You are here

Kuehn, Margarethe Joanna

Overview:

Enterotoxigenic E. coli (ETEC) causes traveler's diarrhea and infant mortality in underdeveloped countries, and Pseudomonas aeruginosa is an opportunistic pathogen for immunocompromised patients. Like all gram negative bacteria studied to date, ETEC and P. aeruginosa produce small outer membrane vesicles that can serve as delivery "bombs" to host tissues. Vesicles contain a subset of outer membrane and soluble periplasmic proteins and lipids. In tissues and sera of infected hosts, vesicles have been observed to bud from the pathogen and come in close contact with epithelial cells. Despite their association with disease, the ability of pathogenic bacteria to distribute an arsenal of virulence factors to the host cells via vesicles remains relatively unexplored.

In our lab, we focus on the genetic, biochemical and functional features of bacterial vesicle production. Using a genetic screen, we have identified genes essential in the vesiculation process, we have identified specific proteins that are enriched in vesicles, and we have identified critical molecules that govern the internalization of vesicles into host cells. Using biochemical analysis of purified vesicles from cell-free culture supernatants, we have found that heat-labile enterotoxin, an important virulence factor of ETEC, is exported from the cells bound to the external surface of vesicles. Presented in this context, it is able to mediate the entry of the entire ETEC vesicle into human colorectal tissue culture cells. We have also discovered that the ability of vesicles to bind to specific cell types depends on their strain of origin: for example, P. aeruginosa vesicles produced by a strain that was cultured from the lungs of a patient with Cystic Fibrosis adhered better to lung than to gut epithelial cells, whereas a strain that was isolated from sera showed no such preference for lung cells. The vesicles stimulate epithelial cells and macrophages to elicit a cytokine response that is distinct from that of LPS (a major component of the vesicles) alone.

These studies will provide new insights into the membrane dynamics of gram-negative bacteria and consequently aid in the identification of new therapeutic targets for important human pathogens.

Positions:

Associate Professor of Biochemistry

Biochemistry
School of Medicine

Associate Professor in Molecular Genetics and Microbiology

Molecular Genetics and Microbiology
School of Medicine

Member of the Duke Cancer Institute

Duke Cancer Institute
School of Medicine

Education:

B.S. 1986

B.S. — University of Washington

Ph.D. 1993

Ph.D. — Washington University

Grants:

Structural and Mechanistic Characterization of MraY Catalysis and Inhibition

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Collaborator
Start Date
January 01, 2017
End Date
November 30, 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

Triggers and Consequences of Bacterial Envelope Stress

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
April 01, 2012
End Date
March 31, 2017

Regulation of Host Defense by IRG Proteins

Administered By
Medicine, Geriatrics
AwardedBy
National Institutes of Health
Role
Advisor
Start Date
March 01, 2004
End Date
May 31, 2016

Enterotoxin targeting and delivery mechanisms

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
June 15, 2006
End Date
May 31, 2012

Duke PREP: Minority Recruitment into Biomedical Sciences

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Advisor
Start Date
August 01, 2003
End Date
July 31, 2008

Heat-Labile Enterotoxin Secretion

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
April 01, 2005
End Date
March 31, 2008

Detection of in vivo ETEC vesicle production

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
March 01, 2003
End Date
February 28, 2006

Production and function of E Coli Vesicles

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
September 01, 2002
End Date
August 31, 2004
Show More

Awards:

Thomas Davis Research Award. American Lung Association of North Carolina.

Type
State
Awarded By
American Lung Association of North Carolina
Date
January 01, 2003

Investigator in Pathogenesis of Infectious Diseases. Burroughs Wellcome Fund.

Type
National
Awarded By
Burroughs Wellcome Fund
Date
January 01, 2002

Burroughs Wellcome Career Award. Burroughs Wellcome.

Type
National
Awarded By
Burroughs Wellcome
Date
January 01, 1997

Damon Runyon-Walter Winchell Postdoctoral Research Fellow. Damon Runyon-Walter Winchell Foundation.

Type
National
Awarded By
Damon Runyon-Walter Winchell Foundation
Date
January 01, 1994

Publications:

Environmentally controlled bacterial vesicle-mediated export.

