433.
PMID
16087747
Source
scival
Published In
Eukaryotic cell
Volume
4
Issue
8
Publish Date
2005
Start Page
1420
End Page
1433
DOI
10.1128/EC.4.8.1420-1433.2005

Galleria mellonella as a model system to study Cryptococcus neoformans pathogenesis

Evaluation of Cryptococcus neoformans virulence in a number of nonmammalian hosts suggests that C. neoformans is a nonspecific pathogen. We used the killing of Galleria mellonella (the greater wax moth) caterpillar by C. neoformans to develop an invertebrate host model system that can be used to study cryptococcal virulence, host immune responses to infection, and the effects of antifungal compounds. All varieties of C. neoformans killed G. mellonella. After injection into the insect hemocoel, C. neoformans proliferated and, despite successful phagocytosis by host hemocytes, killed caterpillars both at 37°C and 30°C. The rate and extent of killing depended on the cryptococcal strain and the number of fungal cells injected. The sequenced C. neoformans clinical strain H99 was the most virulent of the strains tested and killed caterpillars with inocula as low as 20 CFU/caterpillar. Several C. neoformans genes previously shown to be involved in mammalian virulence (CAP59, GPA1, RAS1, and PKA1) also played a role in G. mellonella killing. Combination antifungal therapy (amphotericin B plus flucytosine) administered before or after inoculation was more effective than monotherapy in prolonging survival and in decreasing the tissue burden of cryptococci in the hemocoel. The G. mellonella-C. neoformans pathogenicity model may be a substitute for mammalian models of infection with C. neoformans and may facilitate the in vivo study of fungal virulence and efficacy of antifungal therapies. Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Authors
Mylonakis, E; Moreno, R; Khoury, JBE; Idnurm, A; Heitman, J; Calderwood, SB; Ausubel, FM; Diener, A
MLA Citation
Mylonakis, E, Moreno, R, Khoury, JBE, Idnurm, A, Heitman, J, Calderwood, SB, Ausubel, FM, and Diener, A. "Galleria mellonella as a model system to study Cryptococcus neoformans pathogenesis." Infection and Immunity 73.7 (2005): 3842-3850.
PMID
15972469
Source
scival
Published In
Infection and Immunity
Volume
73
Issue
7
Publish Date
2005
Start Page
3842
End Page
3850
DOI
10.1128/IAI.73.7.3842-3850.2005

Calcium- and calcineurin-independent roles for calmodulin in Cryptococcus neoformans morphogenesis and high-temperature growth

The function of calcium as a signaling molecule is conserved in eukaryotes from fungi to humans. Previous studies have identified the calcium-activated phosphatase calcineurin as a critical factor in governing growth of the human pathogenic fungus Cryptococcus neoformans at mammalian body temperature. Here, we employed insertional mutagenesis to identify new genes required for growth at 37°C. One insertion mutant, cam1-ts, that displayed a growth defect at 37°C and hypersensitivity to the calcineurin inhibitor FK506 at 25°C was isolated. Both phenotypes were linked to the dominant marker in genetic crosses, and molecular analysis revealed that the insertion occurred in the 3′ untranslated region of the gene encoding the calcineurin activator calmodulin (CAM1) and impairs growth at 37°C by significantly reducing calmodulin mRNA abundance. The CAM1 gene was demonstrated to be essential using genetic analysis of a CAM1/cam1Δ diploid strain. In the absence of calcineurin function, the cam1-ts mutant displayed a severe morphological defect with impaired bud formation. Expression of a calmodulin-independent calcineurin mutant did not suppress the growth defect of the cam1-ts mutant at 37°C, indicating that calmodulin promotes growth at high temperature via calcineurin-dependent and -independent pathways. In addition, a Ca 2+-binding-defective allele of CAM1 complemented the 37°C growth defect, FK506 hypersensitivity, and morphogenesis defect of the cam1-ts mutant. Our findings reveal that calmodulin performs Ca2+- and calcineurin-independent and -dependent roles in controlling C. neoformans morphogenesis and high-temperature growth. Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Authors
Kraus, PR; Nichols, CB; Heitman, J
MLA Citation
Kraus, PR, Nichols, CB, and Heitman, J. "Calcium- and calcineurin-independent roles for calmodulin in Cryptococcus neoformans morphogenesis and high-temperature growth." Eukaryotic Cell 4.6 (2005): 1079-1087.
PMID
15947200
Source
scival
Published In
Eukaryotic Cell
Volume
4
Issue
6
Publish Date
2005
Start Page
1079
End Page
1087
DOI
10.1128/EC.4.6.1079-1087.2005

Sexual reproduction between partners of the same mating type in Cryptococcus neoformans

Cryptococcus neoformans is a globally distributed human fungal pathogen that causes life-threatening meningoencephalitis in immunocompromised patients1. It has a defined sexual cycle involving haploid cells of α and a mating types2, yet the vast majority of environmental and clinical isolates are α (ref. 3). Sexual recombination is normally expected to occur between isolates of opposite mating type in organisms with two mating types (or sexes). How sexual reproductive potential can be maintained in an organism with a largely unisexual, nearly clonal population genetic structure is unknown. One clue, however, is that α strains undergo fruiting, a process that resembles sexual mating4 but is thought to be strictly mitotic and asexual. We report here that hallmarks of mating occur during fruiting, including diploidization and meiosis. Pheromone response pathway elements and the key meiotic regulator Dmc1 are required for efficient fruiting. Furthermore, fusion and meiosis can occur between non-isogenic α strains, enabling genetic exchange. These studies reveal how sexual reproduction can occur between partners of the same mating type. These findings have implications for the evolution of microbial pathogens, as well as for parthenogenesis, cell fusion events and transitions between self-fertilizing and outcrossing modes of reproduction observed in both fungi and other kingdoms.

Authors
Lin, X; Hull, CM; Heitman, J
MLA Citation
Lin, X, Hull, CM, and Heitman, J. "Sexual reproduction between partners of the same mating type in Cryptococcus neoformans." Nature 434.7036 (2005): 1017-1021.
PMID
15846346
Source
scival
Published In
Nature
Volume
434
Issue
7036
Publish Date
2005
Start Page
1017
End Page
1021
DOI
10.1038/nature03448

Light controls growth and development via a conserved pathway in the fungal kingdom.

Light inhibits mating and haploid fruiting of the human fungal pathogen Cryptococcus neoformans, but the mechanisms involved were unknown. Two genes controlling light responses were discovered through candidate gene and insertional mutagenesis approaches. Deletion of candidate genes encoding a predicted opsin or phytochrome had no effect on mating, while strains mutated in the white collar 1 homolog gene BWC1 mated equally well in the light or the dark. The predicted Bwc1 protein shares identity with Neurospora crassa WC-1, but lacks the zinc finger DNA binding domain. BWC1 regulates cell fusion and repression of hyphal development after fusion in response to blue light. In addition, bwc1 mutant strains are hypersensitive to ultraviolet light. To identify other components required for responses to light, a novel self-fertile haploid strain was created and subjected to Agrobacterium-mediated insertional mutagenesis. One UV-sensitive mutant that filaments equally well in the light and the dark was identified and found to have an insertion in the BWC2 gene, whose product is structurally similar to N. crassa WC-2. The C. neoformans Bwc1 and Bwc2 proteins interact in the yeast two-hybrid assay. Deletion of BWC1 or BWC2 reduces the virulence of C. neoformans in a murine model of infection; the Bwc1-Bwc2 system thus represents a novel protein complex that influences both development and virulence in a pathogenic fungus. These results demonstrate that a role for blue/UV light in controlling development is an ancient process that predates the divergence of the fungi into the ascomycete and basidiomycete phyla.

Authors
Idnurm, A; Heitman, J
MLA Citation
Idnurm, A, and Heitman, J. "Light controls growth and development via a conserved pathway in the fungal kingdom." PLoS biology. 3.4 (2005): e95-.
PMID
15760278
Source
scival
Published In
PLoS biology
Volume
3
Issue
4
Publish Date
2005
Start Page
e95
DOI
10.1371/journal.pbio.0030095

Light controls growth and development via a conserved pathway in the fungal kingdom

Light inhibits mating and haploid fruiting of the human fungal pathogen Cryptococcus neoformans, but the mechanisms involved were unknown. Two genes controlling light responses were discovered through candidate gene and insertional mutagenesis approaches. Deletion of candidate genes encoding a predicted opsin or phytochrome had no effect on mating, while strains mutated in the white collar 1 homolog gene BWC1 mated equally well in the light or the dark. The predicted Bwc1 protein shares identity with Neurospora crassa WC-1, but lacks the zinc finger DNA binding domain. BWC1 regulates cell fusion and repression of hyphal development after fusion in response to blue light. In addition, bwc1 mutant strains are hypersensitive to ultraviolet light. To identify other components required for responses to light, a novel self-fertile haploid strain was created and subjected to Agrobacterium-mediated insertional mutagenesis. One UV-sensitive mutant that filaments equally well in the light and the dark was identified and found to have an insertion in the BWC2 gene, whose product is structurally similar to N. crassa WC-2. The C. neoformans Bwc1 and Bwc2 proteins interact in the yeast two-hybrid assay. Deletion of BWC1 or BWC2 reduces the virulence of C. neoformans in a murine model of infection; the Bwc1-Bwc2 system thus represents a novel protein complex that influences both development and virulence in a pathogenic fungus. These results demonstrate that a role for blue/UV light in controlling development is an ancient process that predates the divergence of the fungi into the ascomycete and basidiomycete phyla. © 2005 Idnurm and Heitman.

Authors
Idnurm, A; Heitman, J
MLA Citation
Idnurm, A, and Heitman, J. "Light controls growth and development via a conserved pathway in the fungal kingdom." PLoS Biology 3.4 (2005): 0615-0626.
Source
scival
Published In
PLoS Biology
Volume
3
Issue
4
Publish Date
2005
Start Page
0615
End Page
0626
DOI
10.1371/journal.pbio.0030095

Sex-specific homeodomain proteins Sxi1α and Sxi2a coordinately regulate sexual development in Cryptococcus neoformans

Homeodomain proteins are central regulators of development in eukaryotes. In fungi, homeodomain proteins have been shown to control cell identity and sexual development. Cryptococcus neoformans is a human fungal pathogen with a defined sexual cycle that produces spores, the suspected infectious particles. Previously, only a single homeodomain regulatory protein involved in sexual development, Sxi1α, had been identified. Here we present the discovery of Sxi2a, a predicted but heretofore elusive cell-type-specific homeodomain protein essential for the regulation of sexual development. Our studies reveal that Sxi2a is necessary for proper sexual development and sufficient to drive this development in otherwise haploid α cells. We further show that Sxi1α and Sxi2a interact with one another and impart similar expression patterns for two key mating genes. The discovery of Sxi2a and its relationship with Sxi1α leads to a new model for how the sexual cycle is controlled in C. neoformans, with implications for virulence. Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Authors
Hull, CM; Boily, M-J; Heitman, J
MLA Citation
Hull, CM, Boily, M-J, and Heitman, J. "Sex-specific homeodomain proteins Sxi1α and Sxi2a coordinately regulate sexual development in Cryptococcus neoformans." Eukaryotic Cell 4.3 (2005): 526-535.
PMID
15755915
Source
scival
Published In
Eukaryotic Cell
Volume
4
Issue
3
Publish Date
2005
Start Page
526
End Page
535
DOI
10.1128/EC.4.3.526-535.2005

The cyclophilins

Cyclophilins (Enzyme Commission (EC) number 5.1.2.8) belong to a group of proteins that have peptidyl-prolyl cis-trans isomerase activity; such proteins are collectively known as immunophilins and also include the FK-506-binding proteins and the parvulins. Cyclophilins are found in all cells of all organisms studied, in both prokaryotes and eukaryotes; humans have a total of 16 cyclophilin proteins, Arabidopsis up to 29 and Saccharomyces 8. The first member of the cyclophilins to be identified in mammals, cyclophilin A, is the major cellular target for, and thus mediates the actions of, the immunosuppressive drug cyclosporin A. Cyclophilin A forms a ternary complex with cyclosporin A and the calcium-calmodulin-activated serine/threonine-specific protein phosphatase calcineurin; formation of this complex prevents calcineurin from regulating cytokine gene transcription. Recent studies have implicated a diverse array of additional cellular functions for cyclophilins, including roles as chaperones and in cell signaling. © 2005 BioMed Central Ltd.

Authors
Wang, P; Heitman, J
MLA Citation
Wang, P, and Heitman, J. "The cyclophilins." Genome Biology 6.7 (2005).
PMID
15998457
Source
scival
Published In
Genome Biology
Volume
6
Issue
7
Publish Date
2005
DOI
10.1186/gb-2005-6-7-226

Cyclophilin A is localized to the nucleus and controls meiosis in Saccharomyces cerevisiae

Cyclophilin A is conserved from yeast to humans and mediates the ability of cyclosporine to perturb signal transduction cascades via inhibition of calcineurin. Cyclophilin A also catalyzes cis-trans peptidyl-prolyl isomerization during protein folding or conformational changes; however, cyclophilin A is not essential in yeast or human cells, and the true biological functions of this highly conserved enzyme have remained enigmatic. In Saccharomyces cerevisiae, cyclophilin A becomes essential in cells compromised for the nuclear prolyl-isomerase Ess1, and cyclophilin A physically interacts with two nuclear histone deacetylase complexes, Sin3-Rpd3 and Set3C, which both control meiosis. Here we show that cyclophilin A is localized to the nucleus in yeast cells and governs the meiotic gene program to promote efficient sporulation. The prolyl-isomerase activity of cyclophilin A is required for this meiotic function. We document that cyclophilin A physically associates with the Set3C histone deacetylase and analyze in detail the structure of this protein-protein complex. Genetic studies support a model in which cyclophilin A controls meiosis via Set3C and an additional target. Our findings reveal a novel nuclear role for cyclophilin A in governing the transcriptional program required for the vegetative to meiotic developmental switch in budding yeast.

Authors
Arévalo-Rodríguez, M; Heitman, J
MLA Citation
Arévalo-Rodríguez, M, and Heitman, J. "Cyclophilin A is localized to the nucleus and controls meiosis in Saccharomyces cerevisiae." Eukaryotic Cell 4.1 (2005): 17-29.
PMID
15643056
Source
scival
Published In
Eukaryotic cell
Volume
4
Issue
1
Publish Date
2005
Start Page
17
End Page
29
DOI
10.1128/EC.4.1.17-29.2005

In vitro interactions between antifungals and immunosuppressants against Aspergillus fumigatus isolates from transplant and nontransplant patients.

We performed in vitro antifungal checkerboard testing on 12 Aspergillus fumigatus clinical isolates (6 transplant recipients and 6 nontransplant patients) with three antifungal agents (amphotericin B, voriconazole, and caspofungin) and three immunosuppressants (FK506, cyclosporine, and rapamycin). We were not able to detect a difference in calcineurin inhibitor antifungal activity against isolates from transplant recipients and nontransplant patients.

Authors
Steinbach, WJ; Singh, N; Miller, JL; Benjamin, DK; Schell, WA; Heitman, J; Perfect, JR
MLA Citation
Steinbach, WJ, Singh, N, Miller, JL, Benjamin, DK, Schell, WA, Heitman, J, and Perfect, JR. "In vitro interactions between antifungals and immunosuppressants against Aspergillus fumigatus isolates from transplant and nontransplant patients." Antimicrob Agents Chemother 48.12 (December 2004): 4922-4925.
PMID
15561883
Source
pubmed
Published In
Antimicrobial agents and chemotherapy
Volume
48
Issue
12
Publish Date
2004
Start Page
4922
End Page
4925
DOI
10.1128/AAC.48.12.4922-4925.2004

Adenylyl cyclase-associated protein Aca1 regulates virulence and differentiation of Cryptococcus neoformans via the cyclic AMP-protein kinase A cascade.

The evolutionarily conserved cyclic AMP (cAMP) signaling pathway controls cell functions in response to environmental cues in organisms as diverse as yeast and mammals. In the basidiomycetous human pathogenic fungus Cryptococcus neoformans, the cAMP pathway governs virulence and morphological differentiation. Here we identified and characterized adenylyl cyclase-associated protein, Aca1, which functions in parallel with the Galpha subunit Gpa1 to control the adenylyl cyclase (Cac1). Aca1 interacted with the C terminus of Cac1 in the yeast two-hybrid system. By molecular and genetic approaches, Aca1 was shown to play a critical role in mating by regulating cell fusion and filamentous growth in a cAMP-dependent manner. Aca1 also regulates melanin and capsule production via the Cac1-cAMP-protein kinase A pathway. Genetic epistasis studies support models in which Aca1 and Gpa1 are necessary and sufficient components that cooperate to activate adenylyl cyclase. Taken together, these studies further define the cAMP signaling cascade controlling virulence of this ubiquitous human fungal pathogen.

Authors
Bahn, Y-S; Hicks, JK; Giles, SS; Cox, GM; Heitman, J
MLA Citation
Bahn, Y-S, Hicks, JK, Giles, SS, Cox, GM, and Heitman, J. "Adenylyl cyclase-associated protein Aca1 regulates virulence and differentiation of Cryptococcus neoformans via the cyclic AMP-protein kinase A cascade." Eukaryot Cell 3.6 (December 2004): 1476-1491.
PMID
15590822
Source
pubmed
Published In
Eukaryotic cell
Volume
3
Issue
6
Publish Date
2004
Start Page
1476
End Page
1491
DOI
10.1128/EC.3.6.1476-1491.2004

Convergent evolution of chromosomal sex-determining regions in the animal and fungal kingdoms.

Sexual identity is governed by sex chromosomes in plants and animals, and by mating type (MAT) loci in fungi. Comparative analysis of the MAT locus from a species cluster of the human fungal pathogen Cryptococcus revealed sequential evolutionary events that fashioned this large, highly unusual region. We hypothesize that MAT evolved via four main steps, beginning with acquisition of genes into two unlinked sex-determining regions, forming independent gene clusters that then fused via chromosomal translocation. A transitional tripolar intermediate state then converted to a bipolar system via gene conversion or recombination between the linked and unlinked sex-determining regions. MAT was subsequently subjected to intra- and interallelic gene conversion and inversions that suppress recombination. These events resemble those that shaped mammalian sex chromosomes, illustrating convergent evolution in sex-determining structures in the animal and fungal kingdoms.

Authors
Fraser, JA; Diezmann, S; Subaran, RL; Allen, A; Lengeler, KB; Dietrich, FS; Heitman, J
MLA Citation
Fraser, JA, Diezmann, S, Subaran, RL, Allen, A, Lengeler, KB, Dietrich, FS, and Heitman, J. "Convergent evolution of chromosomal sex-determining regions in the animal and fungal kingdoms." PLoS Biol 2.12 (December 2004): e384-.
PMID
15538538
Source
pubmed
Published In
PLoS biology
Volume
2
Issue
12
Publish Date
2004
Start Page
e384
DOI
10.1371/journal.pbio.0020384

The calcineurin target, Crz1, functions in azole tolerance but is not required for virulence of Candida albicans.

In Candida albicans, calcineurin is essential for virulence and survival during membrane perturbation by azoles. Crz1 is a proposed downstream target of calcineurin based on studies of Saccharomyces cerevisiae. However, the in vitro phenotypes of C. albicans crz1/crz1 and calcineurin mutants differ and Crz1 is not required for virulence.

Authors
Onyewu, C; Wormley, FL; Perfect, JR; Heitman, J
MLA Citation
Onyewu, C, Wormley, FL, Perfect, JR, and Heitman, J. "The calcineurin target, Crz1, functions in azole tolerance but is not required for virulence of Candida albicans." Infect Immun 72.12 (December 2004): 7330-7333.
PMID
15557662
Source
pubmed
Published In
Infection and immunity
Volume
72
Issue
12
Publish Date
2004
Start Page
7330
End Page
7333
DOI
10.1128/IAI.72.12.7330-7333.2004

A Sch9 protein kinase homologue controlling virulence independently of the cAMP pathway in Cryptococcus neoformans.

The polysaccharide capsule is one of the established virulence factors in Cryptococcus neoformans that provides a barrier against the host-mediated immune response. Mutation of the gene encoding the Saccharomyces cerevisiae Sch9 protein kinase homologue resulted in cells with enlarged capsules in C. neoformans. Capsule production was abrogated in sch9 pka1 double mutants, indicating that protein kinase A (PKA) signaling is still necessary for capsule formation in sch9 mutants. The sch9 mutant also exhibited increased thermal tolerance, a phenotype similar to sch9 mutant strains of S. cerevisiae. In addition, the sch9 mutant was attenuated in mating and the highly encapsulated cells were attenuated in virulence, in contrast to the pkr1 mutant, lacking the regulatory subunit of protein kinase A, that produced similarly enlarged capsules yet was increased in virulence. Interestingly, the virulence for the sch9 mutant strain could be restored by introduction of a pkr1 mutation; and the sch9 pkr1 mutant strain was dramatically increased in size and capsule thickness, suggesting that Sch9 and PKA function via different targets involved in virulence. Our findings support a model in which Sch9 modulates capsule formation and contributes to the virulence of C. neoformans both independently of and in conjunction with the cAMP-PKA pathway.

Authors
Wang, P; Cox, GM; Heitman, J
MLA Citation
Wang, P, Cox, GM, and Heitman, J. "A Sch9 protein kinase homologue controlling virulence independently of the cAMP pathway in Cryptococcus neoformans." Curr Genet 46.5 (November 2004): 247-255.
PMID
15503029
Source
pubmed
Published In
Current Genetics
Volume
46
Issue
5
Publish Date
2004
Start Page
247
End Page
255
DOI
10.1007/s00294-004-0529-1

Identification of Cryptococcus neoformans temperature-regulated genes with a genomic-DNA microarray.

The ability to survive and proliferate at 37 degrees C is an essential virulence attribute of pathogenic microorganisms. A partial-genome microarray was used to profile gene expression in the human-pathogenic fungus Cryptococcus neoformans during growth at 37 degrees C. Genes with orthologs involved in stress responses were induced during growth at 37 degrees C, suggesting that a conserved transcriptional program is used by C. neoformans to alter gene expression during stressful conditions. A gene encoding the transcription factor homolog Mga2 was induced at 37 degrees C and found to be important for high-temperature growth. Genes encoding fatty acid biosynthetic enzymes were identified as potential targets of Mga2, suggesting that membrane remodeling is an important component of adaptation to high growth temperatures. mga2Delta mutants were extremely sensitive to the ergosterol synthesis inhibitor fluconazole, indicating a coordination of the synthesis of membrane component precursors. Unexpectedly, genes involved in amino acid and pyrimidine biosynthesis were repressed at 37 degrees C, but components of these pathways were found to be required for high-temperature growth. Our findings demonstrate the utility of even partial-genome microarrays for delineating regulatory cascades that contribute to microbial pathogenesis.

Authors
Kraus, PR; Boily, M-J; Giles, SS; Stajich, JE; Allen, A; Cox, GM; Dietrich, FS; Perfect, JR; Heitman, J
MLA Citation
Kraus, PR, Boily, M-J, Giles, SS, Stajich, JE, Allen, A, Cox, GM, Dietrich, FS, Perfect, JR, and Heitman, J. "Identification of Cryptococcus neoformans temperature-regulated genes with a genomic-DNA microarray." Eukaryot Cell 3.5 (October 2004): 1249-1260.
PMID
15470254
Source
pubmed
Published In
Eukaryotic cell
Volume
3
Issue
5
Publish Date
2004
Start Page
1249
End Page
1260
DOI
10.1128/EC.3.5.1249-1260.2004

Cryptococcus neoformans mitochondrial genomes from serotype A and D strains do not influence virulence.

Cryptococcus neoformans is an encapsulated pathogenic yeast producing meningoencephalitis. Two primary strains in genetic studies, serotype A H99 and serotype D JEC21, possess dramatic differences in virulence. Since it has been shown that mitochondrial gene expression is prominent at the site of the infection and there are significant differences between mitochondrial gene structure and regulation between the serotype A and D strains, this study used AD hybrids to move serotype A and D mitochondria under different genomic influences. When the serotype D MATa strain is involved in the mating crosses, there is uniparental transmission of mitochondrial DNA, but with the serotype A MATa strain, mitochondrial DNA can be inherited from either parent and recombination in the mitochondrial genome may also occur. In virulence studies between serotype A and D strains, it was found that the primary genetic control of the virulence composite for growth in the central nervous system is encoded in the nuclear DNA and not through mitochondrial DNA.

Authors
Toffaletti, DL; Nielsen, K; Dietrich, F; Heitman, J; Perfect, JR
MLA Citation
Toffaletti, DL, Nielsen, K, Dietrich, F, Heitman, J, and Perfect, JR. "Cryptococcus neoformans mitochondrial genomes from serotype A and D strains do not influence virulence." Curr Genet 46.4 (October 2004): 193-204.
PMID
15309505
Source
pubmed
Published In
Current Genetics
Volume
46
Issue
4
Publish Date
2004
Start Page
193
End Page
204
DOI
10.1007/s00294-004-0521-9

Investigation of the basis of virulence in serotype A strains of Cryptococcus neoformans from apparently immunocompetent individuals.

Cryptococcus neoformans serotype A strains commonly infect immunocompromised patients to cause fungal meningitis. To understand the basis of serotype A cryptococcal infections in apparently immunocompetent patients, we tested two hypotheses: the strains were naturally occurring hypervirulent pkr1 (PKA regulatory subunit) mutants, or the strains were hybrids with C. neoformans var. gattii strains that normally infect immunocompetent individuals. Analysis of clinical isolates obtained from apparently immunocompetent individuals from three continents revealed that none were pkr1 mutants, but several exhibited phenotypes consistent with perturbations in cAMP signaling. Additionally, none of the strains were unusual hybrids with gattii strains. Except for one strain that was an AD hybrid, all others were serotype A (var. grubii) isolates. Taken together, our findings indicate that the ability of these clinical isolates to infect apparently normal individuals may be attributable to mutations other than pkr1 and/or underlying immune system impairment in patients.

Authors
D'Souza, CA; Hagen, F; Boekhout, T; Cox, GM; Heitman, J
MLA Citation
D'Souza, CA, Hagen, F, Boekhout, T, Cox, GM, and Heitman, J. "Investigation of the basis of virulence in serotype A strains of Cryptococcus neoformans from apparently immunocompetent individuals." Curr Genet 46.2 (August 2004): 92-102.
PMID
15150669
Source
pubmed
Published In
Current Genetics
Volume
46
Issue
2
Publish Date
2004
Start Page
92
End Page
102
DOI
10.1007/s00294-004-0511-y

A genetic linkage map of Cryptococcus neoformans variety neoformans serotype D (Filobasidiella neoformans).

To construct a genetic linkage map of the heterothallic yeast, Cryptococcus neoformans (Filobasidiella neoformans), we crossed two mating-compatible strains and analyzed 94 progeny for the segregation of 301 polymorphic markers, consisting of 228 restriction site polymorphisms, 63 microsatellites, two indels, and eight mating-type (MAT)-associated markers. All but six markers showed no significant (P < 0.05) segregation distortion. At a minimum LOD score of 6.0 and a maximum recombination frequency of 0.30, 20 linkage groups were resolved, resulting in a map length of approximately 1500 cM. Average marker density is 5.4 cM (range 1-28.7 cM). Hybridization of selected markers to blots of electrophoretic karyotypes unambiguously assigned all linkage groups to chromosomes and led us to conclude that the C. neoformans genome is approximately 20.2 Mb, comprising 14 chromosomes ranging in size from 0.8 to 2.3 Mb, with a ratio of approximately 13.2 kb/cM averaged across the genome. However, only 2 of 12 ungrouped markers hybridized to chromosome 10. The hybridizations revealed at least one possible reciprocal translocation involving chromosomes 8, 9, and 12. This map has been critical to genome sequence assembly and will be essential for future studies of quantitative trait inheritance.

Authors
Marra, RE; Huang, JC; Fung, E; Nielsen, K; Heitman, J; Vilgalys, R; Mitchell, TG
MLA Citation
Marra, RE, Huang, JC, Fung, E, Nielsen, K, Heitman, J, Vilgalys, R, and Mitchell, TG. "A genetic linkage map of Cryptococcus neoformans variety neoformans serotype D (Filobasidiella neoformans)." Genetics 167.2 (June 2004): 619-631.
PMID
15238516
Source
pubmed
Published In
Genetics
Volume
167
Issue
2
Publish Date
2004
Start Page
619
End Page
631
DOI
10.1534/genetics.103.023408

The alpha-specific cell identity factor Sxi1alpha is not required for virulence of Cryptococcus neoformans.

Cryptococcus neoformans is a human fungal pathogen that has two mating types (a and alpha). Experiments have shown that in some backgrounds alpha strains are more virulent than a strains. Our studies reveal that the only known alpha-specific factor, SXI1alpha, is not necessary for virulence.

Authors
Hull, CM; Cox, GM; Heitman, J
MLA Citation
Hull, CM, Cox, GM, and Heitman, J. "The alpha-specific cell identity factor Sxi1alpha is not required for virulence of Cryptococcus neoformans." Infect Immun 72.6 (June 2004): 3643-3645.
PMID
15155676
Source
pubmed
Published In
Infection and immunity
Volume
72
Issue
6
Publish Date
2004
Start Page
3643
End Page
3645
DOI
10.1128/IAI.72.6.3643-3645.2004

In vitro interactions between antifungals and immunosuppressants against Aspergillus fumigatus.

The optimal treatment for invasive aspergillosis remains elusive, despite the increased efficacy of newer agents. The immunosuppressants cyclosporine (CY), tacrolimus (FK506), and sirolimus (formerly called rapamycin) exhibit in vitro and in vivo activity against Candida albicans, Cryptococcus neoformans, and Saccharomyces cerevisiae, including fungicidal synergy with azole antifungals. We report here that both FK506 and CY exhibit a clear in vitro positive interaction with caspofungin against Aspergillus fumigatus by disk diffusion, microdilution checkerboard, and gross and microscopic morphological analyses. Microscopic morphological analyses indicate that the calcineurin inhibitors delay filamentation, and in combination with caspofungin there is a positive interaction. Our findings suggest a potential role for combination therapy with calcineurin pathway inhibitors and existing antifungal agents to augment activity against A. fumigatus.

Authors
Steinbach, WJ; Schell, WA; Blankenship, JR; Onyewu, C; Heitman, J; Perfect, JR
MLA Citation
Steinbach, WJ, Schell, WA, Blankenship, JR, Onyewu, C, Heitman, J, and Perfect, JR. "In vitro interactions between antifungals and immunosuppressants against Aspergillus fumigatus." Antimicrob Agents Chemother 48.5 (May 2004): 1664-1669.
PMID
15105118
Source
pubmed
Published In
Antimicrobial agents and chemotherapy
Volume
48
Issue
5
Publish Date
2004
Start Page
1664
End Page
1669

Cryptococcus neoformans shows a remarkable genotypic diversity in Brazil.

The genotypic diversity of Brazilian Cryptococcus neoformans strains was analyzed. The majority of the samples were alphaA (65%), followed by alphaB (17.5%), alphaD (9%), alphaAaD hybrids (5%), and alphaC (3.5%). A considerable genotypic diversity occurred within C. neoformans var. grubii, and a new amplified fragment length polymorphism genotype, 1B, was recognized.

Authors
Barreto de Oliveira, MT; Boekhout, T; Theelen, B; Hagen, F; Baroni, FA; Lazera, MS; Lengeler, KB; Heitman, J; Rivera, ING; Paula, CR
MLA Citation
Barreto de Oliveira, MT, Boekhout, T, Theelen, B, Hagen, F, Baroni, FA, Lazera, MS, Lengeler, KB, Heitman, J, Rivera, ING, and Paula, CR. "Cryptococcus neoformans shows a remarkable genotypic diversity in Brazil." J Clin Microbiol 42.3 (March 2004): 1356-1359.
PMID
15004118
Source
pubmed
Published In
Journal of clinical microbiology
Volume
42
Issue
3
Publish Date
2004
Start Page
1356
End Page
1359

Cyclic AMP-dependent protein kinase catalytic subunits have divergent roles in virulence factor production in two varieties of the fungal pathogen Cryptococcus neoformans.

Our earlier findings established that cyclic AMP-dependent protein kinase functions in a signaling cascade that regulates mating and virulence of Cryptococcus neoformans var. grubii (serotype A). Mutants lacking the serotype A protein kinase A (PKA) catalytic subunit Pka1 are unable to mate, fail to produce melanin or capsule, and are avirulent in animal models, whereas mutants lacking the PKA regulatory subunit Pkr1 overproduce capsule and are hypervirulent. Because other mutations have been observed to confer different phenotypes in two diverged varieties of C. neoformans (grubii variety [serotype A] and neoformans variety [serotype D]), we analyzed the functions of the PKA genes in the serotype D neoformans variety. Surprisingly, the Pka1 catalytic subunit was not required for mating, haploid fruiting, or melanin or capsule production of serotype D strains. Here we identify a second PKA catalytic subunit gene, PKA2, that is present in both serotype A and D strains of C. neoformans. The divergent Pka2 catalytic subunit was found to regulate mating, haploid fruiting, and virulence factor production in serotype D strains. In contrast, Pka2 has no role in mating, melanin production, or capsule formation in serotype A strains. Our studies illustrate how different components of signaling pathways can be co-opted and functionally specialized during the evolution of related but distinct varieties or subspecies of a human fungal pathogen.

Authors
Hicks, JK; D'Souza, CA; Cox, GM; Heitman, J
MLA Citation
Hicks, JK, D'Souza, CA, Cox, GM, and Heitman, J. "Cyclic AMP-dependent protein kinase catalytic subunits have divergent roles in virulence factor production in two varieties of the fungal pathogen Cryptococcus neoformans." Eukaryot Cell 3.1 (February 2004): 14-26.
PMID
14871933
Source
pubmed
Published In
Eukaryotic cell
Volume
3
Issue
1
Publish Date
2004
Start Page
14
End Page
26

Evolution of fungal sex chromosomes.

Sexual reproduction enables organisms to shuffle two parental genomes to produce recombinant progeny, and to purge the genome of deleterious mutations. Sex is conserved in virtually all organisms, from bacteria and fungi to plants and animals, and yet the mechanisms by which sexual identity are established share both conserved general features and are remarkably diverse. In animals, sexual identity is established by dimorphic sex chromosomes, whereas in fungi a specialized region of the genome, known as the mating-type locus, governs the establishment of cell type identity and differs in DNA sequence between cells of different mating-types. Recent studies on the mating-type loci of fungi and algae reveal features shared with the mammalian X and Y chromosomes, suggesting that these represent early steps in the evolution of sex chromosomes.

Authors
Fraser, JA; Heitman, J
MLA Citation
Fraser, JA, and Heitman, J. "Evolution of fungal sex chromosomes." Mol Microbiol 51.2 (January 2004): 299-306. (Review)
PMID
14756773
Source
pubmed
Published In
Molecular Microbiology
Volume
51
Issue
2
Publish Date
2004
Start Page
299
End Page
306
DOI
10.1046/j.1365-2958.2003.03874.x

Cryptococcus neoformans virulence gene discovery through insertional mutagenesis

Insertional mutagenesis was applied to Cryptococcus neoformans to identify genes associated with virulence attributes. Using biolistic transformation, we generated 4,300 nourseothricin (NAT)-resistant strains, of which 590 exhibited stable resistance. We focused on mutants with defects in established virulence factors and identified two with reduced growth at 37°C, four with reduced production of the antioxidant pigment melanin, and two with an increased sensitivity to nitric oxide (NO). The NAT insertion and mutant phenotypes were genetically linked in five of eight mutants, and the DNA flanking the insertions was characterized. For the strains with altered growth at 37°C and altered melanin production, mutations were in previously uncharacterized genes, while the two NO-sensitive strains bore insertions in the flavohemoglobin gene FHB1, whose product counters NO stress. Because of the frequent instability of nourseothricin resistance associated with biolistic transformation, Agrobacterium-mediated transformation was tested. This transkingdom DNA delivery approach produced 100% stable nourseothricin-resistant transformants, and three melanin-defective strains were identified from 576 transformants, of which 2 were linked to NAT in segregation analysis. One of these mutants contained a T-DNA insertion in the promoter of the LAC1 (laccase) gene, which encodes a key enzyme required for melanin production, while the second contained an insertion in the promoter of the CLC1 gene, encoding a voltage-gated chloride channel. Clc1 and its homologs are required for ion homeostasis, and in their absence Cu+ transport into the secretory pathway is compromised, depriving laccase and other Cu+-dependent proteins of their essential cofactor. The NAT resistance cassette was optimized for cryptococcal codon usage and GC content and was then used to disrupt a mitogen-activated protein kinase gene, a predicted gene, and two putative chloride channel genes to analyze their contributions to fungal physiology. Our findings demonstrate that both insertional mutagenesis methods can be applied to gene identification, but Agrobacterium-mediated transformation is more efficient and generates exclusively stable insertion mutations.