Over the past two decades, researchers studying both microbial and host cell communities have gained an appreciation for the ability of bacteria to produce, regulate, and functionally utilize outer membrane vesicles (OMVs) as a means to survive and interact with their cellular and acellular environments. Common ground has emerged, as it appears that vesicle production is an environmentally controlled and specific secretion process; however, it has been challenging to discover the principles that govern fundamentals of vesicle-mediated transport. Namely, there does not appear to be a single mechanism modulating OMV export, nor universal "markers" for OMV cargo incorporation, nor particular host cell responses common to treatment with all OMVs. Given the diversity of species studied, their differences in envelope architecture and composition, the diversity of environmentally regulated bacterial processes, and the variety of interactions between bacteria and their abiotic and biotic environments, this is hardly surprising. Nevertheless, the ability of bacteria to control exported material in the context of a packaged insoluble particle, a vesicle, is emerging as a significant contribution to bacterial viability, biofilm communities, and bacterial-host interactions. In this review, we focus on detailing important, recent findings regarding the content and functional differences in bacterially secreted vesicles that are influenced by growth conditions.

Authors
Orench-Rivera, N; Kuehn, MJ
MLA Citation
Orench-Rivera, N, and Kuehn, MJ. "Environmentally controlled bacterial vesicle-mediated export." Cellular microbiology 18.11 (November 2016): 1525-1536.
Website
http://hdl.handle.net/10161/13025
PMID
27673272
Source
epmc
Published In
Cellular Microbiology
Volume
18
Issue
11
Publish Date
2016
Start Page
1525
End Page
1536
DOI
10.1111/cmi.12676

Outer Membrane Vesicle Production Facilitates LPS Remodeling and Outer Membrane Maintenance in Salmonella during Environmental Transitions.

The ability of Gram-negative bacteria to carefully modulate outer membrane (OM) composition is essential to their survival. However, the asymmetric and heterogeneous structure of the Gram-negative OM poses unique challenges to the cell's successful adaption to rapid environmental transitions. Although mechanisms to recycle and degrade OM phospholipid material exist, there is no known mechanism by which to remove unfavorable lipopolysaccharide (LPS) glycoforms, except slow dilution through cell growth. As all Gram-negative bacteria constitutively shed OM vesicles (OMVs), we propose that cells may utilize OMV formation as a way to selectively remove environmentally disadvantageous LPS species. We examined the native kinetics of OM composition during physiologically relevant environmental changes in Salmonella enterica, a well-characterized model system for activation of PhoP/Q and PmrA/B two-component systems (TCSs). In response to acidic pH, toxic metals, antimicrobial peptides, and lack of divalent cations, these TCSs modify the LPS lipid A and core, lengthen the O antigen, and upregulate specific OM proteins. An environmental change to PhoP/Q- and PmrA/B-activating conditions simultaneously induced the addition of modified species of LPS to the OM, downregulation of previously dominant species of LPS, greater OMV production, and increased OMV diameter. Comparison of the relative abundance of lipid A species present in the OM and the newly budded OMVs following two sets of rapid environmental shifts revealed the retention of lipid A species with modified phosphate moieties in the OM concomitant with the selective loss of palmitoylated species via vesiculation following exposure to moderately acidic environmental conditions.All Gram-negative bacteria alter the structural composition of LPS present in their OM in response to various environmental stimuli. We developed a system to track the native dynamics of lipid A change in Salmonella enterica serovar Typhimurium following an environmental shift to PhoP/Q- and PmrA/B-inducing conditions. We show that growth conditions influence OMV production, size, and lipid A content. We further demonstrate that the lipid A content of OMVs does not fit a stochastic model of content selection, revealing the significant retention of lipid A species containing covalent modifications that mask their 1- and 4'-phosphate moieties under host-like conditions. Furthermore, palmitoylation of the lipid A to form hepta-acylated species substantially increases the likelihood of its incorporation into OMVs. These results highlight a role for the OMV response in OM remodeling and maintenance processes in Gram-negative bacteria.

Authors
Bonnington, KE; Kuehn, MJ
MLA Citation
Bonnington, KE, and Kuehn, MJ. "Outer Membrane Vesicle Production Facilitates LPS Remodeling and Outer Membrane Maintenance in Salmonella during Environmental Transitions." mBio 7.5 (October 18, 2016).
Website
http://hdl.handle.net/10161/13027
PMID
27795394
Source
epmc
Published In
mBio
Volume
7
Issue
5
Publish Date
2016
DOI
10.1128/mbio.01532-16

A standardized method to determine the concentration of extracellular vesicles using tunable resistive pulse sensing.