Authors
Idnurm, A; Reedy, JL; Nussbaum, JC; Heitman, J
MLA Citation
Idnurm, A, Reedy, JL, Nussbaum, JC, and Heitman, J. "Cryptococcus neoformans virulence gene discovery through insertional mutagenesis." Eukaryotic Cell 3.2 (2004): 420-429.
PMID
15075272
Source
scival
Published In
Eukaryotic Cell
Volume
3
Issue
2
Publish Date
2004
Start Page
420
End Page
429
DOI
10.1128/EC.3.2.420-429.2004

Challenge of Drosophila melanogaster with Cryptococcus neoformans and role of the innate immune response

We found that the ingestion of Cryptococcus neoformans by Drosophila melanogaster resulted in the death of the fly but that the ingestion of Saccharomyces cerevisiae or the nonpathogenic Cryptococcus kuetzingii or Cryptococcus laurentii did not The C. neoformans protein kinase A and RAS signal transduction pathways, previously shown to be involved in virulence in mammals, also played a role in killing Drosophila. Mutation of the Toll immune response pathway, the predominant antifungal pathway of the fly, did not play a role in Drosophila defense following ingestion of the yeast. However, the Toll pathway was necessary for the clearance of C. neoformans introduced directly into the hemolymph of D. melanogaster and for the survival of systemically infected flies.

Authors
Apidianakis, Y; Rahme, LG; Heitman, J; Ausubel, FM; Calderwood, SB; Mylonakis, E
MLA Citation
Apidianakis, Y, Rahme, LG, Heitman, J, Ausubel, FM, Calderwood, SB, and Mylonakis, E. "Challenge of Drosophila melanogaster with Cryptococcus neoformans and role of the innate immune response." Eukaryotic Cell 3.2 (2004): 413-419.
PMID
15075271
Source
scival
Published In
Eukaryotic Cell
Volume
3
Issue
2
Publish Date
2004
Start Page
413
End Page
419
DOI
10.1128/EC.3.2.413-419.2004

Cyptococcus neoformans Shows a Remarkable Genotypic Diversity in Brazil

The genotypic diversity of Brazilian Cryptococcus neoformans strains was analyzed. The majority of the samples were αA (65%), followed by αB (17.5%), αD (9%), αAaD hybrids (5%), and αC (3.5%). A considerable genotypic diversity occurred within C. neoformans var. grubii, and a new amplified fragment length polymorphism genotype, 1B, was recognized.

Authors
Oliveira, MTBD; Boekhout, T; Theelen, B; Hagen, F; Baroni, FA; Lazera, MS; Lengeler, KB; Heitman, J; Rivera, ING; Paula, CR
MLA Citation
Oliveira, MTBD, Boekhout, T, Theelen, B, Hagen, F, Baroni, FA, Lazera, MS, Lengeler, KB, Heitman, J, Rivera, ING, and Paula, CR. "Cyptococcus neoformans Shows a Remarkable Genotypic Diversity in Brazil." Journal of Clinical Microbiology 42.3 (2004): 1356-1359.
Source
scival
Published In
Journal of Clinical Microbiology
Volume
42
Issue
3
Publish Date
2004
Start Page
1356
End Page
1359
DOI
10.1128/JCM.42.3.1356-1359.2004

Antifungal attributes of immunosuppressive agents: New paradigms in management and elucidating the pathophysiologic basis of opportunistic mycoses in organ transplant recipients

The currently available immunosuppressive agents cyclosporine A, tacrolimus, and rapamycin have potent antifungal activity against a number of opportunistic fungi in organ transplant recipients, most notably, C. neoformans, Candida, and Aspergillus species. The targets of their antifungal activity are fungal homologs of the signaling molecules that mediate their immunosuppressive action in humans, which has implications for further unraveling the pathogenesis of these infections. Corroborative clinical data suggest that despite the apparent paradox between the antifungal activity of the immunosuppressive agents and the occurrence of fungal infections during their administration, the antifungal attributes of these drugs may influence the spectrum and clinical characteristics of these infections after organ transplantation. Finally, the potent synergistic interactions between the immunosuppressive agents and antifungal drugs against many pathogenic fungi, including those that are typically resistant to traditional antifungal agents, could potentially have a role in devising novel therapeutic strategies for opportunistic mycoses in transplant recipients.

Authors
Singh, N; Heitman, J
MLA Citation
Singh, N, and Heitman, J. "Antifungal attributes of immunosuppressive agents: New paradigms in management and elucidating the pathophysiologic basis of opportunistic mycoses in organ transplant recipients." Transplantation 77.6 (2004): 795-800.
PMID
15077015
Source
scival
Published In
Transplantation
Volume
77
Issue
6
Publish Date
2004
Start Page
795
End Page
800
DOI
10.1097/01.TP.0000117252.75651.D6

FKBP12 controls aspartate pathway flux in Saccharomyces cerevisiae to prevent toxic intermediate accumulation

FKBP12 is a conserved member of the prolyl-isomerase enzyme family and serves as the intracellular receptor for FK506 that mediates immunosuppression in mammals and antimicrobial actions in fungi. To investigate the cellular functions of FKBP12 in Saccharomyces cerevisiae, we employed a high-throughput assay to identify mutations that are synthetically lethal with a mutation in the FPR1 gene, which encodes FKBP12. This screen identified a mutation in the HOM6 gene, which encodes homoserine dehydrogenase, the enzyme catalyzing the last step in conversion of aspartic acid into homoserine, the common precursor in threonine and methionine synthesis. Lethality of fpr1 hom6 double mutants was suppressed by null mutations in HOM3 or HOM2, encoding aspartokinase and aspartate β-semialdehyde dehydrogenase, respectively, supporting the hypothesis that fpr1 hom6 double mutants are inviable because of toxic accumulation of aspartate β-semialdehyde, the substrate of homoserine dehydrogenase. Our findings also indicate that mutation or inhibition of FKBP12 dysregulates the homoserine synthetic pathway by perturbing aspartokinase feedback inhibition by threonine. Because this pathway is conserved in fungi but not in mammals, our findings suggest a facile route to synergistic antifungal drug development via concomitant inhibition of FKBP12 and Hom6.

Authors
Arévalo-Rodríguez, M; Pan, X; Boeke, JD; Heitman, J
MLA Citation
Arévalo-Rodríguez, M, Pan, X, Boeke, JD, and Heitman, J. "FKBP12 controls aspartate pathway flux in Saccharomyces cerevisiae to prevent toxic intermediate accumulation." Eukaryotic Cell 3.5 (2004): 1287-1296.
PMID
15470257
Source
scival
Published In
Eukaryotic Cell
Volume
3
Issue
5
Publish Date
2004
Start Page
1287
End Page
1296
DOI
10.1128/EC.3.5.1287-1296.2004

Cryptococcus neoformans Kin1 protein kinase homologue, identified through a Caenorhabditis elegans screen, promotes virulence in mammals

Cryptococcal infections are a global cause of significant morbidity and mortality. Recent studies support the hypothesis that virulence of Cryptococcus neoformans may have evolved via survival selection in environmental hosts, such as amoebae and free-living nematodes. We used killing of the nematode Caenorhabditis elegans by C. neoformans as an assay to screen a library of random C. neoformans insertion mutants. Of 350 mutants tested, seven were identified with attenuated virulence that persisted after crossing the mutation back into a wild-type strain. Genetic analysis of one strain revealed an insertion in a gene homologous to Saccharomyces cerevisiae KIN1, which encodes a serine/threonine protein kinase. C. neoformans kin1 mutants exhibited significant defects in virulence in murine inhalation and haematogenous infection models and displayed increased binding to alveolar and peritoneal macrophages. The kin1 mutant phenotypes were complemented by the wild-type KIN1 gene. These findings show that the C. neoformans Kin1 kinase homologue is required for full virulence in disparate hosts and that C. elegans can be used as a substitute host to identify novel factors involved in fungal pathogenesis in mammals.

Authors
Mylonakis, E; Idnurm, A; Moreno, R; Khoury, JE; Rottman, JB; Ausubel, FM; Heitman, J; Calderwood, SB
MLA Citation
Mylonakis, E, Idnurm, A, Moreno, R, Khoury, JE, Rottman, JB, Ausubel, FM, Heitman, J, and Calderwood, SB. "Cryptococcus neoformans Kin1 protein kinase homologue, identified through a Caenorhabditis elegans screen, promotes virulence in mammals." Molecular Microbiology 54.2 (2004): 407-419.
PMID
15469513
Source
scival
Published In
Molecular Microbiology
Volume
54
Issue
2
Publish Date
2004
Start Page
407
End Page
419
DOI
10.1111/j.1365-2958.2004.04310.x

PAK kinases Ste20 and Pak1 govern cell polarity at different stages of mating in Cryptococcus neoformans

Sexual identity and mating are linked to virulence of the fungal pathogen Cryptococcus neoformans. Cells of the α mating type are more prevalent and can be more virulent than a cells, and basidiospores are thought to be the infectious propagule. Mating in C. neoformans involves cell-cell fusion and the generation of dikaryotic hyphae, processes that involve substantial changes in cell polarity. Two p21-activated kinase (PAK) kinases, Pak1 and Ste20, are required for both mating and virulence in C. neoformans. We show here that Ste20 and Pak1 play crucial roles in polarized morphogenesis at different steps during mating: Pak1 functions during cell fusion, whereas Ste20 fulfills a distinct morphogenic role and is required to maintain polarity in the heterokaryotic mating filament. In conclusion, our studies demonstrate that PAK kinases are necessary for polar growth during mating and that polarity establishment is necessary for mating and may contribute to virulence of C. neoformans.

Authors
Nichols, CB; Fraser, JA; Heitman, J
MLA Citation
Nichols, CB, Fraser, JA, and Heitman, J. "PAK kinases Ste20 and Pak1 govern cell polarity at different stages of mating in Cryptococcus neoformans." Molecular Biology of the Cell 15.10 (2004): 4476-4489.
PMID
15282344
Source
scival
Published In
Molecular Biology of the Cell
Volume
15
Issue
10
Publish Date
2004
Start Page
4476
End Page
4489
DOI
10.1091/mbc.E04-05-0370

SXI1α controls uniparental mitochondrial inheritance in Cryptococcus neoformans

Authors
Yan, Z; Hull, CM; Heitman, J; Sun, S; Xu, J
MLA Citation
Yan, Z, Hull, CM, Heitman, J, Sun, S, and Xu, J. "SXI1α controls uniparental mitochondrial inheritance in Cryptococcus neoformans." Current Biology 14.18 (2004): R743-R744.
PMID
15380081
Source
scival
Published In
Current Biology
Volume
14
Issue
18
Publish Date
2004
Start Page
R743
End Page
R744
DOI
10.1016/j.cub.2004.09.008

Prolyl isomerases in yeast.

Prolyl isomerases are enzymes that catalyze cis-trans isomerization of peptidyl-prolyl bonds and span three structurally unrelated protein families: the cyclophilins, FKBPs, and parvulins. The genome of the budding yeast Saccharomyces cerevisiae encodes eight different cyclophilins (Cpr1 to Cpr8), four FKBPs (Fpr1 to Fpr4), and a single parvulin (Ess1). Remarkably, two of these proteins, cyclophilin A and FKBP12, are conserved from yeast to humans and mediate virtually all of the intracellular actions of the immunosuppressive antifungal drugs cyclosporin A, FK506, and rapamycin. The study of prolyl isomerases in S. cerevisiae has proven invaluable to understand the elusive functions of these proteins, and continues to provide new insights into their diverse cellular roles. Here we review the current state of knowledge about prolyl-isomerases in this model organism.

Authors
Arevalo-Rodriguez, M; Wu, X; Hanes, SD; Heitman, J
MLA Citation
Arevalo-Rodriguez, M, Wu, X, Hanes, SD, and Heitman, J. "Prolyl isomerases in yeast." Frontiers in bioscience : a journal and virtual library. 9 (2004): 2420-2446.
PMID
15353296
Source
scival
Published In
Frontiers in bioscience : a journal and virtual library
Volume
9
Publish Date
2004
Start Page
2420
End Page
2446

Calcineurin: A central controller of signalling in eukaryotes. Workshop on the calcium/calcineurin/NFAT pathway: Regulation and function

Authors
Aramburu, J; Heitman, J; Crabtree, GR
MLA Citation
Aramburu, J, Heitman, J, and Crabtree, GR. "Calcineurin: A central controller of signalling in eukaryotes. Workshop on the calcium/calcineurin/NFAT pathway: Regulation and function." EMBO Reports 5.4 (2004): 343-348.
PMID
15060569
Source
scival
Published In
EMBO Reports
Volume
5
Issue
4
Publish Date
2004
Start Page
343
End Page
348
DOI
10.1038/sj.embor.7400133

Evidence of sexual recombination among Cryptococcus neoformans serotype A isolates in sub-Saharan Africa.

The most common cause of fungal meningitis in humans, Cryptococcus neoformans serotype A, is a basidiomycetous yeast with a bipolar mating system. However, the vast majority (>99.9%) of C. neoformans serotype A isolates possess only one of the two mating type alleles (MATalpha). Isolates with the other allele (MATa) were recently discovered and proven to mate in the laboratory. It has been a mystery whether and where C. neoformans strains undergo sexual reproduction. Here, we applied population genetic approaches to demonstrate that a population of C. neoformans serotype A clinical isolates from Botswana contains an unprecedented proportion of fertile MATa isolates and exhibits evidence of both clonal expansion and recombination within two partially genetically isolated subgroups. Our findings provide evidence for sexual recombination among some populations of C. neoformans serotype A from sub-Saharan Africa, which may have a direct impact on their evolution.

Authors
Litvintseva, AP; Marra, RE; Nielsen, K; Heitman, J; Vilgalys, R; Mitchell, TG
MLA Citation
Litvintseva, AP, Marra, RE, Nielsen, K, Heitman, J, Vilgalys, R, and Mitchell, TG. "Evidence of sexual recombination among Cryptococcus neoformans serotype A isolates in sub-Saharan Africa." Eukaryot Cell 2.6 (December 2003): 1162-1168.
PMID
14665451
Source
pubmed
Published In
Eukaryotic cell
Volume
2
Issue
6
Publish Date
2003
Start Page
1162
End Page
1168

Coping with stress: calmodulin and calcineurin in model and pathogenic fungi.

Calcium signaling via calmodulin and calcineurin is critical for the regulation of stress responses in fungi. The functions of calmodulin and calcineurin are largely conserved among pathogenic fungi and model fungi, however, the mechanisms of action have diverged. Saccharomyces cerevisiae is an excellent model for understanding the framework of calcium-mediated signal transduction pathways, and considerable progress has been made in understanding the details of how Ca(2+)-calmodulin and calcineurin control adaptation to environmental stress. Studies using the divergent human pathogenic fungi Candida albicans and Cryptococcus neoformans reveal that calcineurin is critical for virulence, yet it acts via distinct mechanisms in each fungus. These differences in function may reflect the requirements of each pathogen to survive inside the host, and illustrate that studies must be conducted in each organism in order to elucidate the details of the molecular mechanisms of calmodulin and calcineurin-mediated signaling pathways.

Authors
Kraus, PR; Heitman, J
MLA Citation
Kraus, PR, and Heitman, J. "Coping with stress: calmodulin and calcineurin in model and pathogenic fungi." Biochem Biophys Res Commun 311.4 (November 28, 2003): 1151-1157. (Review)
PMID
14623301
Source
pubmed
Published In
Biochemical and Biophysical Research Communications
Volume
311
Issue
4
Publish Date
2003
Start Page
1151
End Page
1157

Enzymes that counteract nitrosative stress promote fungal virulence.

Enzymes that protect cells from reactive oxygen species (superoxide dismutase, catalase, peroxidase) have well-established roles in mammalian biology and microbial pathogenesis. Two recently identified enzymes detoxify nitric oxide (NO)-related molecules; flavohemoglobin denitrosylase consumes NO, and S-nitrosoglutathione (GSNO) reductase metabolizes GSNO. Although both enzymes protect microorganisms from nitrosative challenge in vitro, their relevance has not been established in physiological contexts. Here we studied their biological functions in Cryptococcus neoformans, an established human fungal pathogen that replicates in macrophages and whose growth in vitro and in infected animals is controlled by NO bioactivity. We show that both flavohemoglobin denitrosylase and GSNO reductase contribute to C. neoformans pathogenesis. FHB1 and GNO1 mutations abolished NO- and GSNO-consuming activity, respectively. Growth of fhb1 mutant cells was inhibited by nitrosative challenge, whereas that of gno1 mutants was not. fhb1 mutants showed attenuated virulence in a murine model, and virulence was restored in iNOS(-/-) animals. Survival of the fhb1 mutant was also reduced in activated macrophages and restored to wild-type by inhibition of NOS activity. Combining mutations in flavohemoglobin and GSNO reductase, or flavohemoglobin and superoxide dismutase, further attenuated virulence. These studies illustrate that fungal pathogens elaborate enzymatic defenses against nitrosative stress mounted by the host.

Authors
de Jesús-Berríos, M; Liu, L; Nussbaum, JC; Cox, GM; Stamler, JS; Heitman, J
MLA Citation
de Jesús-Berríos, M, Liu, L, Nussbaum, JC, Cox, GM, Stamler, JS, and Heitman, J. "Enzymes that counteract nitrosative stress promote fungal virulence." Curr Biol 13.22 (November 11, 2003): 1963-1968.
PMID
14614821
Source
pubmed
Published In
Current Biology
Volume
13
Issue
22
Publish Date
2003
Start Page
1963
End Page
1968

Fungal mating-type loci.

Authors
Fraser, JA; Heitman, J
MLA Citation
Fraser, JA, and Heitman, J. "Fungal mating-type loci." Curr Biol 13.20 (October 14, 2003): R792-R795. (Review)
PMID
14561417
Source
pubmed
Published In
Current Biology
Volume
13
Issue
20
Publish Date
2003
Start Page
R792
End Page
R795

Recapitulation of the sexual cycle of the primary fungal pathogen Cryptococcus neoformans var. gattii: implications for an outbreak on Vancouver Island, Canada.

Cryptococcus neoformans is a human fungal pathogen that exists as three distinct varieties or sibling species: the predominantly opportunistic pathogens C. neoformans var. neoformans (serotype D) and C. neoformans var. grubii (serotype A) and the primary pathogen C. neoformans var. gattii (serotypes B and C). While serotypes A and D are cosmopolitan, serotypes B and C are typically restricted to tropical regions. However, serotype B isolates of C. neoformans var. gattii have recently caused an outbreak on Vancouver Island in Canada, highlighting the threat of this fungus and its capacity to infect immunocompetent individuals. Here we report a large-scale analysis of the mating abilities of serotype B and C isolates from diverse sources and identify unusual strains that mate robustly and are suitable for further genetic analysis. Unlike most isolates, which are of both the a and alpha mating types but are predominantly sterile, the majority of the Vancouver outbreak strains are exclusively of the alpha mating type and the majority are fertile. In an effort to enhance mating of these isolates, we identified and disrupted the CRG1 gene encoding the GTPase-activating protein involved in attenuating pheromone response. crg1 mutations dramatically increased mating efficiency and enabled mating with otherwise sterile isolates. Our studies provide a genetic and molecular foundation for further studies of this primary pathogen and reveal that the Vancouver Island outbreak may be attributable to a recent recombination event.

Authors
Fraser, JA; Subaran, RL; Nichols, CB; Heitman, J
MLA Citation
Fraser, JA, Subaran, RL, Nichols, CB, and Heitman, J. "Recapitulation of the sexual cycle of the primary fungal pathogen Cryptococcus neoformans var. gattii: implications for an outbreak on Vancouver Island, Canada." Eukaryot Cell 2.5 (October 2003): 1036-1045.
PMID
14555486
Source
pubmed
Published In
Eukaryotic cell
Volume
2
Issue
5
Publish Date
2003
Start Page
1036
End Page
1045

Phospholipid-binding protein Cts1 controls septation and functions coordinately with calcineurin in Cryptococcus neoformans.

Cryptococcus neoformans is an opportunistic fungal pathogen that causes life-threatening meningoencephalitis in immunocompromised patients. The Ca(2+)-calmodulin-activated protein phosphatase calcineurin is necessary for virulence of C. neoformans. Mutants lacking the calcineurin catalytic (Cna1) or regulatory (Cnb1) subunit fail to grow at elevated temperature and are defective in virulence and hyphal elongation. Here we isolated a multicopy suppressor gene, CTS1, which restores growth of a calcineurin mutant strain at 37 degrees C. The CTS1 gene (for calcineurin temperature suppressor 1) encodes a protein containing a C2 domain and a leucine zipper motif that may function as an effector of calcineurin. The CTS1 gene was disrupted by homologous recombination, and cts1 mutants were viable but exhibited defects in cell separation, growth, mating, and haploid fruiting. In addition, cts1 mutants were inviable when calcineurin was mutated or inhibited. Taken together, these findings suggest that calcineurin and Cts1 function in parallel pathways that regulate growth, cell separation, and hyphal elongation.

Authors
Fox, DS; Cox, GM; Heitman, J
MLA Citation
Fox, DS, Cox, GM, and Heitman, J. "Phospholipid-binding protein Cts1 controls septation and functions coordinately with calcineurin in Cryptococcus neoformans." Eukaryot Cell 2.5 (October 2003): 1025-1035.
PMID
14555485
Source
pubmed
Published In
Eukaryotic cell
Volume
2
Issue
5
Publish Date
2003
Start Page
1025
End Page
1035

Disruption of ergosterol biosynthesis confers resistance to amphotericin B in Candida lusitaniae.

Candida lusitaniae is an emerging human pathogen that, unlike other fungal pathogens, frequently develops resistance to the commonly used antifungal agent amphotericin B. Amphotericin B is a member of the polyene class of antifungal drugs, which impair fungal cell membrane integrity. Here we analyzed mechanisms contributing to amphotericin B resistance in C. lusitaniae. Sensitivity to polyenes in the related fungi Saccharomyces cerevisiae and Candida albicans requires the ergosterol biosynthetic gene ERG6. In an effort to understand the mechanisms contributing to amphotericin B resistance in C. lusitaniae, we isolated the ERG6 gene and created a C. lusitaniae erg6 delta strain. This mutant strain exhibited a growth defect, was resistant to amphotericin B, and was hypersensitive to other sterol inhibitors. Based on the similarities between the phenotypes of the erg6 delta mutant and clinical isolates of C. lusitaniae resistant to amphotericin B, we analyzed ERG6 expression levels and ergosterol content in multiple clinical isolates. C. lusitaniae amphotericin B-resistant isolates were found to have increased levels of ERG6 transcript as well as reduced ergosterol content. These changes suggest that another gene in the ergosterol biosynthetic pathway could be mutated or misregulated. Further transcript analysis showed that expression of the ERG3 gene, which encodes C-5 sterol desaturase, was reduced in two amphotericin B-resistant isolates. Our findings reveal that mutation or altered expression of ergosterol biosynthetic genes can result in resistance to amphotericin B in C. lusitaniae.

Authors
Young, LY; Hull, CM; Heitman, J
MLA Citation
Young, LY, Hull, CM, and Heitman, J. "Disruption of ergosterol biosynthesis confers resistance to amphotericin B in Candida lusitaniae." Antimicrob Agents Chemother 47.9 (September 2003): 2717-2724.
PMID
12936965
Source
pubmed
Published In
Antimicrobial agents and chemotherapy
Volume
47
Issue
9
Publish Date
2003
Start Page
2717
End Page
2724

Sexual cycle of Cryptococcus neoformans var. grubii and virulence of congenic a and alpha isolates.

Cryptococcus neoformans is a human-pathogenic fungus that has evolved into three distinct varieties that infect most prominently the central nervous system. A sexual cycle involving haploid cells of a and alpha mating types has been reported for two varieties (C. neoformans var. neoformans, serotype D, and C. neoformans var. gattii, serotypes B and C), yet the vast majority of infections involve a distinct variety (C. neoformans var. grubii, serotype A) that has been thought to be clonal and restricted to the alpha mating type. We recently identified the first serotype A isolate of the a mating type which had been thought to be extinct (strain 125.91). Here we report that this unusual strain can mate with a subset of pathogenic serotype A strains to produce a filamentous dikaryon with fused clamp connections, basidia, and viable recombinant basidiospores. One meiotic segregant mated poorly with the serotype A reference strain H99 but robustly with a crg1 mutant that lacks a regulator of G protein signaling and is hyperresponsive to mating pheromone. This meiotic segregant was used to create congenic a and alpha mating type serotype A strains. Virulence tests with rabbit and murine models of cryptococcal meningitis showed that the serotype A congenic a and alpha mating type strains had equivalent virulence in animal models, in contrast to previous studies linking the alpha mating type to increased virulence in congenic serotype D strains. Our studies highlight a role for sexual recombination in the evolution of a human fungal pathogen and provide a robust genetic platform to establish the molecular determinants of virulence.

Authors
Nielsen, K; Cox, GM; Wang, P; Toffaletti, DL; Perfect, JR; Heitman, J
MLA Citation
Nielsen, K, Cox, GM, Wang, P, Toffaletti, DL, Perfect, JR, and Heitman, J. "Sexual cycle of Cryptococcus neoformans var. grubii and virulence of congenic a and alpha isolates." Infect Immun 71.9 (September 2003): 4831-4841.
PMID
12933823
Source
pubmed
Published In
Infection and immunity
Volume
71
Issue
9
Publish Date
2003
Start Page
4831
End Page
4841

A new class of heterotrimeric G protein governs yeast pseudohyphal differentiation.

Authors
Harashima, T; Heitman, J
MLA Citation
Harashima, T, and Heitman, J. "A new class of heterotrimeric G protein governs yeast pseudohyphal differentiation." July 2003.
Source
wos-lite
Published In
Yeast
Volume
20
Publish Date
2003
Start Page
S77
End Page
S77

Cyclophilin A is nuclear localized and controls the sporulation gene program of Saccharomyces cerevisiae.

Authors
Arevalo-Rodriguez, M; Heitman, J
MLA Citation
Arevalo-Rodriguez, M, and Heitman, J. "Cyclophilin A is nuclear localized and controls the sporulation gene program of Saccharomyces cerevisiae." July 2003.
Source
wos-lite
Published In
Yeast
Volume
20
Publish Date
2003
Start Page
S171
End Page
S171

Saccharomyces cerevisiae as a model of human fungal pathogens.

Authors
Heitman, J
MLA Citation
Heitman, J. "Saccharomyces cerevisiae as a model of human fungal pathogens." July 2003.
Source
wos-lite
Published In
Yeast
Volume
20
Publish Date
2003
Start Page
S17
End Page
S17

A MAP kinase cascade composed of cell type specific and non-specific elements controls mating and differentiation of the fungal pathogen Cryptococcus neoformans.

Cryptococcus neoformans is an opportunistic fungal pathogen with a defined sexual cycle in which the alpha allele of the mating type locus is linked to virulence and haploid differentiation. Here we analysed a conserved MAP kinase cascade composed of mating-type specific (Ste11alpha, Ste12alpha) and non-specific (Ste7, Cpk1) elements. Gene disruption experiments demonstrate that this specialized MAP kinase pathway is required for both mating and cell type-specific differentiation but not for virulence. The Ste11alpha, Ste7 and Cpk1 kinases were found to act as a co-ordinate signalling module, whereas the Ste12alpha transcription factor functions with a redundant partner or in a branched or parallel signalling pathway. Our studies illustrate how MAP kinase cascades can be constructed from cell type-specific and non-specific components, yielding pathways that contribute to cell type-specific patterns of signalling and differentiation.

Authors
Davidson, RC; Nichols, CB; Cox, GM; Perfect, JR; Heitman, J
MLA Citation
Davidson, RC, Nichols, CB, Cox, GM, Perfect, JR, and Heitman, J. "A MAP kinase cascade composed of cell type specific and non-specific elements controls mating and differentiation of the fungal pathogen Cryptococcus neoformans." Mol Microbiol 49.2 (July 2003): 469-485.
PMID
12828643
Source
pubmed
Published In
Molecular Microbiology
Volume
49
Issue
2
Publish Date
2003
Start Page
469
End Page
485

Calcineurin is essential for Candida albicans survival in serum and virulence.

Calcineurin is a calcium-activated protein phosphatase that is the target of the immunosuppressants cyclosporin A and FK506. In T cells, calcineurin controls nuclear import of the NF-AT transcription factor and gene activation. In plants and fungi, calcineurin functions in stress responses (e.g., temperature, cations, and pH) and is necessary for the virulence of the fungal pathogen Cryptococcus neoformans. Here we show that calcineurin is also required for the virulence of another major fungus that is pathogenic to humans, Candida albicans. C. albicans calcineurin mutants had significantly reduced virulence in a murine model of systemic infection. In contrast to its role in C. neoformans, calcineurin was not required for C. albicans survival at 37 degrees C. Moreover, C. albicans calcineurin mutant strains exhibited no defects in known Candida virulence traits associated with host invasion, including filamentous growth, germ tube formation, and adherence to and injury of mammalian cells. C. albicans calcineurin mutant strains failed to colonize and grow in the kidneys of infected animals and were unable to survive when exposed to serum in vitro. Our studies illustrate that calcineurin has evolved to control aspects of the virulence of two divergent fungal pathogens via distinct mechanisms that can be targeted to achieve broad-spectrum antifungal action.

Authors
Blankenship, JR; Wormley, FL; Boyce, MK; Schell, WA; Filler, SG; Perfect, JR; Heitman, J
MLA Citation
Blankenship, JR, Wormley, FL, Boyce, MK, Schell, WA, Filler, SG, Perfect, JR, and Heitman, J. "Calcineurin is essential for Candida albicans survival in serum and virulence." Eukaryot Cell 2.3 (June 2003): 422-430.
PMID
12796287
Source
pubmed
Published In
Eukaryotic cell
Volume
2
Issue
3
Publish Date
2003
Start Page
422
End Page
430

The Cryptococcus neoformans MAP kinase Mpk1 regulates cell integrity in response to antifungal drugs and loss of calcineurin function.

Cell wall integrity is crucial for fungal growth, development and stress survival. In the model yeast Saccharomyces cerevisiae, the cell integrity Mpk1/Slt2 MAP kinase and calcineurin pathways monitor cell wall integrity and promote cell wall remodelling under stress conditions. We have identified the Cryptococcus neoformans homologue of the S. cerevisiae Mpk1/Slt2 MAP kinase and have characterized its role in the maintenance of cell integrity in response to elevated growth temperature and in the presence of cell wall synthesis inhibitors. C. neoformans Mpk1 is required for growth at 37 degrees C in vitro, and this growth defect is suppressed by osmotic stabilization. C. neoformans mutants lacking Mpk1 are attenuated for virulence in the mouse model of cryptococcosis. Phosphorylation of Mpk1 is induced in response to perturbations of cell wall biosynthesis by the antifungal drugs nikkomycin Z (a chitin synthase inhibitor), caspofungin (a beta-1,3-glucan synthase inhibitor), or FK506 (a calcineurin inhibitor), and mutants lacking Mpk1 display enhanced sensitivity to nikkomycin Z and caspofungin. Lastly, we show that calcineurin and Mpk1 play complementing roles in regulating cell integrity in C. neoformans. Our studies demonstrate that pharmacological inhibition of the cell integrity pathway would enhance the activity of antifungal drugs that target the cell wall.

Authors
Kraus, PR; Fox, DS; Cox, GM; Heitman, J
MLA Citation
Kraus, PR, Fox, DS, Cox, GM, and Heitman, J. "The Cryptococcus neoformans MAP kinase Mpk1 regulates cell integrity in response to antifungal drugs and loss of calcineurin function." Mol Microbiol 48.5 (June 2003): 1377-1387.
PMID
12787363
Source
pubmed
Published In
Molecular Microbiology
Volume
48
Issue
5
Publish Date
2003
Start Page
1377
End Page
1387

Ergosterol biosynthesis inhibitors become fungicidal when combined with calcineurin inhibitors against Candida albicans, Candida glabrata, and Candida krusei.

Azoles target the ergosterol biosynthetic enzyme lanosterol 14alpha-demethylase and are a widely applied class of antifungal agents because of their broad therapeutic window, wide spectrum of activity, and low toxicity. Unfortunately, azoles are generally fungistatic and resistance to fluconazole is emerging in several fungal pathogens. We recently established that the protein phosphatase calcineurin allows survival of Candida albicans during the membrane stress exerted by azoles. The calcineurin inhibitors cyclosporine A (CsA) and tacrolimus (FK506) are dramatically synergistic with azoles, resulting in potent fungicidal activity, and mutant strains lacking calcineurin are markedly hypersensitive to azoles. Here we establish that drugs targeting other enzymes in the ergosterol biosynthetic pathway (terbinafine and fenpropimorph) also exhibit dramatic synergistic antifungal activity against wild-type C. albicans when used in conjunction with CsA and FK506. Similarly, C. albicans mutant strains lacking calcineurin B are markedly hypersensitive to terbinafine and fenpropimorph. The FK506 binding protein FKBP12 is required for FK506 synergism with ergosterol biosynthesis inhibitors, and a calcineurin mutation that confers FK506 resistance abolishes drug synergism. Additionally, we provide evidence of drug synergy between the nonimmunosuppressive FK506 analog L-685,818 and fenpropimorph or terbinafine against wild-type C. albicans. These drug combinations also exert synergistic effects against two other Candida species, C. glabrata and C. krusei, which are known for intrinsic or rapidly acquired resistance to azoles. These studies demonstrate that the activity of non-azole antifungal agents that target ergosterol biosynthesis can be enhanced by inhibition of the calcineurin signaling pathway, extending their spectrum of action and providing an alternative approach by which to overcome antifungal drug resistance.

Authors
Onyewu, C; Blankenship, JR; Del Poeta, M; Heitman, J
MLA Citation
Onyewu, C, Blankenship, JR, Del Poeta, M, and Heitman, J. "Ergosterol biosynthesis inhibitors become fungicidal when combined with calcineurin inhibitors against Candida albicans, Candida glabrata, and Candida krusei." Antimicrob Agents Chemother 47.3 (March 2003): 956-964.
PMID
12604527
Source
pubmed
Published In
Antimicrobial agents and chemotherapy
Volume
47
Issue
3
Publish Date
2003
Start Page
956
End Page
964

Ras1 controls pheromone expression and response during mating in Cryptococcus neoformans.

The Cryptococcus neoformans Ras1 signal transduction pathway controls mating, hyphal differentiation, and the ability of this opportunistic human fungal pathogen to grow at elevated temperatures. To further elucidate how Ras1 signals in this organism, the RAS1 gene was disrupted in the congenic serotype D strain background. Genetic epistasis experiments indicated that Ras1 regulates the mating response through the MAP kinase/pheromone response pathway. In fact, Ras1 is required for the transcriptional induction of elements of the pheromone response pathway. However, the ability of C. neoformans Ras1 to allow growth at 37 degrees C is mediated by a separate signaling pathway. Therefore a single Ras protein may differentially activate distinct downstream targets in response to different signals within the same organism. This conserved signaling motif has been coopted in C. neoformans to regulate mating and morphogenesis in addition to being required for its pathogenic potential.

Authors
Waugh, MS; Vallim, MA; Heitman, J; Alspaugh, JA
MLA Citation
Waugh, MS, Vallim, MA, Heitman, J, and Alspaugh, JA. "Ras1 controls pheromone expression and response during mating in Cryptococcus neoformans." Fungal Genet Biol 38.1 (February 2003): 110-121.
PMID
12553941
Source
pubmed
Published In
Fungal Genetics and Biology
Volume
38
Issue
1
Publish Date
2003
Start Page
110
End Page
121

Teaching old drugs new tricks: reincarnating immunosuppressants as antifungal drugs.

Invasive fungal infections are rising worldwide as the number of immunocompromised patients increases. Unfortunately, our armamentarium of antifungal drugs is limited. Although current therapies are effective in treating some of the most prevalent infections, the development of novel treatments is vital because of emerging drug-resistant strains and species and because of the toxicity of certain current therapies. The immunosuppressive drugs CsA (cyclosporin A), FK-506 (tacrolimus) and rapamycin (sirolimus) exert potent antifungal effects against a variety of pathogenic fungi. These compounds are all currently in clinical use as immunosuppressive therapy to treat and prevent rejection of transplanted organs. Rapamycin is also in clinical trials as an antiproliferative agent for chemotherapy and invasive cardiology. Recent studies reveal a potent fungicidal synergism between azoles and the calcineurin inhibitors CsA and FK-506, and animal studies demonstrate that the CsA-fluconazole synergistic combination has therapeutic benefit. Less immunosuppressive analogs have been identified with potential to enhance current therapies, or as monotherapy without deleterious effects on the immune system. In summary, these highly successful pharmaceutical agents may find an even broader clinical application in combating infectious diseases.

Authors
Blankenship, JR; Steinbach, WJ; Perfect, JR; Heitman, J
MLA Citation
Blankenship, JR, Steinbach, WJ, Perfect, JR, and Heitman, J. "Teaching old drugs new tricks: reincarnating immunosuppressants as antifungal drugs." Curr Opin Investig Drugs 4.2 (February 2003): 192-199. (Review)
PMID
12669381
Source
pubmed
Published In
Current Opinion in Investigational Drugs
Volume
4
Issue
2
Publish Date
2003
Start Page
192
End Page
199

A nomenclature for restriction enzymes, DNA methyltransferases, homing endonucleases and their genes

A nomenclature is described for restriction endonucleases, DNA methyltransferases, homing endonucleases and related genes and gene products. It provides explicit categories for the many different Type II enzymes now identified and provides a system for naming the putative genes found by sequence analysis of microbial genomes.