Understanding the pathogenic role of extracellular vesicles (EVs) in disease and their potential diagnostic and therapeutic utility is extremely reliant on in-depth quantification, measurement and identification of EV sub-populations. Quantification of EVs has presented several challenges, predominantly due to the small size of vesicles such as exosomes and the availability of various technologies to measure nanosized particles, each technology having its own limitations.A standardized methodology to measure the concentration of extracellular vesicles (EVs) has been developed and tested. The method is based on measuring the EV concentration as a function of a defined size range. Blood plasma EVs are isolated and purified using size exclusion columns (qEV) and consecutively measured with tunable resistive pulse sensing (TRPS). Six independent research groups measured liposome and EV samples with the aim to evaluate the developed methodology. Each group measured identical samples using up to 5 nanopores with 3 repeat measurements per pore. Descriptive statistics and unsupervised multivariate data analysis with principal component analysis (PCA) were used to evaluate reproducibility across the groups and to explore and visualise possible patterns and outliers in EV and liposome data sets.PCA revealed good reproducibility within and between laboratories, with few minor outlying samples. Measured mean liposome (not filtered with qEV) and EV (filtered with qEV) concentrations had coefficients of variance of 23.9% and 52.5%, respectively. The increased variance of the EV concentration measurements could be attributed to the use of qEVs and the polydisperse nature of EVs.The results of this study demonstrate the feasibility of this standardized methodology to facilitate comparable and reproducible EV concentration measurements.

Authors
Vogel, R; Coumans, FAW; Maltesen, RG; Böing, AN; Bonnington, KE; Broekman, ML; Broom, MF; Buzás, EI; Christiansen, G; Hajji, N; Kristensen, SR; Kuehn, MJ; Lund, SM; Maas, SLN; Nieuwland, R; Osteikoetxea, X; Schnoor, R; Scicluna, BJ; Shambrook, M; de Vrij, J; Mann, SI; Hill, AF; Pedersen, S
MLA Citation
Vogel, R, Coumans, FAW, Maltesen, RG, Böing, AN, Bonnington, KE, Broekman, ML, Broom, MF, Buzás, EI, Christiansen, G, Hajji, N, Kristensen, SR, Kuehn, MJ, Lund, SM, Maas, SLN, Nieuwland, R, Osteikoetxea, X, Schnoor, R, Scicluna, BJ, Shambrook, M, de Vrij, J, Mann, SI, Hill, AF, and Pedersen, S. "A standardized method to determine the concentration of extracellular vesicles using tunable resistive pulse sensing." Journal of extracellular vesicles 5 (January 2016): 31242-.
Website
http://hdl.handle.net/10161/13026
PMID
27680301
Source
epmc
Published In
Journal of Extracellular Vesicles
Volume
5
Publish Date
2016
Start Page
31242
DOI
10.3402/jev.v5.31242

Outer-membrane vesicles from Gram-negative bacteria: biogenesis and functions.

Outer-membrane vesicles (OMVs) are spherical buds of the outer membrane filled with periplasmic content and are commonly produced by Gram-negative bacteria. The production of OMVs allows bacteria to interact with their environment, and OMVs have been found to mediate diverse functions, including promoting pathogenesis, enabling bacterial survival during stress conditions and regulating microbial interactions within bacterial communities. Additionally, because of this functional versatility, researchers have begun to explore OMVs as a platform for bioengineering applications. In this Review, we discuss recent advances in the study of OMVs, focusing on new insights into the mechanisms of biogenesis and the functions of these vesicles.

Authors
Schwechheimer, C; Kuehn, MJ
MLA Citation
Schwechheimer, C, and Kuehn, MJ. "Outer-membrane vesicles from Gram-negative bacteria: biogenesis and functions." Nature reviews. Microbiology 13.10 (October 2015): 605-619. (Review)
PMID
26373371
Source
epmc
Published In
Nature Reviews Microbiology
Volume
13
Issue
10
Publish Date
2015
Start Page
605
End Page
619
DOI
10.1038/nrmicro3525

NlpI-mediated modulation of outer membrane vesicle production through peptidoglycan dynamics in Escherichia coli.