Authors
Roberts, RJ; Belfort, M; Bestor, T; Bhagwat, AS; Bickle, TA; Bitinaite, J; Blumenthal, RM; Degtyarev, SK; Dryden, DTF; Dybvig, K; Firman, K; Gromova, ES; Gumport, RI; Halford, SE; Hattman, S; Heitman, J; Hornby, DP; Janulaitis, A; Jeltsch, A; Josephsen, J; Kiss, A; Klaenhammer, TR; Kobayashi, I; Kong, H; Krüger, DH; Lacks, S; Marinus, MG; Miyahara, M; Morgan, RD; Murray, NE; Nagaraja, V; Piekarowicz, A; Pingoud, A; Raleigh, E; Rao, DN; Reich, N; Repin, VE; Selker, EU; Shaw, P-C; Stein, DC et al.
MLA Citation
Roberts, RJ, Belfort, M, Bestor, T, Bhagwat, AS, Bickle, TA, Bitinaite, J, Blumenthal, RM, Degtyarev, SK, Dryden, DTF, Dybvig, K, Firman, K, Gromova, ES, Gumport, RI, Halford, SE, Hattman, S, Heitman, J, Hornby, DP, Janulaitis, A, Jeltsch, A, Josephsen, J, Kiss, A, Klaenhammer, TR, Kobayashi, I, Kong, H, Krüger, DH, Lacks, S, Marinus, MG, Miyahara, M, Morgan, RD, Murray, NE, Nagaraja, V, Piekarowicz, A, Pingoud, A, Raleigh, E, Rao, DN, Reich, N, Repin, VE, Selker, EU, Shaw, P-C, and Stein, DC et al. "A nomenclature for restriction enzymes, DNA methyltransferases, homing endonucleases and their genes." Nucleic Acids Research 31.7 (2003): 1805-1812.
PMID
12654995
Source
scival
Published In
Nucleic Acids Research
Volume
31
Issue
7
Publish Date
2003
Start Page
1805
End Page
1812
DOI
10.1093/nar/gkg274

Cell identity and sexual development in Cryptococcus neoformans are controlled by the mating-type-specific homeodomain protein Sxi1alpha.

Virulence in the human fungal pathogen Cryptococcus neoformans is associated with the alpha mating type. Studies to identify the properties of alpha cells that enhance pathogenesis have led to the identification of a mating-type locus of unusually large size and distinct architecture. Here, we demonstrate that the previously identified MATalpha components are insufficient to regulate sexual differentiation, and we identify a novel alpha-specific regulator, SXI1alpha. Our data show that SXI1alpha establishes alpha cell identity and controls progression through the sexual cycle, and we discover that ectopic expression of SXI1alpha in a cells is sufficient to drive a/alpha sexual development. SXI1alpha is the first example of a key regulator of cell identity and sexual differentiation in C. neoformans, and its identification and characterization lead to a new model of how cell fate and the sexual cycle are controlled in C. neoformans.

Authors
Hull, CM; Davidson, RC; Heitman, J
MLA Citation
Hull, CM, Davidson, RC, and Heitman, J. "Cell identity and sexual development in Cryptococcus neoformans are controlled by the mating-type-specific homeodomain protein Sxi1alpha." Genes Dev 16.23 (December 1, 2002): 3046-3060.
PMID
12464634
Source
pubmed
Published In
Genes & development
Volume
16
Issue
23
Publish Date
2002
Start Page
3046
End Page
3060
DOI
10.1101/gad.1041402

Killing of Caenorhabditis elegans by Cryptococcus neoformans as a model of yeast pathogenesis.

We found that the well-studied nematode Caenorhabditis elegans can use various yeasts, including Cryptococcus laurentii and Cryptococcus kuetzingii, as a sole source of food, producing similar brood sizes compared with growth on its usual laboratory food source Escherichia coli OP50. C. elegans grown on these yeasts had a life span similar to (C. laurentii) or longer than (C. kuetzingii) those fed on E. coli. However, the human pathogenic yeast Cryptococcus neoformans killed C. elegans, and the C. neoformans polysaccharide capsule as well as several C. neoformans genes previously shown to be involved in mammalian virulence were also shown to play a role in C. elegans killing. These included genes associated with signal transduction pathways (GPA1, PKA1, PKR1, and RAS1), laccase production (LAC1), and the alpha mating type. C. neoformans adenine auxotrophs, which are less virulent in mammals, were also less virulent in C. elegans. These results support the model that mammalian pathogenesis of C. neoformans may be a consequence of adaptations that have evolved during the interaction of C. neoformans with environmental predators such as free-living nematodes and amoebae and suggest that C. elegans can be used as a simple model host in which C. neoformans pathogenesis can be readily studied.

Authors
Mylonakis, E; Ausubel, FM; Perfect, JR; Heitman, J; Calderwood, SB
MLA Citation
Mylonakis, E, Ausubel, FM, Perfect, JR, Heitman, J, and Calderwood, SB. "Killing of Caenorhabditis elegans by Cryptococcus neoformans as a model of yeast pathogenesis." Proc Natl Acad Sci U S A 99.24 (November 26, 2002): 15675-15680.
PMID
12438649
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
99
Issue
24
Publish Date
2002
Start Page
15675
End Page
15680
DOI
10.1073/pnas.232568599

Fungal mating: Candida albicans flips a switch to get in the mood.

The fungal pathogen Candida albicans can mate under highly controlled conditions. It can also undergo phenotypic switching. A recent discovery joins these disparate processes to reveal that 'opaque' switch variants mate 10(6) times better than 'white' variants.

Authors
Hull, CM; Heitman, J
MLA Citation
Hull, CM, and Heitman, J. "Fungal mating: Candida albicans flips a switch to get in the mood." Curr Biol 12.22 (November 19, 2002): R782-R784. (Review)
PMID
12445405
Source
pubmed
Published In
Current Biology
Volume
12
Issue
22
Publish Date
2002
Start Page
R782
End Page
R784

The homeodomain protein Sxi1 alpha is a key regulator of cell identity and sexual differentiation in the pathogenic fungus Cryptococcus neoformans

Authors
Hull, CM; Davidson, RC; Heitman, J
MLA Citation
Hull, CM, Davidson, RC, and Heitman, J. "The homeodomain protein Sxi1 alpha is a key regulator of cell identity and sexual differentiation in the pathogenic fungus Cryptococcus neoformans." November 2002.
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
13
Publish Date
2002
Start Page
521A
End Page
521A

Good fungi gone bad: the corruption of calcineurin.

Calcineurin is a Ca(2+)/calmodulin-activated protein phosphatase that is conserved in eukaryotes, from yeast to humans, and is the conserved target of the immunosuppressive drugs cyclosporin A (CsA) and FK506. Genetic studies in yeast and fungi established the molecular basis of calcineurin inhibition by the cyclophilin A-CsA and FKBP12-FK506 complexes. Calcineurin also functions in fungi to control a myriad of physiological processes including cell cycle progression, cation homeostasis, and morphogenesis. Recent investigations into the molecular mechanisms of pathogenesis in Candida albicans and Cryptococcus neoformans, two fungi that cause life-threatening infections in humans, have revealed an essential role for calcineurin in morphogenesis, virulence, and antifungal drug action. Novel non-immunosuppressive analogs of the calcineurin inhibitors CsA and FK506 that retain antifungal activity have been identified and hold promise as candidate antifungal drugs. In addition, comparisons of calcineurin function in both fungi and humans may identify fungal-specific components of calcineurin-signaling pathways that could be targeted for therapy, as well as conserved elements of calcium signaling events.

Authors
Fox, DS; Heitman, J
MLA Citation
Fox, DS, and Heitman, J. "Good fungi gone bad: the corruption of calcineurin." Bioessays 24.10 (October 2002): 894-903. (Review)
PMID
12325122
Source
pubmed
Published In
Bioessays
Volume
24
Issue
10
Publish Date
2002
Start Page
894
End Page
903
DOI
10.1002/bies.10157

Mating-type locus of Cryptococcus neoformans: a step in the evolution of sex chromosomes.

The sexual development and virulence of the fungal pathogen Cryptococcus neoformans is controlled by a bipolar mating system determined by a single locus that exists in two alleles, alpha and a. The alpha and a mating-type alleles from two divergent varieties were cloned and sequenced. The C. neoformans mating-type locus is unique, spans >100 kb, and contains more than 20 genes. MAT-encoded products include homologs of regulators of sexual development in other fungi, pheromone and pheromone receptors, divergent components of a MAP kinase cascade, and other proteins with no obvious function in mating. The alpha and a alleles of the mating-type locus have extensively rearranged during evolution and strain divergence but are stable during genetic crosses and in the population. The C. neoformans mating-type locus is strikingly different from the other known fungal mating-type loci, sharing features with the self-incompatibility systems and sex chromosomes of algae, plants, and animals. Our study establishes a new paradigm for mating-type loci in fungi with implications for the evolution of cell identity and self/nonself recognition.

Authors
Lengeler, KB; Fox, DS; Fraser, JA; Allen, A; Forrester, K; Dietrich, FS; Heitman, J
MLA Citation
Lengeler, KB, Fox, DS, Fraser, JA, Allen, A, Forrester, K, Dietrich, FS, and Heitman, J. "Mating-type locus of Cryptococcus neoformans: a step in the evolution of sex chromosomes." Eukaryot Cell 1.5 (October 2002): 704-718.
PMID
12455690
Source
pubmed
Published In
Eukaryotic cell
Volume
1
Issue
5
Publish Date
2002
Start Page
704
End Page
718

Physical maps for genome analysis of serotype A and D strains of the fungal pathogen Cryptococcus neoformans.

The basidiomycete fungus Cryptococcus neoformans is an important opportunistic pathogen of humans that poses a significant threat to immunocompromised individuals. Isolates of C. neoformans are classified into serotypes (A, B, C, D, and AD) based on antigenic differences in the polysaccharide capsule that surrounds the fungal cells. Genomic and EST sequencing projects are underway for the serotype D strain JEC21 and the serotype A strain H99. As part of a genomics program for C. neoformans, we have constructed fingerprinted bacterial artificial chromosome (BAC) clone physical maps for strains H99 and JEC21 to support the genomic sequencing efforts and to provide an initial comparison of the two genomes. The BAC clones represented an estimated 10-fold redundant coverage of the genomes of each serotype and allowed the assembly of 20 contigs each for H99 and JEC21. We found that the genomes of the two strains are sufficiently distinct to prevent coassembly of the two maps when combined fingerprint data are used to construct contigs. Hybridization experiments placed 82 markers on the JEC21 map and 102 markers on the H99 map, enabling contigs to be linked with specific chromosomes identified by electrophoretic karyotyping. These markers revealed both extensive similarity in gene order (conservation of synteny) between JEC21 and H99 as well as examples of chromosomal rearrangements including inversions and translocations. Sequencing reads were generated from the ends of the BAC clones to allow correlation of genomic shotgun sequence data with physical map contigs. The BAC maps therefore represent a valuable resource for the generation, assembly, and finishing of the genomic sequence of both JEC21 and H99. The physical maps also serve as a link between map-based and sequence-based data, providing a powerful resource for continued genomic studies

Authors
Schein, JE; Tangen, KL; Chiu, R; Shin, H; Lengeler, KB; MacDonald, WK; Bosdet, I; Heitman, J; Jones, SJM; Marra, MA; Kronstad, JW
MLA Citation
Schein, JE, Tangen, KL, Chiu, R, Shin, H, Lengeler, KB, MacDonald, WK, Bosdet, I, Heitman, J, Jones, SJM, Marra, MA, and Kronstad, JW. "Physical maps for genome analysis of serotype A and D strains of the fungal pathogen Cryptococcus neoformans." Genome Res 12.9 (September 2002): 1445-1453.
PMID
12213782
Source
pubmed
Published In
Genome research
Volume
12
Issue
9
Publish Date
2002
Start Page
1445
End Page
1453
DOI
10.1101/gr.81002

A PCR-based strategy to generate integrative targeting alleles with large regions of homology.

Cryptococcus neoformans is an opportunistic fungal pathogen with a defined sexual cycle for which genetic and molecular techniques are well developed. The entire genome sequence of one C. neoformans strain is nearing completion. The efficient use of this sequence is dependent upon the development of methods to perform more rapid genetic analysis including gene-disruption techniques. A modified PCR overlap technique to generate targeting constructs for gene disruption that contain large regions of gene homology is described. This technique was used to disrupt or delete more than a dozen genes with efficiencies comparable to those previously reported using cloning technology to generate targeting constructs. Moreover, it is shown that disruptions can be made using this technique in a variety of strain backgrounds, including the pathogenic serotype A isolate H99 and recently characterized stable diploid strains. In combination with the availability of the complete genomic sequence, this gene-disruption technique should pave the way for higher throughput genetic analysis of this important pathogenic fungus.

Authors
Davidson, RC; Blankenship, JR; Kraus, PR; de Jesus Berrios, M; Hull, CM; D'Souza, C; Wang, P; Heitman, J
MLA Citation
Davidson, RC, Blankenship, JR, Kraus, PR, de Jesus Berrios, M, Hull, CM, D'Souza, C, Wang, P, and Heitman, J. "A PCR-based strategy to generate integrative targeting alleles with large regions of homology." Microbiology 148.Pt 8 (August 2002): 2607-2615.
PMID
12177355
Source
pubmed
Published In
Microbiology (Reading, England)
Volume
148
Issue
Pt 8
Publish Date
2002
Start Page
2607
End Page
2615
DOI
10.1099/00221287-148-8-2607

The Galpha protein Gpa2 controls yeast differentiation by interacting with kelch repeat proteins that mimic Gbeta subunits.

G protein coupled receptors (GPCR) sense diverse ligands and signal via heterotrimeric G proteins. The Saccharomyces cerevisiae GPCR Gpr1 senses glucose and controls filamentous growth via an unusual Galpha protein, Gpa2, which lacks any known Gbetagamma subunits. Our genetic and biochemical studies identify Gpa2 interaction partners (Gpb1/2, Gpg1) and provide evidence that these proteins function as G protein subunit mimics and signaling effectors. Gpb1 and Gpb2 lack the seven WD-40 repeats found in Gbeta subunits and instead contain seven kelch repeats implicated in protein-protein interactions. Gbeta subunits and the kelch repeat protein galactose oxidase fold into strikingly similar seven-bladed beta propellers. Our studies demonstrate that Gpa2 signals in conjunction with Gbeta structural mimics and that homologous G protein subunits or effectors may be conserved in multicellular eukaryotes.

Authors
Harashima, T; Heitman, J
MLA Citation
Harashima, T, and Heitman, J. "The Galpha protein Gpa2 controls yeast differentiation by interacting with kelch repeat proteins that mimic Gbeta subunits." Mol Cell 10.1 (July 2002): 163-173.
PMID
12150916
Source
pubmed
Published In
Molecular Cell
Volume
10
Issue
1
Publish Date
2002
Start Page
163
End Page
173

FKBP12 is the only FK506 binding protein mediating T-cell inhibition by the immunosuppressant FK506.

BACKGROUND: FK506-binding proteins (FKBP) are immunophilins that interact with the immunosuppressive drugs FK506 and rapamycin. Several FKBP family members such as FKBP12, FKBP12.6, and FKBP51 are expressed in T cells. It has been speculated that these FKBPs are possibly redundant in the immunosuppressant-induced T-cell inactivation. To determine the pharmacological relevance of multiple FKBP members in the immunosuppressant-induced T-cell inactivation, we have investigated the physiological responses of FKBP12-deficient and FKBP12.6-deficient mutant T cells to the immunosuppressive agent FK506. METHODS: FKBP12-deficient and FKBP12.6-deficient T cells were isolated from genetically engineered FKBP12-deficient and FKBP12.6-deficient mice, respectively. T-cell growth inhibitory assay was used to assess their responses to immunosuppressant FK506 treatments. RESULTS: We found that growth inhibition induced by FK506 is abolished in FKBP12-deficient cells but not in FKBP12.6-deficient cells. CONCLUSIONS: FKBP12 is the only FKBP family member that plays a key role in immunosuppressant-mediated immunosuppression.

Authors
Xu, X; Su, B; Barndt, RJ; Chen, H; Xin, H; Yan, G; Chen, L; Cheng, D; Heitman, J; Zhuang, Y; Fleischer, S; Shou, W
MLA Citation
Xu, X, Su, B, Barndt, RJ, Chen, H, Xin, H, Yan, G, Chen, L, Cheng, D, Heitman, J, Zhuang, Y, Fleischer, S, and Shou, W. "FKBP12 is the only FK506 binding protein mediating T-cell inhibition by the immunosuppressant FK506." Transplantation 73.11 (June 15, 2002): 1835-1838.
PMID
12085010
Source
pubmed
Published In
Transplantation
Volume
73
Issue
11
Publish Date
2002
Start Page
1835
End Page
1838

Protein kinase A operates a molecular switch that governs yeast pseudohyphal differentiation.

The yeast Saccharomyces cerevisiae undergoes a dimorphic filamentous transition in response to nutrient cues that is affected by both mitogen-activated protein kinase and cyclic AMP-protein kinase A signaling cascades. Here two transcriptional regulators, Flo8 and Sfl1, are shown to be the direct molecular targets of protein kinase A. Flo8 and Sfl1 antagonistically control expression of the cell adhesin Flo11 via a common promoter element. Phosphorylation by the protein kinase A catalytic subunit Tpk2 promotes Flo8 binding and activation of the Flo11 promoter and relieves repression by prohibiting dimerization and DNA binding by Sfl1. Our studies illustrate in molecular detail how protein kinase A combinatorially effects a key developmental switch. Similar mechanisms may operate in pathogenic fungi and more complex multicellular eukaryotic organisms.

Authors
Pan, X; Heitman, J
MLA Citation
Pan, X, and Heitman, J. "Protein kinase A operates a molecular switch that governs yeast pseudohyphal differentiation." Mol Cell Biol 22.12 (June 2002): 3981-3993.
PMID
12024012
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
22
Issue
12
Publish Date
2002
Start Page
3981
End Page
3993

Pheromones stimulate mating and differentiation via paracrine and autocrine signaling in Cryptococcus neoformans.

Cryptococcus neoformans is a pathogenic fungus with a defined sexual cycle involving haploid MATalpha and MATa cells. Interestingly, MATalpha strains are more common, are more virulent than congenic MATa strains, and undergo haploid fruiting in response to nitrogen limitation or MATa cells. Three genes encoding the MFalpha pheromone were identified in the MATalpha mating-type locus and shown to be transcriptionally induced by limiting nutrients and coculture with MATa cells. The MFalpha1, MFalpha2, and MFalpha3 genes were mutated, individually and in combination. MATalpha strains lacking MFalpha pheromone failed to induce morphological changes in MATa cells. Pheromoneless MATalpha mutants were fusion and mating impaired but not sterile and mated at approximately 1% the wild-type level. The pheromoneless MATalpha mutants were also partially defective in haploid fruiting, and overexpression of MFalpha pheromone enhanced haploid fruiting. Overexpression of MFa pheromone also enhanced haploid fruiting of MATalpha cells and stimulated conjugation tube formation in MATa cells. A conserved G-protein activated mitogen-activated protein kinase signaling pathway was found to be required for both induction and response to mating pheromones. The MFalpha pheromone was not essential for virulence of C. neoformans but does contribute to the overall virulence composite. These studies define paracrine and autocrine pheromone response pathways that signal mating and differentiation of this pathogenic fungus.

Authors
Shen, W-C; Davidson, RC; Cox, GM; Heitman, J
MLA Citation
Shen, W-C, Davidson, RC, Cox, GM, and Heitman, J. "Pheromones stimulate mating and differentiation via paracrine and autocrine signaling in Cryptococcus neoformans." Eukaryot Cell 1.3 (June 2002): 366-377.
PMID
12455985
Source
pubmed
Published In
Eukaryotic cell
Volume
1
Issue
3
Publish Date
2002
Start Page
366
End Page
377

Mating-type-specific and nonspecific PAK kinases play shared and divergent roles in Cryptococcus neoformans.

Cryptococcus neoformans is an opportunistic fungal pathogen with a defined sexual cycle involving fusion of haploid MATalpha and MATa cells. Virulence has been linked to the mating type, and MATalpha cells are more virulent than congenic MATa cells. To study the link between the mating type and virulence, we functionally analyzed three genes encoding homologs of the p21-activated protein kinase family: STE20alpha, STE20a, and PAK1. In contrast to the STE20 genes that were previously shown to be in the mating-type locus, the PAK1 gene is unlinked to the mating type. The STE20alpha, STE20a, and PAK1 genes were disrupted in serotype A and D strains of C. neoformans, revealing central but distinct roles in mating, differentiation, cytokinesis, and virulence. ste20alpha pak1 and ste20a pak1 double mutants were synthetically lethal, indicating that these related kinases share an essential function. In summary, our studies identify an association between the STE20alpha gene, the MATalpha locus, and virulence in a serotype A clinical isolate and provide evidence that PAK kinases function in a MAP kinase signaling cascade controlling the mating, differentiation, and virulence of this fungal pathogen.

Authors
Wang, P; Nichols, CB; Lengeler, KB; Cardenas, ME; Cox, GM; Perfect, JR; Heitman, J
MLA Citation
Wang, P, Nichols, CB, Lengeler, KB, Cardenas, ME, Cox, GM, Perfect, JR, and Heitman, J. "Mating-type-specific and nonspecific PAK kinases play shared and divergent roles in Cryptococcus neoformans." Eukaryot Cell 1.2 (April 2002): 257-272.
PMID
12455960
Source
pubmed
Published In
Eukaryotic cell
Volume
1
Issue
2
Publish Date
2002
Start Page
257
End Page
272

Yeasts as Model-T cells

Authors
Heitman, J
MLA Citation
Heitman, J. "Yeasts as Model-T cells." FASEB JOURNAL 16.5 (March 22, 2002): A891-A891.
Source
wos-lite
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
16
Issue
5
Publish Date
2002
Start Page
A891
End Page
A891

Calcineurin is essential for survival during membrane stress in Candida albicans.

The immunosuppressants cyclosporin A (CsA) and FK506 inhibit the protein phosphatase calcineurin and block T-cell activation and transplant rejection. Calcineurin is conserved in microorganisms and plays a general role in stress survival. CsA and FK506 are toxic to several fungi, but the common human fungal pathogen Candida albicans is resistant. However, combination of either CsA or FK506 with the antifungal drug fluconazole that perturbs synthesis of the membrane lipid ergosterol results in potent, synergistic fungicidal activity. Here we show that the C.albicans FK506 binding protein FKBP12 homolog is required for FK506 synergistic action with fluconazole. A mutation in the calcineurin B regulatory subunit that confers dominant FK506 resistance (CNB1-1/CNB1) abolished FK506-fluconazole synergism. Candida albicans mutants lacking calcineurin B (cnb1/cnb1) were found to be viable and markedly hypersensitive to fluconazole or membrane perturbation with SDS. FK506 was synergistic with fluconazole against azole-resistant C.albicans mutants, against other Candida species, or when combined with different azoles. We propose that calcineurin is part of a membrane stress survival pathway that could be targeted for therapy.

Authors
Cruz, MC; Goldstein, AL; Blankenship, JR; Del Poeta, M; Davis, D; Cardenas, ME; Perfect, JR; McCusker, JH; Heitman, J
MLA Citation
Cruz, MC, Goldstein, AL, Blankenship, JR, Del Poeta, M, Davis, D, Cardenas, ME, Perfect, JR, McCusker, JH, and Heitman, J. "Calcineurin is essential for survival during membrane stress in Candida albicans." EMBO J 21.4 (February 15, 2002): 546-559.
PMID
11847103
Source
pubmed
Published In
EMBO Journal
Volume
21
Issue
4
Publish Date
2002
Start Page
546
End Page
559

Adenylyl cyclase functions downstream of the Galpha protein Gpa1 and controls mating and pathogenicity of Cryptococcus neoformans.

The signaling molecule cyclic AMP (cAMP) is a ubiquitous second messenger that enables cells to detect and respond to extracellular signals. cAMP is generated by the enzyme adenylyl cyclase, which is activated or inhibited by the Galpha subunits of heterotrimeric G proteins in response to ligand-activated G-protein-coupled receptors. Here we identified the unique gene (CAC1) encoding adenylyl cyclase in the opportunistic fungal pathogen Cryptococcus neoformans. The CAC1 gene was disrupted by transformation and homologous recombination. In stark contrast to the situation for Saccharomyces cerevisiae, in which adenylyl cyclase is essential, C. neoformans cac1 mutant strains were viable and had no vegetative growth defect. Furthermore, cac1 mutants maintained the yeast-like morphology of wild-type cells, in contrast to the constitutively filamentous phenotype found upon the loss of adenylyl cyclase in another basidiomycete pathogen, Ustilago maydis. Like C. neoformans mutants lacking the Galpha protein Gpal, cac1 mutants were mating defective and failed to produce two inducible virulence factors: capsule and melanin. As a consequence, cac1 mutant strains were avirulent in animal models of cryptococcal meningitis. Reintroduction of the wild-type CAC1 gene or the addition of exogenous cAMP suppressed cac1 mutant phenotypes. Moreover, the overexpression of adenylyl cyclase restored mating and virulence factor production in gpal mutant strains. Physiological studies revealed that the Galpha protein Gpa1 and adenylyl cyclase controlled cAMP production in response to glucose, and no cAMP was detectable in extracts from cac1 or gpa1 mutant strains. These findings provide direct evidence that Gpal and adenylyl cyclase function in a conserved signal transduction pathway controlling cAMP production, hyphal differentiation, and virulence of this human fungal pathogen.

Authors
Alspaugh, JA; Pukkila-Worley, R; Harashima, T; Cavallo, LM; Funnell, D; Cox, GM; Perfect, JR; Kronstad, JW; Heitman, J
MLA Citation
Alspaugh, JA, Pukkila-Worley, R, Harashima, T, Cavallo, LM, Funnell, D, Cox, GM, Perfect, JR, Kronstad, JW, and Heitman, J. "Adenylyl cyclase functions downstream of the Galpha protein Gpa1 and controls mating and pathogenicity of Cryptococcus neoformans." Eukaryot Cell 1.1 (February 2002): 75-84.
PMID
12455973
Source
pubmed
Published In
Eukaryotic cell
Volume
1
Issue
1
Publish Date
2002
Start Page
75
End Page
84

Ras1 and Ras2 contribute shared and unique roles in physiology and virulence of Cryptococcus neoformans.

The Ras1 signal transduction pathway controls the ability of the pathogenic fungus Cryptococcus neoformans to grow at high temperatures and to mate. A second RAS gene was identified in this organism. RAS2 is expressed at a very low level compared to RAS1, and a ras2 mutation caused no alterations in vegetative growth rate, differentiation or virulence factor expression. The ras2 mutant strain was equally virulent to the wild-type strain in the murine inhalational model of cryptococcosis. Although a ras1 ras2 double mutant strain is viable, mutation of both RAS genes results in a decreased growth rate at all temperatures compared to strains with either single mutation. Overexpression of the RAS2 gene completely suppressed the ras1 mutant mating defect and partially suppressed its high temperature growth defect. After prolonged incubation at a restrictive temperature, the ras1 mutant demonstrated actin polarity defects that were also partially suppressed by RAS2 overexpression. These studies indicate that the C. neoformans Ras1 and Ras2 proteins share overlapping functions, but also play distinct signalling roles. Our findings also suggest a mechanism by which Ras1 controls growth of this pathogenic fungus at 37 degrees C, supporting a conserved role for Ras homologues in microbial cellular differentiation, morphogenesis and virulence.

Authors
Waugh, MS; Nichols, CB; DeCesare, CM; Cox, GM; Heitman, J; Alspaugh, JA
MLA Citation
Waugh, MS, Nichols, CB, DeCesare, CM, Cox, GM, Heitman, J, and Alspaugh, JA. "Ras1 and Ras2 contribute shared and unique roles in physiology and virulence of Cryptococcus neoformans." Microbiology 148.Pt 1 (January 2002): 191-201.
PMID
11782511
Source
pubmed
Published In
Microbiology (Reading, England)
Volume
148
Issue
Pt 1
Publish Date
2002
Start Page
191
End Page
201
DOI
10.1099/00221287-148-1-191

Genetics of Cryptococcus neoformans.

Cryptococcus neoformans is a pathogenic fungus that primarily afflicts immunocompromised patients, infecting the central nervous system to cause meningoencephalitis that is uniformly fatal if untreated. C. neoformans is a basidiomycetous fungus with a defined sexual cycle that has been linked to differentiation and virulence. Recent advances in classical and molecular genetic approaches have allowed molecular descriptions of the pathways that control cell type and virulence. An ongoing genome sequencing project promises to reveal much about the evolution of this human fungal pathogen into three distinct varieties or species. C. neoformans shares features with both model ascomycetous yeasts (Saccharomyces cerevisiae, Schizosaccharomyces pombe) and basidiomycetous pathogens and mushrooms (Ustilago maydis, Coprinus cinereus, Schizophyllum commune), yet ongoing studies reveal unique features associated with virulence and the arrangement of the mating type locus. These advances have catapulted C. neoformans to center stage as a model of both fungal pathogenesis and the interesting approaches to life that the kingdom of fungi has adopted.

Authors
Hull, CM; Heitman, J
MLA Citation
Hull, CM, and Heitman, J. "Genetics of Cryptococcus neoformans." Annu Rev Genet 36 (2002): 557-615. (Review)
PMID
12429703
Source
pubmed
Published In
Annual Review of Genetics
Volume
36
Publish Date
2002
Start Page
557
End Page
615
DOI
10.1146/annurev.genet.36.052402.152652

A PCR-based strategy to generate integrative targeting alleles with large regions of homology

Cryptococcus neoformans is an opportunistic fungal pathogen with a defined sexual cycle for which genetic and molecular techniques are well developed. The entire genome sequence of one C. neoformans strain is nearing completion. The efficient use of this sequence is dependent upon the development of methods to perform more rapid genetic analysis including gene-disruption techniques. A modified PCR overlap technique to generate targeting constructs for gene disruption that contain large regions of gene homology is described. This technique was used to disrupt or delete more than a dozen genes with efficiencies comparable to those previously reported using cloning technology to generate targeting constructs. Moreover, it is shown that disruptions can be made using this technique in a variety of strain backgrounds, including the pathogenic serotype A isolate H99 and recently characterized stable diploid strains. In combination with the availability of the complete genomic sequence, this gene-disruption technique should pave the way for higher throughput genetic analysis of this important pathogenic fungus.

Authors
Davidson, RC; Blankenship, JR; Kraus, PR; Berrios, MDJ; Hull, CM; D'Souza, C; Wang, P; Heitman, J
MLA Citation
Davidson, RC, Blankenship, JR, Kraus, PR, Berrios, MDJ, Hull, CM, D'Souza, C, Wang, P, and Heitman, J. "A PCR-based strategy to generate integrative targeting alleles with large regions of homology." Microbiology 148.8 (2002): 2607-2615.
Source
scival
Published In
Microbiology
Volume
148
Issue
8
Publish Date
2002
Start Page
2607
End Page
2615

The Gα protein Gpa2 controls yeast differentiation by interacting with kelch repeat proteins that mimic Gβ subunits

G protein coupled receptors (GPCR) sense diverse ligands and signal via heterotrimeric G proteins. The Saccharomyces cerevisiae GPCR Gpr1 senses glucose and controls filamentous growth via an unusual Gα protein, Gpa2, which lacks any known Gβγ subunits. Our genetic and biochemical studies identify Gpa2 interaction partners (Gpb1/2, Gpg1) and provide evidence that these proteins function as G protein subunit mimics and signaling effectors. Gpb1 and Gpb2 lack the seven WD-40 repeats found in Gβ subunits and instead contain seven kelch repeats implicated in protein-protein interactions. Gβ subunits and the kelch repeat protein galactose oxidase fold into strikingly similar seven-bladed β propellers. Our studies demonstrate that Gpa2 signals in conjunction with Gβ structural mimics and that homologous G protein subunits or effectors may be conserved in multicellular eukaryotes.

Authors
Harashima, T; Heitman, J
MLA Citation
Harashima, T, and Heitman, J. "The Gα protein Gpa2 controls yeast differentiation by interacting with kelch repeat proteins that mimic Gβ subunits." Molecular Cell 10.1 (2002): 163-173.
Source
scival
Published In
Molecular Cell
Volume
10
Issue
1
Publish Date
2002
Start Page
163
End Page
173
DOI
10.1016/S1097-2765(02)00569-5

Ras1 and Ras2 contribute shared and unique roles in physiology and virulence of Cryptococcus neoformans

The Ras1 signal transduction pathway controls the ability of the pathogenic fungus Cryptococcus neoformans to grow at high temperatures and to mate. A second RAS gene was identified in this organism. RAS2 is expressed at a very low level compared to RAS1, and a ras2 mutation caused no alterations in vegetative growth rate, differentiation or virulence factor expression. The ras2 mutant strain was equally virulent to the wild-type strain in the murine inhalational model of cryptococcosis. Although a ras1 ras2 double mutant strain is viable, mutation of both RAS genes results in a decreased growth rate at all temperatures compared to strains with either single mutation. Overexpression of the RAS2 gene completely suppressed the ras1 mutant mating defect and partially suppressed its high temperature growth defect. After prolonged incubation at a restrictive temperature, the ras1 mutant demonstrated actin polarity defects that were also partially suppressed by RAS2 overexpression. These studies indicate that the C. neoformans Ras1 and Ras2 proteins share overlapping functions, but also play distinct signalling roles. Our findings also suggest a mechanism by which Ras1 controls growth of this pathogenic fungus at 37 °C, supporting a conserved role for Ras homologues in microbial cellular differentiation, morphogenesis and virulence.

Authors
Waugh, MS; Nichols, CB; DeCesare, CM; Cox, GM; Heitman, J; Alspaugh, JA
MLA Citation
Waugh, MS, Nichols, CB, DeCesare, CM, Cox, GM, Heitman, J, and Alspaugh, JA. "Ras1 and Ras2 contribute shared and unique roles in physiology and virulence of Cryptococcus neoformans." Microbiology 148.1 (2002): 191-201.
Source
scival
Published In
Microbiology
Volume
148
Issue
1
Publish Date
2002
Start Page
191
End Page
201

It infects me, it infects me not: phenotypic switching in the fungal pathogen Cryptococcus neoformans.

Authors
D'Souza, CA; Heitman, J
MLA Citation
D'Souza, CA, and Heitman, J. "It infects me, it infects me not: phenotypic switching in the fungal pathogen Cryptococcus neoformans." J Clin Invest 108.11 (December 2001): 1577-1578.
PMID
11733551
Source
pubmed
Published In
Journal of Clinical Investigation
Volume
108
Issue
11
Publish Date
2001
Start Page
1577
End Page
1578
DOI
10.1172/JCI14497

The TOR signal transduction cascade controls cellular differentiation in response to nutrients.

Rapamycin binds and inhibits the Tor protein kinases, which function in a nutrient-sensing signal transduction pathway that has been conserved from the yeast Saccharomyces cerevisiae to humans. In yeast cells, the Tor pathway has been implicated in regulating cellular responses to nutrients, including proliferation, translation, transcription, autophagy, and ribosome biogenesis. We report here that rapamycin inhibits pseudohyphal filamentous differentiation of S. cerevisiae in response to nitrogen limitation. Overexpression of Tap42, a protein phosphatase regulatory subunit, restored pseudohyphal growth in cells exposed to rapamycin. The tap42-11 mutation compromised pseudohyphal differentiation and rendered it resistant to rapamycin. Cells lacking the Tap42-regulated protein phosphatase Sit4 exhibited a pseudohyphal growth defect and were markedly hypersensitive to rapamycin. Mutations in other Tap42-regulated phosphatases had no effect on pseudohyphal differentiation. Our findings support a model in which pseudohyphal differentiation is controlled by a nutrient-sensing pathway involving the Tor protein kinases and the Tap42-Sit4 protein phosphatase. Activation of the MAP kinase or cAMP pathways, or mutation of the Sok2 repressor, restored filamentation in rapamycin treated cells, supporting models in which the Tor pathway acts in parallel with these known pathways. Filamentous differentiation of diverse fungi was also blocked by rapamycin, demonstrating that the Tor signaling cascade plays a conserved role in regulating filamentous differentiation in response to nutrients.

Authors
Cutler, NS; Pan, X; Heitman, J; Cardenas, ME
MLA Citation
Cutler, NS, Pan, X, Heitman, J, and Cardenas, ME. "The TOR signal transduction cascade controls cellular differentiation in response to nutrients." Mol Biol Cell 12.12 (December 2001): 4103-4113.
PMID
11739804
Source
pubmed
Published In
Molecular Biology of the Cell
Volume
12
Issue
12
Publish Date
2001
Start Page
4103
End Page
4113

Rapamycin and less immunosuppressive analogs are toxic to Candida albicans and Cryptococcus neoformans via FKBP12-dependent inhibition of TOR.

Candida albicans and Cryptococcus neoformans cause both superficial and disseminated infections in humans. Current antifungal therapies for deep-seated infections are limited to amphotericin B, flucytosine, and azoles. A limitation is that commonly used azoles are fungistatic in vitro and in vivo. Our studies address the mechanisms of antifungal activity of the immunosuppressive drug rapamycin (sirolimus) and its analogs with decreased immunosuppressive activity. C. albicans rbp1/rbp1 mutant strains lacking a homolog of the FK506-rapamycin target protein FKBP12 were found to be viable and resistant to rapamycin and its analogs. Rapamycin and analogs promoted FKBP12 binding to the wild-type Tor1 kinase but not to a rapamycin-resistant Tor1 mutant kinase (S1972R). FKBP12 and TOR mutations conferred resistance to rapamycin and its analogs in C. albicans, C. neoformans, and Saccharomyces cerevisiae. Our findings demonstrate the antifungal activity of rapamycin and rapamycin analogs is mediated via conserved complexes with FKBP12 and Tor kinase homologs in divergent yeasts. Taken together with our observations that rapamycin and its analogs are fungicidal and that spontaneous drug resistance occurs at a low rate, these mechanistic findings support continued investigation of rapamycin analogs as novel antifungal agents.