Outer membrane vesicles (OMVs) are ubiquitously secreted from the outer membrane (OM) of Gram-negative bacteria. These heterogeneous structures are composed of OM filled with periplasmic content from the site of budding. By analyzing mutants that have vesicle production phenotypes, we can gain insight into the mechanism of OMV budding in wild-type cells, which has thus far remained elusive. In this study, we present data demonstrating that the hypervesiculation phenotype of the nlpI deletion mutant of Escherichia coli correlates with changes in peptidoglycan (PG) dynamics. Our data indicate that in stationary phase cultures the nlpI mutant exhibits increased PG synthesis that is dependent on spr, consistent with a model in which NlpI controls the activity of the PG endopeptidase Spr. In log phase, the nlpI mutation was suppressed by a dacB mutation, suggesting that NlpI regulates penicillin-binding protein 4 (PBP4) during exponential growth. The data support a model in which NlpI negatively regulates PBP4 activity during log phase, and Spr activity during stationary phase, and that in the absence of NlpI, the cell survives by increasing PG synthesis. Further, the nlpI mutant exhibited a significant decrease in covalent outer membrane (OM-PG) envelope stabilizing cross-links, consistent with its high level of OMV production. Based on these results, we propose that one mechanism wild-type Gram-negative bacteria can use to modulate vesiculation is by altering PG-OM cross-linking via localized modulation of PG degradation and synthesis.

Authors
Schwechheimer, C; Rodriguez, DL; Kuehn, MJ
MLA Citation
Schwechheimer, C, Rodriguez, DL, and Kuehn, MJ. "NlpI-mediated modulation of outer membrane vesicle production through peptidoglycan dynamics in Escherichia coli." MicrobiologyOpen 4.3 (June 2015): 375-389.
Website
http://hdl.handle.net/10161/10653
PMID
25755088
Source
epmc
Published In
MicrobiologyOpen
Volume
4
Issue
3
Publish Date
2015
Start Page
375
End Page
389
DOI
10.1002/mbo3.244

Genome-Wide Assessment of Outer Membrane Vesicle Production in Escherichia coli.

The production of outer membrane vesicles by Gram-negative bacteria has been well documented; however, the mechanism behind the biogenesis of these vesicles remains unclear. Here a high-throughput experimental method and systems-scale analysis was conducted to determine vesiculation values for the whole genome knockout library of Escherichia coli mutant strains (Keio collection). The resultant dataset quantitatively recapitulates previously observed phenotypes and implicates nearly 150 new genes in the process of vesiculation. Gene functional and biochemical pathway analyses suggest that mutations that truncate outer membrane structures such as lipopolysaccharide and enterobacterial common antigen lead to hypervesiculation, whereas mutants in oxidative stress response pathways result in lower levels. This study expands and refines the current knowledge regarding the cellular pathways required for outer membrane vesiculation in E. coli.

Authors
Kulp, AJ; Sun, B; Ai, T; Manning, AJ; Orench-Rivera, N; Schmid, AK; Kuehn, MJ
MLA Citation
Kulp, AJ, Sun, B, Ai, T, Manning, AJ, Orench-Rivera, N, Schmid, AK, and Kuehn, MJ. "Genome-Wide Assessment of Outer Membrane Vesicle Production in Escherichia coli." PloS one 10.9 (January 2015): e0139200-.
Website
http://hdl.handle.net/10161/11279
PMID
26406465
Source
epmc
Published In
PloS one
Volume
10
Issue
9
Publish Date
2015
Start Page
e0139200
DOI
10.1371/journal.pone.0139200

Modulation of bacterial outer membrane vesicle production by envelope structure and content.

Vesiculation is a ubiquitous secretion process of Gram-negative bacteria, where outer membrane vesicles (OMVs) are small spherical particles on the order of 50 to 250 nm composed of outer membrane (OM) and lumenal periplasmic content. Vesicle functions have been elucidated in some detail, showing their importance in virulence factor secretion, bacterial survival, and biofilm formation in pathogenesis. Furthermore, OMVs serve as an envelope stress response, protecting the secreting bacteria from internal protein misfolding stress, as well as external envelope stressors. Despite their important functional roles very little is known about the regulation and mechanism of vesicle production. Based on the envelope architecture and prior characterization of the hypervesiculation phenotypes for mutants lacking the lipoprotein, Lpp, which is involved in the covalent OM-peptidoglycan (PG) crosslinks, it is expected that an inverse relationship exists between OMV production and PG-crosslinked Lpp.In this study, we found that subtle modifications of PG remodeling and crosslinking modulate OMV production, inversely correlating with bound Lpp levels. However, this inverse relationship was not found in strains in which OMV production is driven by an increase in "periplasmic pressure" resulting from the accumulation of protein, PG fragments, or lipopolysaccharide. In addition, the characterization of an nlpA deletion in backgrounds lacking either Lpp- or OmpA-mediated envelope crosslinks demonstrated a novel role for NlpA in envelope architecture.From this work, we conclude that OMV production can be driven by distinct Lpp concentration-dependent and Lpp concentration-independent pathways.