Authors
Cruz, MC; Goldstein, AL; Blankenship, J; Del Poeta, M; Perfect, JR; McCusker, JH; Bennani, YL; Cardenas, ME; Heitman, J
MLA Citation
Cruz, MC, Goldstein, AL, Blankenship, J, Del Poeta, M, Perfect, JR, McCusker, JH, Bennani, YL, Cardenas, ME, and Heitman, J. "Rapamycin and less immunosuppressive analogs are toxic to Candida albicans and Cryptococcus neoformans via FKBP12-dependent inhibition of TOR." Antimicrob Agents Chemother 45.11 (November 2001): 3162-3170.
PMID
11600372
Source
pubmed
Published In
Antimicrobial agents and chemotherapy
Volume
45
Issue
11
Publish Date
2001
Start Page
3162
End Page
3170
DOI
10.1128/AAC.45.11.3162-3170.2001

Phytosphingosine as a specific inhibitor of growth and nutrient import in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, we have demonstrated a necessary role for sphingolipids in the heat stress response through inhibition of nutrient import (Chung, N., Jenkins, G. M., Hannun, Y. A., Heitman, J., and Obeid, L. M. (2000) J. Biol. Chem. 275, 17229-17232). In this study, we used a combination of pharmacological and genetic approaches to determine which endogenous sphingolipid is the likely mediator of growth inhibition. When cells were treated with exogenous phytosphingosine (PHS, 20 microm) or structurally similar or metabolically related molecules, including 3-ketodihydrosphingosine, dihydrosphingosine, C(2)-phytoceramide (PHC), and stearylamine, only PHS inhibited growth. Also, PHS was shown to inhibit uptake of uracil, tryptophan, leucine, and histidine. Again this effect was specific to PHS. Because of the dynamic nature of sphingolipid metabolism, however, it was difficult to conclude that growth inhibition was caused by PHS itself. By using mutant yeast strains defective in various steps in sphingolipid metabolism, we further determined the specificity of PHS. The elo2Delta strain, which is defective in the conversion of PHS to PHC, was shown to have slower biosynthesis of ceramides and to be hypersensitive to PHS (5 microm), suggesting that PHS does not need to be converted to PHC. The lcb4Delta lcb5Delta strain is defective in the conversion of PHS to PHS 1-phosphate, and it was as sensitive to PHS as the wild-type strain. The syr2Delta mutant strain was defective in the conversion of DHS to PHS. Interestingly, this strain was resistant to high concentrations of DHS (40 microm) that inhibited the growth of an isogenic wild-type strain, demonstrating that DHS needs to be converted to PHS to inhibit growth. Together, these data demonstrate that the active sphingolipid species that inhibits yeast growth is PHS or a closely related and yet unidentified metabolite.

Authors
Chung, N; Mao, C; Heitman, J; Hannun, YA; Obeid, LM
MLA Citation
Chung, N, Mao, C, Heitman, J, Hannun, YA, and Obeid, LM. "Phytosphingosine as a specific inhibitor of growth and nutrient import in Saccharomyces cerevisiae." J Biol Chem 276.38 (September 21, 2001): 35614-35621.
PMID
11468289
Source
pubmed
Published In
The Journal of biological chemistry
Volume
276
Issue
38
Publish Date
2001
Start Page
35614
End Page
35621
DOI
10.1074/jbc.M105653200

Two cyclophilin A homologs with shared and distinct functions important for growth and virulence of Cryptococcus neoformans.

Cyclophilin A is the target of the immunosuppressant cyclosporin A (CsA) and is encoded by a single unique gene conserved from yeast to humans. In the pathogenic fungus Cryptococcus neoformans, two homologous linked genes, CPA1 and CPA2, were found to encode two conserved cyclophilin A proteins. In contrast to Saccharomyces cerevisiae, in which cyclophilin A mutations confer CsA resistance but few other phenotypes, cyclophilin A mutations conferred dramatic phenotypes in C. neoformans. The Cpa1 and Cpa2 cyclophilin A proteins play a shared role in cell growth, mating, virulence and CsA toxicity. The Cpa1 and Cpa2 proteins also have divergent functions. cpa1 mutants are inviable at 39 degrees C and attenuated for virulence, whereas cpa2 mutants are viable at 39 degrees C and fully virulent. cpa1 cpa2 double mutants exhibited synthetic defects in growth and virulence. Cyclophilin A active site mutants restored growth of cpa1 cpa2 mutants at ambient but not at higher temperatures, suggesting that the prolyl isomerase activity of cyclophilin A has an in vivo function.

Authors
Wang, P; Cardenas, ME; Cox, GM; Perfect, JR; Heitman, J
MLA Citation
Wang, P, Cardenas, ME, Cox, GM, Perfect, JR, and Heitman, J. "Two cyclophilin A homologs with shared and distinct functions important for growth and virulence of Cryptococcus neoformans." EMBO Rep 2.6 (June 2001): 511-518.
PMID
11415984
Source
pubmed
Published In
EMBO Reports
Volume
2
Issue
6
Publish Date
2001
Start Page
511
End Page
518
DOI
10.1093/embo-reports/kve109

Conserved cAMP signaling cascades regulate fungal development and virulence.

Two well characterized signal transduction cascades regulating fungal development and virulence are the MAP kinase and cAMP signaling cascades. Here we review the current state of knowledge on cAMP signaling cascades in fungi. While the processes regulated by cAMP signaling in fungi are as diverse as the fungi themselves, the components involved in signal transduction are remarkably conserved. Fungal cAMP signaling cascades are also quite versatile, which is apparent from the differential regulation of similar biological processes. In this review we compare and contrast cAMP signaling pathways that regulate development in the budding yeast Saccharomyces cerevisiae, the fission yeast Schizosaccharomyces pombe, and differentiation and virulence in the human pathogen Cryptococcus neoformans and the plant pathogen Ustilago maydis. We also present examples of interaction between the cAMP and MAP kinase signaling cascades in the regulation of fungal development and virulence.

Authors
D'Souza, CA; Heitman, J
MLA Citation
D'Souza, CA, and Heitman, J. "Conserved cAMP signaling cascades regulate fungal development and virulence." FEMS Microbiol Rev 25.3 (May 2001): 349-364. (Review)
PMID
11348689
Source
pubmed
Published In
Fems Microbiology Reviews
Volume
25
Issue
3
Publish Date
2001
Start Page
349
End Page
364

Cyclic AMP-dependent protein kinase controls virulence of the fungal pathogen Cryptococcus neoformans.

Cryptococcus neoformans is an opportunistic fungal pathogen that infects the human central nervous system. This pathogen elaborates two specialized virulence factors: the antioxidant melanin and an antiphagocytic immunosuppressive polysaccharide capsule. A signaling cascade controlling mating and virulence was identified. The PKA1 gene encoding the major cyclic AMP (cAMP)-dependent protein kinase catalytic subunit was identified and disrupted. pka1 mutant strains were sterile, failed to produce melanin or capsule, and were avirulent. The PKR1 gene encoding the protein kinase A (PKA) regulatory subunit was also identified and disrupted. pkr1 mutant strains overproduced capsule and were hypervirulent in animal models of cryptococcosis. pkr1 pka1 double mutant strains exhibited phenotypes similar to that of pka1 mutants, providing epistasis evidence that the Pka1 catalytic subunit functions downstream of the Pkr1 regulatory subunit. The PKA pathway was also shown to function downstream of the Galpha protein Gpa1 and to regulate cAMP production by feedback inhibition. These findings define a Galpha protein-cAMP-PKA signaling pathway regulating differentiation and virulence of a human fungal pathogen.

Authors
D'Souza, CA; Alspaugh, JA; Yue, C; Harashima, T; Cox, GM; Perfect, JR; Heitman, J
MLA Citation
D'Souza, CA, Alspaugh, JA, Yue, C, Harashima, T, Cox, GM, Perfect, JR, and Heitman, J. "Cyclic AMP-dependent protein kinase controls virulence of the fungal pathogen Cryptococcus neoformans." Mol Cell Biol 21.9 (May 2001): 3179-3191.
PMID
11287622
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
21
Issue
9
Publish Date
2001
Start Page
3179
End Page
3191
DOI
10.1128/MCB.21.9.3179-3191.2001

The TOR kinases link nutrient sensing to cell growth.

Rapamycin is an immunosuppressive natural product that inhibits the proliferation of T-cells in response to nutrients and growth factors. Rapamycin binds to the peptidyl-prolyl isomerase FKBP12 and forms protein-drug complexes that inhibit signal transduction by the TOR kinases. The FKBP12 and TOR proteins are conserved from fungi to humans, and in both organisms the TOR signaling pathway plays a role in nutrient sensing. In response to nitrogen sources or amino acids, TOR regulates both transcription and translation, enabling cells to appropriately respond to growth-promoting signals. Rapamycin is having a profound impact on clinical medicine and was approved as an immunosuppressant for transplant recipients in 1999. Ongoing clinical studies address new clinical applications for rapamycin as an antiproliferative drug for chemotherapy and invasive cardiology.

Authors
Rohde, J; Heitman, J; Cardenas, ME
MLA Citation
Rohde, J, Heitman, J, and Cardenas, ME. "The TOR kinases link nutrient sensing to cell growth." J Biol Chem 276.13 (March 30, 2001): 9583-9586. (Review)
PMID
11266435
Source
pubmed
Published In
The Journal of biological chemistry
Volume
276
Issue
13
Publish Date
2001
Start Page
9583
End Page
9586
DOI
10.1074/jbc.R000034200

A metabolic enzyme for S-nitrosothiol conserved from bacteria to humans.

Considerable evidence indicates that NO biology involves a family of NO-related molecules and that S-nitrosothiols (SNOs) are central to signal transduction and host defence. It is unknown, however, how cells switch off the signals or protect themselves from the SNOs produced for defence purposes. Here we have purified a single activity from Escherichia coli, Saccharomyces cerevisiae and mouse macrophages that metabolizes S-nitrosoglutathione (GSNO), and show that it is the glutathione-dependent formaldehyde dehydrogenase. Although the enzyme is highly specific for GSNO, it controls intracellular levels of both GSNO and S-nitrosylated proteins. Such 'GSNO reductase' activity is widely distributed in mammals. Deleting the reductase gene in yeast and mice abolishes the GSNO-consuming activity, and increases the cellular quantity of both GSNO and protein SNO. Furthermore, mutant yeast cells show increased susceptibility to a nitrosative challenge, whereas their resistance to oxidative stress is unimpaired. We conclude that GSNO reductase is evolutionarily conserved from bacteria to humans, is critical for SNO homeostasis, and protects against nitrosative stress.

Authors
Liu, L; Hausladen, A; Zeng, M; Que, L; Heitman, J; Stamler, JS
MLA Citation
Liu, L, Hausladen, A, Zeng, M, Que, L, Heitman, J, and Stamler, JS. "A metabolic enzyme for S-nitrosothiol conserved from bacteria to humans." Nature 410.6827 (March 22, 2001): 490-494.
PMID
11260719
Source
pubmed
Published In
Nature
Volume
410
Issue
6827
Publish Date
2001
Start Page
490
End Page
494
DOI
10.1038/35068596

Calcineurin is required for hyphal elongation during mating and haploid fruiting in Cryptococcus neoformans.

Cryptococcus neoformans is a fungal pathogen that causes meningitis in immunocompromised patients. Its growth is sensitive to the immunosuppressants FK506 and cyclosporin, which inhibit the Ca2+- calmodulin-activated protein phosphatase calcineurin. Calcineurin is required for growth at 37 degrees C and virulence of C.neoformans. We found that calcineurin is also required for mating. FK506 blocks mating of C.neoformans via FKBP12-dependent inhibition of calcineurin, and mutants lacking calcineurin are bilaterally sterile. Calcineurin is not essential for the initial fusion event, but is required for hyphal elongation and survival of the heterokaryon produced by cell fusion. It is also required for hyphal elongation in diploid strains and during asexual haploid fruiting of MATalpha cells in response to nitrogen limitation. Because mating and haploid fruiting produce infectious basidiospores, our studies suggest a second link between calcineurin and virulence of C.neoformans. Calcine urin regulates filamentation and 37 degrees C growth via distinct pathways. Together with studies revealing that calcineurin mediates neurite extension and neutrophil migration in mammals, our findings indicate that calcineurin plays a conserved role in the control of cell morphology.

Authors
Cruz, MC; Fox, DS; Heitman, J
MLA Citation
Cruz, MC, Fox, DS, and Heitman, J. "Calcineurin is required for hyphal elongation during mating and haploid fruiting in Cryptococcus neoformans." EMBO J 20.5 (March 1, 2001): 1020-1032.
PMID
11230126
Source
pubmed
Published In
EMBO Journal
Volume
20
Issue
5
Publish Date
2001
Start Page
1020
End Page
1032
DOI
10.1093/emboj/20.5.1020

Dismantling the Cryptococcus coat - Response

Authors
D'Souza, CA; Heitman, J
MLA Citation
D'Souza, CA, and Heitman, J. "Dismantling the Cryptococcus coat - Response." TRENDS IN MICROBIOLOGY 9.3 (March 2001): 113-113.
Source
wos-lite
Published In
Trends in Microbiology
Volume
9
Issue
3
Publish Date
2001
Start Page
113
End Page
113
DOI
10.1016/S0966-842X(00)01946-6

Dismantling the Cryptococcus coat.

Authors
D'Souza, CA; Heitman, J
MLA Citation
D'Souza, CA, and Heitman, J. "Dismantling the Cryptococcus coat." Trends Microbiol 9.3 (March 2001): 112-113. (Letter)
PMID
11303499
Source
pubmed
Published In
Trends in Microbiology
Volume
9
Issue
3
Publish Date
2001
Start Page
112
End Page
113

Calcineurin regulatory subunit is essential for virulence and mediates interactions with FKBP12-FK506 in Cryptococcus neoformans.

Calcineurin is a Ca2+-calmodulin-regulated protein phosphatase that is the target of the immunosuppressive drugs cyclosporin A and FK506. Calcineurin is a heterodimer composed of a catalytic A and a regulatory B subunit. In previous studies, the calcineurin A homologue was identified and shown to be required for growth at 37 degrees C and hence for virulence of the pathogenic fungus Cryptococcus neoformans. Here, we identify the gene encoding the calcineurin B regulatory subunit and demonstrate that calcineurin B is also required for growth at elevated temperature and virulence. We show that the FKR1-1 mutation, which confers dominant FK506 resistance, results from a 6 bp duplication generating a two-amino-acid insertion in the latch region of calcineurin B. This mutation was found to reduce FKBP12-FK506 binding to calcineurin both in vivo and in vitro. Molecular modelling based on the FKBP12-FK506-calcineurin crystal structure illustrates how this mutation perturbs drug interactions with the phosphatase target. In summary, our studies reveal a central role for calcineurin B in virulence and antifungal drug action in the human fungal pathogen C. neoformans.

Authors
Fox, DS; Cruz, MC; Sia, RA; Ke, H; Cox, GM; Cardenas, ME; Heitman, J
MLA Citation
Fox, DS, Cruz, MC, Sia, RA, Ke, H, Cox, GM, Cardenas, ME, and Heitman, J. "Calcineurin regulatory subunit is essential for virulence and mediates interactions with FKBP12-FK506 in Cryptococcus neoformans." Mol Microbiol 39.4 (February 2001): 835-849.
PMID
11251806
Source
pubmed
Published In
Molecular Microbiology
Volume
39
Issue
4
Publish Date
2001
Start Page
835
End Page
849

Serotype AD strains of Cryptococcus neoformans are diploid or aneuploid and are heterozygous at the mating-type locus.

Cryptococcus neoformans is a pathogenic basidiomycete with a defined sexual cycle involving mating between haploid yeast cells with a transient diploid state. C. neoformans occurs in four predominant serotypes (A, B, C, and D), which represent different varieties or species. Rare clinical and environmental isolates with an unusual AD serotype have been reported and suggested to be diploid. We found by fluorescence-activated cell sorter analysis that serotype AD strains are aneuploid or diploid. PCR analysis with primers specific for serotype A or D alleles of the CNA1, CLA4, and GPA1 genes revealed that both alleles are often present in serotype AD strains. PCR analysis with primers specific for genes in the MATa or MATalpha mating-type loci revealed that serotype AD strains are heterozygous for the mating-type locus. Interestingly, in several serotype AD strains, the MATalpha locus was derived from the serotype D parent and the MATa locus was inherited from a serotype A parent that has been thought to be extinct. Basidiospores from a self-fertile serotype AD strain bearing the putative serotype A MATa locus showed a very low viability ( approximately 5%), and no fertile serotype A MATa strain could be recovered. Serotype AD strains were virulent in a murine model. Hybrid AD strains could readily be isolated following a laboratory cross between a serotype A strain and a serotype D strain. In summary, serotype AD strains of C. neoformans are unusual aneuploid or diploid strains that result from matings between serotype A and D strains. Self-fertile isolates fail to undergo normal meiosis because of genetic divergence. Our findings further suggest that serotype A MATa strains may exist in nature.

Authors
Lengeler, KB; Cox, GM; Heitman, J
MLA Citation
Lengeler, KB, Cox, GM, and Heitman, J. "Serotype AD strains of Cryptococcus neoformans are diploid or aneuploid and are heterozygous at the mating-type locus." Infect Immun 69.1 (January 2001): 115-122.
PMID
11119496
Source
pubmed
Published In
Infection and immunity
Volume
69
Issue
1
Publish Date
2001
Start Page
115
End Page
122
DOI
10.1128/IAI.69.1.115-122.2001

Dismantling the Cryptococcus coat [1] (multiple letters)

Authors
D'Souza, CA; Heitman, J
MLA Citation
D'Souza, CA, and Heitman, J. "Dismantling the Cryptococcus coat [1] (multiple letters)." Trends in Microbiology 9.3 (2001): 112-113.
Source
scival
Published In
Trends in Microbiology
Volume
9
Issue
3
Publish Date
2001
Start Page
112
End Page
113

Identification of the MATa mating-type locus of Cryptococcus neoformans reveals a serotype A MATa strain thought to have been extinct.

Cryptococcus neoformans is an opportunistic fungal pathogen with a defined sexual cycle involving mating between haploid MATa and MATalpha cells. Here we describe the isolation of part of the MATa mating-type locus encoding a Ste20 kinase homolog, Ste20a. We show that the STE20a gene cosegregates with the MATa mating type in genetic crosses, maps within the mating-type locus on a 1.8-Mb chromosome, and is allelic with the MATalpha locus. We identify the first MATa isolate of the most common pathogenic variety of C. neoformans (serotype A, variety grubii) which had been thought to be extinct. This serotype A MATa strain is sterile, fails to produce mating pheromone, and is less virulent than a serotype A MATalpha strain in an animal model. Our studies illustrate an association of mating type with virulence and suggest that, like Candida albicans, pathogenic isolates of C. neoformans may be largely asexual.

Authors
Lengeler, KB; Wang, P; Cox, GM; Perfect, JR; Heitman, J
MLA Citation
Lengeler, KB, Wang, P, Cox, GM, Perfect, JR, and Heitman, J. "Identification of the MATa mating-type locus of Cryptococcus neoformans reveals a serotype A MATa strain thought to have been extinct." Proc Natl Acad Sci U S A 97.26 (December 19, 2000): 14455-14460.
PMID
11121047
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
97
Issue
26
Publish Date
2000
Start Page
14455
End Page
14460
DOI
10.1073/pnas.97.26.14455

Characterization of the MFalpha pheromone of the human fungal pathogen cryptococcus neoformans.

Cryptococcus neoformans is an important human pathogenic fungus with a defined sexual cycle and well-developed molecular and genetic approaches. C. neoformans is predominantly haploid and has two mating types, MATa and MATalpha. Mating is known to be regulated by nutritional limitation and thought also to be regulated by pheromones. Previously, a portion of the MATalpha locus was cloned, and a presumptive pheromone gene, MFalpha1, was identified by its ability to induce conjugation tube-like filaments when introduced by transformation into MATa cells. Here, the ability of the MFalpha1 gene to induce these morphological changes in MATa cells was used as a phenotypic assay to perform a structure-function analysis of the gene. We show that the MFalpha1 open reading frame is required for the morphological response of MATa cells. We also find that the cysteine residue of the C-terminal CAAX motif is required for activity of the MFalpha1 pheromone. In addition, we use a reporter system to measure the expression levels of the MFalpha1 pheromone gene and find that two signals, nutrient starvation and the presence of factors secreted by mating partner cells, impinge on this promoter and regulate MFalpha1 expression. We identify a second pheromone gene, MFalpha2, and show phenotypically that this gene is also expressed. Finally, we have synthesized the MFalpha1 pheromone and show that only the predicted mature modified form of the alpha-factor peptide triggers morphological responses in MATa cells.

Authors
Davidson, RC; Moore, TD; Odom, AR; Heitman, J
MLA Citation
Davidson, RC, Moore, TD, Odom, AR, and Heitman, J. "Characterization of the MFalpha pheromone of the human fungal pathogen cryptococcus neoformans." Mol Microbiol 38.5 (December 2000): 1017-1026.
PMID
11123675
Source
pubmed
Published In
Molecular Microbiology
Volume
38
Issue
5
Publish Date
2000
Start Page
1017
End Page
1026

Signal transduction cascades regulating fungal development and virulence.

Cellular differentiation, mating, and filamentous growth are regulated in many fungi by environmental and nutritional signals. For example, in response to nitrogen limitation, diploid cells of the yeast Saccharomyces cerevisiae undergo a dimorphic transition to filamentous growth referred to as pseudohyphal differentiation. Yeast filamentous growth is regulated, in part, by two conserved signal transduction cascades: a mitogen-activated protein kinase cascade and a G-protein regulated cyclic AMP signaling pathway. Related signaling cascades play an analogous role in regulating mating and virulence in the plant fungal pathogen Ustilago maydis and the human fungal pathogens Cryptococcus neoformans and Candida albicans. We review here studies on the signaling cascades that regulate development of these and other fungi. This analysis illustrates both how the model yeast S. cerevisiae can serve as a paradigm for signaling in other organisms and also how studies in other fungi provide insights into conserved signaling pathways that operate in many divergent organisms.

Authors
Lengeler, KB; Davidson, RC; D'souza, C; Harashima, T; Shen, WC; Wang, P; Pan, X; Waugh, M; Heitman, J
MLA Citation
Lengeler, KB, Davidson, RC, D'souza, C, Harashima, T, Shen, WC, Wang, P, Pan, X, Waugh, M, and Heitman, J. "Signal transduction cascades regulating fungal development and virulence." Microbiol Mol Biol Rev 64.4 (December 2000): 746-785. (Review)
PMID
11104818
Source
pubmed
Published In
Microbiology and molecular biology reviews : MMBR
Volume
64
Issue
4
Publish Date
2000
Start Page
746
End Page
785

Signal transduction cascades regulating pseudohyphal differentiation of Saccharomyces cerevisiae.

In response to nitrogen limitation, diploid cells of the yeast Saccharomyces cerevisiae undergo a dimorphic transition to filamentous pseudohyphal growth. At least two signaling pathways regulate filamentation. One involves components of the MAP kinase cascade that also regulates mating of haploid cells. The second involves a nutrient-sensing G-protein-coupled receptor that signals via an unusual G(alpha) protein, cAMP and protein kinase A. Recent studies reveal crosstalk between these pathways during pseudohyphal growth. Related MAP kinase and cAMP pathways regulate filamentation and virulence of human and plant fungal pathogens, and represent novel targets for antifungal drug design.

Authors
Pan, X; Harashima, T; Heitman, J
MLA Citation
Pan, X, Harashima, T, and Heitman, J. "Signal transduction cascades regulating pseudohyphal differentiation of Saccharomyces cerevisiae." Curr Opin Microbiol 3.6 (December 2000): 567-572. (Review)
PMID
11121775
Source
pubmed
Published In
Current Opinion in Microbiology
Volume
3
Issue
6
Publish Date
2000
Start Page
567
End Page
572

Sok2 regulates yeast pseudohyphal differentiation via a transcription factor cascade that regulates cell-cell adhesion.

In response to nitrogen limitation, Saccharomyces cerevisiae undergoes a dimorphic transition to filamentous pseudohyphal growth. In previous studies, the transcription factor Sok2 was found to negatively regulate pseudohyphal differentiation. By genome array and Northern analysis, we found that genes encoding the transcription factors Phd1, Ash1, and Swi5 were all induced in sok2/sok2 hyperfilamentous mutants. In accord with previous studies of others, Swi5 was required for ASH1 expression. Phd1 and Ash1 regulated expression of the cell surface protein Flo11, which is required for filamentous growth, and were largely required for filamentation of sok2/sok2 mutant strains. These findings reveal that a complex transcription factor cascade regulates filamentation. These findings also reveal a novel dual role for the transcription factor Swi5 in regulating filamentous growth. Finally, these studies illustrate how mother-daughter cell adhesion can be accomplished by two distinct mechanisms: one involving Flo11 and the other involving regulation of the endochitinase Cts1 and the endoglucanase Egt2 by Swi5.

Authors
Pan, X; Heitman, J
MLA Citation
Pan, X, and Heitman, J. "Sok2 regulates yeast pseudohyphal differentiation via a transcription factor cascade that regulates cell-cell adhesion." Mol Cell Biol 20.22 (November 2000): 8364-8372.
PMID
11046133
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
20
Issue
22
Publish Date
2000
Start Page
8364
End Page
8372

A new face of the Rhesus antigen.

Authors
Heitman, J; Agre, P
MLA Citation
Heitman, J, and Agre, P. "A new face of the Rhesus antigen." Nat Genet 26.3 (November 2000): 258-259.
PMID
11062455
Source
pubmed
Published In
Nature Genetics
Volume
26
Issue
3
Publish Date
2000
Start Page
258
End Page
259
DOI
10.1038/81532

Identification and characterization of a highly conserved calcineurin binding protein, CBP1/calcipressin, in Cryptococcus neoformans.

Calcineurin is the conserved target of the immunosuppressants cyclosporin A and FK506. Using the yeast two-hybrid system, we identified a novel calcineurin binding protein, CBP1, from the pathogenic fungus Cryptococcus neoformans. We show that CBP1 binds to calcineurin in vitro and in vivo, and FKBP12-FK506 inhibits CBP1 binding to calcineurin. Cryptococcus neoformans cbp1 mutant strains exhibit modest defects in growth under stress conditions and virulence, similar to but less severe than the phenotypes of calcineurin mutants. Saccharomyces cerevisiae mutants lacking the CBP1 homolog RCN1 are, like calcineurin mutants, sensitive to lithium cation stress. CBP1 shares a central peptide sequence motif, SPPxSPP, with related proteins in S.CEREVISIAE:, Schizosaccharomyces pombe, Drosophila melanogaster, Caenorhabditis elegans and humans, and peptides containing this motif altered calcineurin activity in vitro. Interestingly, the human CBP1 homolog DSCR1 is encoded by the Down's syndrome candidate region interval on chromosome 21, is highly expressed in the heart and central nervous system, and may play a role in calcineurin functions in heart development, neurite extension and memory.

Authors
Görlach, J; Fox, DS; Cutler, NS; Cox, GM; Perfect, JR; Heitman, J
MLA Citation
Görlach, J, Fox, DS, Cutler, NS, Cox, GM, Perfect, JR, and Heitman, J. "Identification and characterization of a highly conserved calcineurin binding protein, CBP1/calcipressin, in Cryptococcus neoformans." EMBO J 19.14 (July 17, 2000): 3618-3629.
PMID
10899116
Source
pubmed
Published In
EMBO Journal
Volume
19
Issue
14
Publish Date
2000
Start Page
3618
End Page
3629
DOI
10.1093/emboj/19.14.3618

The Ess1 prolyl isomerase is linked to chromatin remodeling complexes and the general transcription machinery.

The Ess1/Pin1 peptidyl-prolyl isomerase (PPIase) is thought to control mitosis by binding to cell cycle regulatory proteins and altering their activity. Here we isolate temperature-sensitive ess1 mutants and identify six multicopy suppressors that rescue their mitotic-lethal phenotype. None are cell cycle regulators. Instead, five encode proteins involved in transcription that bind DNA, modify chromatin structure or are regulatory subunits of RNA polymerase II. A sixth suppressor, cyclophilin A, is a member of a distinct family of PPIases that are targets of immuno suppressive drugs. We show that the expression of some but not all genes is decreased in ess1 mutants, and that Ess1 interacts with the C-terminal domain (CTD) of RNA polymerase II in vitro and in vivo. The results forge a strong link between PPIases and the transcription machinery and suggest a new model for how Ess1/Pin1 controls mitosis. In this model, Ess1 binds and isomerizes the CTD of RNA polymerase II, thus altering its interaction with proteins required for transcription of essential cell cycle genes.

Authors
Wu, X; Wilcox, CB; Devasahayam, G; Hackett, RL; Arévalo-Rodríguez, M; Cardenas, ME; Heitman, J; Hanes, SD
MLA Citation
Wu, X, Wilcox, CB, Devasahayam, G, Hackett, RL, Arévalo-Rodríguez, M, Cardenas, ME, Heitman, J, and Hanes, SD. "The Ess1 prolyl isomerase is linked to chromatin remodeling complexes and the general transcription machinery." EMBO J 19.14 (July 17, 2000): 3727-3738.
PMID
10899126
Source
pubmed
Published In
EMBO Journal
Volume
19
Issue
14
Publish Date
2000
Start Page
3727
End Page
3738
DOI
10.1093/emboj/19.14.3727

Cyclophilin A and Ess1 interact with and regulate silencing by the Sin3-Rpd3 histone deacetylase.

Three families of prolyl isomerases have been identified: cyclophilins, FK506-binding proteins (FKBPs) and parvulins. All 12 cyclophilins and FKBPs are dispensable for growth in yeast, whereas the one parvulin homolog, Ess1, is essential. We report here that cyclophilin A becomes essential when Ess1 function is compromised. We also show that overexpression of cyclophilin A suppresses ess1 conditional and null mutations, and that cyclophilin A enzymatic activity is required for suppression. These results indicate that cyclophilin A and Ess1 function in parallel pathways and act on common targets by a mechanism that requires prolyl isomerization. Using genetic and biochemical approaches, we found that one of these targets is the Sin3-Rpd3 histone deacetylase complex, and that cyclophilin A increases and Ess1 decreases disruption of gene silencing by this complex. We show that conditions that favor acetylation over deacetylation suppress ess1 mutations. Our findings support a model in which Ess1 and cyclophilin A modulate the activity of the Sin3-Rpd3 complex, and excess histone deacetylation causes mitotic arrest in ess1 mutants.

Authors
Arévalo-Rodríguez, M; Cardenas, ME; Wu, X; Hanes, SD; Heitman, J
MLA Citation
Arévalo-Rodríguez, M, Cardenas, ME, Wu, X, Hanes, SD, and Heitman, J. "Cyclophilin A and Ess1 interact with and regulate silencing by the Sin3-Rpd3 histone deacetylase." EMBO J 19.14 (July 17, 2000): 3739-3749.
PMID
10899127
Source
pubmed
Published In
EMBO Journal
Volume
19
Issue
14
Publish Date
2000
Start Page
3739
End Page
3749
DOI
10.1093/emboj/19.14.3739

Sphingolipids signal heat stress-induced ubiquitin-dependent proteolysis.

Sphingolipids are essential eukaryotic membrane lipids that are structurally and metabolically conserved through evolution. Sphingolipids have also been proposed to regulate eukaryotic stress responses as novel second messengers. Here we show that, in Saccharomyces cerevisiae, phytosphingosine, a putative sphingolipid second messenger, mediates heat stress signaling and activates ubiquitin-dependent proteolysis via the endocytosis vacuolar degradation and 26 S proteasome pathways. Inactivation of serine palmitoyltransferase, a key enzyme in generating endogenous phytosphingosine, prevents proteolysis during heat stress. Addition of phytosphingosine bypasses the requirement for serine palmitoyltransferase and restores proteolysis. Phytosphingosine-induced proteolysis requires multiubiquitin chain formation through the stress-responsive lysine 63 residue of ubiquitin. We propose that heat stress increases phytosphingosine and activates ubiquitin-dependent proteolysis.

Authors
Chung, N; Jenkins, G; Hannun, YA; Heitman, J; Obeid, LM
MLA Citation
Chung, N, Jenkins, G, Hannun, YA, Heitman, J, and Obeid, LM. "Sphingolipids signal heat stress-induced ubiquitin-dependent proteolysis." J Biol Chem 275.23 (June 9, 2000): 17229-17232.
PMID
10764732
Source
pubmed
Published In
The Journal of biological chemistry
Volume
275
Issue
23
Publish Date
2000
Start Page
17229
End Page
17232
DOI
10.1074/jbc.C000229200

A STE12 homolog is required for mating but dispensable for filamentation in candida lusitaniae.

Candida lusitaniae is a dimorphic yeast that is emerging as an opportunistic fungal pathogen. In contrast to Candida albicans, which is diploid and asexual, C. lusitaniae has been reported to have a sexual cycle. We have employed genetic approaches to demonstrate that C. lusitaniae is haploid and has a sexual cycle involving mating between MATa and MATalpha cells under nutrient deprivation conditions. By degenerate PCR, we identified a C. lusitaniae homolog (Cls12) of the Ste12 transcription factor that regulates mating, filamentation, and virulence in Saccharomyces cerevisiae, C. albicans, and Cryptococcus neoformans. Comparison of the CLS12 DNA and protein sequences to other STE12 homologs and transformation experiments with selectable markers from S. cerevisiae (URA3, KanMX, HphMX) and C. albicans (CaURA3) provide evidence that the CUG codon encodes serine instead of leucine in C. lusitaniae, as is also the case in C. albicans. The C. lusitaniae CLS12 gene was disrupted by biolistic transformation and homologous recombination. C. lusitaniae cls12 mutant strains were sterile but had no defect in filamentous growth. Our findings reveal both conserved and divergent roles for the C. lusitaniae STE12 homolog in regulating differentiation of this emerging fungal pathogen.

Authors
Young, LY; Lorenz, MC; Heitman, J
MLA Citation
Young, LY, Lorenz, MC, and Heitman, J. "A STE12 homolog is required for mating but dispensable for filamentation in candida lusitaniae." Genetics 155.1 (May 2000): 17-29.
PMID
10790381
Source
pubmed
Published In
Genetics
Volume
155
Issue
1
Publish Date
2000
Start Page
17
End Page
29

Protection from nitrosative stress by yeast flavohemoglobin.

Yeast hemoglobin was discovered close to half a century ago, but its function has remained unknown. Herein, we report that this flavohemoglobin protects Saccharomyces cerevisiae from nitrosative stress. Deletion of the flavohemoglobin gene (YHB1) abolished the nitric oxide (NO)-consuming activity of yeast cells. Levels of protein nitrosylation were more than 10-fold higher in yhb1 mutant yeast than in isogenic wild-type cells after incubation with NO donors. Growth of mutant cells was inhibited by a nitrosative challenge that had little effect on wild-type cells, whereas the resistance of mutant cells to oxidative stress was unimpaired. Protection conferred by yeast flavohemoglobin against NO and S-nitrosothiols was seen under both anaerobic and aerobic conditions, consistent with a primary function in NO detoxification. A phylogenetic analysis indicated that protection from nitrosative stress is likely to be a conserved function among microorganismal flavohemoglobins. Flavohemoglobin is therefore a potential target for antimicrobial therapy.

Authors
Liu, L; Zeng, M; Hausladen, A; Heitman, J; Stamler, JS
MLA Citation
Liu, L, Zeng, M, Hausladen, A, Heitman, J, and Stamler, JS. "Protection from nitrosative stress by yeast flavohemoglobin." Proc Natl Acad Sci U S A 97.9 (April 25, 2000): 4672-4676.
PMID
10758168
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
97
Issue
9
Publish Date
2000
Start Page
4672
End Page
4676
DOI
10.1073/pnas.090083597

Diploid strains of the pathogenic basidiomycete Cryptococcus neoformans are thermally dimorphic.

Cryptococcus neoformans is an opportunistic human pathogenic fungus with a defined sexual cycle. Clinical and environmental isolates of C. neoformans are haploid, and the diploid stage of the lifecycle is thought to be transient and unstable. In contrast, we find that diploid strains are readily obtained following genetic crosses of congenic MATalpha and MATa strains. At 37 degrees C, the diploid strains grow as yeast cells with a single nucleus that is larger than a haploid nucleus, contains a 2n content of DNA by FACS analysis, and is heterozygous for the MATalpha and MATa loci. At 24 degrees C, these diploid self-fertile strains filament and sporulate, producing recombinant haploid progeny in which meiotic segregation has occurred. In contrast to dikaryotic filament cells that are typically linked by fused clamp connections during mating, self-fertile diploid strains produce monokaryotic filament cells with unfused clamp connections. We also show that these diploid strains can be transformed and sporulated and that an integrated selectable marker segregates in a mendelian fashion. The diploid state could play novel roles in the lifecycle and virulence of the organism and can be exploited for the analysis of essential genes.