Authors
Schwechheimer, C; Kulp, A; Kuehn, MJ
MLA Citation
Schwechheimer, C, Kulp, A, and Kuehn, MJ. "Modulation of bacterial outer membrane vesicle production by envelope structure and content." BMC microbiology 14 (December 21, 2014): 324-.
Website
http://hdl.handle.net/10161/10654
PMID
25528573
Source
epmc
Published In
BMC Microbiology
Volume
14
Publish Date
2014
Start Page
324
DOI
10.1186/s12866-014-0324-1

Protein selection and export via outer membrane vesicles.

Outer membrane vesicles (OMVs) are constitutively produced by all Gram-negative bacteria. OMVs form when buds from the outer membrane (OM) of cells encapsulate periplasmic material and pinch off from the OM to form spheroid particles approximately 10 to 300nm in diameter. OMVs accomplish a diversity of functional roles yet the OMV's utility is ultimately determined by its unique composition. Inclusion into OMVs may impart a variety of benefits to the protein cargo, including: protection from proteolytic degradation, enhancement of long-distance delivery, specificity in host-cell targeting, modulation of the immune response, coordinated secretion with other bacterial effectors, and/or exposure to a unique function-promoting environment. Many enriched OMV-associated components are virulence factors, aiding in host cell destruction, immune system evasion, host cell invasion, or antibiotic resistance. Although the mechanistic details of how proteins become enriched as OMV cargo remain elusive, recent data on OM biogenesis and relationships between LPS structure and OMV-cargo inclusion rates shed light on potential models for OM organization and consequent OMV budding. In this review, mechanisms based on pre-existing OM microdomains are proposed to explain how cargo may experience differing levels of enrichment in OMVs and degrees of association with OMVs during extracellular export. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.

Authors
Bonnington, KE; Kuehn, MJ
MLA Citation
Bonnington, KE, and Kuehn, MJ. "Protein selection and export via outer membrane vesicles." Biochim Biophys Acta 1843.8 (August 2014): 1612-1619. (Review)
PMID
24370777
Source
pubmed
Published In
Biochimica et Biophysica Acta: international journal of biochemistry and biophysics
Volume
1843
Issue
8
Publish Date
2014
Start Page
1612
End Page
1619
DOI
10.1016/j.bbamcr.2013.12.011

Synthetic effect between envelope stress and lack of outer membrane vesicle production in Escherichia coli.

Outer membrane vesicles (OMVs) are composed of outer membrane and periplasmic components and are ubiquitously secreted by Gram-negative bacteria. OMVs can disseminate virulence factors for pathogenic bacteria as well as serve as an envelope stress response. From a transposon mutant screen for OMV phenotypes, it was discovered that an nlpA mutant of Escherichia coli produces fewer OMVs than the wild type, whereas a degP mutant produces higher levels of OMVs. NlpA is an inner-membrane-anchored lipoprotein that has a minor role in methionine import. DegP is a periplasmic chaperone/protease for misfolded envelope proteins that is critical when cells are heat shocked. To reveal how these proteins contribute to OMV production, the mutations were combined and the double mutant analyzed. The ΔnlpA ΔdegP strain displayed a high-temperature growth defect that corresponded to the production of fewer OMVs than produced by the ΔdegP strain. This phenotype also pertained to other undervesiculation mutations in a ΔdegP background. The hypovesiculation phenotype of ΔnlpA in the wild-type strain as well as in the degP deletion strain was found to be a stationary-phase phenomenon. The periplasm of the ΔnlpA ΔdegP strain was determined to contain significantly more protein in stationary phase than the wild type. Additionally, misfolded DegP substrate outer membrane porins were detected in ΔdegP mutant-derived OMVs. These data suggest that an accumulation of envelope proteins resulting from decreased vesiculation was toxic and contributed to the growth defect. We conclude that OMV production contributes to relieve the envelope of accumulated toxic proteins and that NlpA plays an important role in the production of vesicles in stationary phase.

Authors
Schwechheimer, C; Kuehn, MJ
MLA Citation
Schwechheimer, C, and Kuehn, MJ. "Synthetic effect between envelope stress and lack of outer membrane vesicle production in Escherichia coli." J Bacteriol 195.18 (September 2013): 4161-4173.
PMID
23852867
Source
pubmed
Published In
Journal of bacteriology
Volume
195
Issue
18
Publish Date
2013
Start Page
4161
End Page
4173
DOI
10.1128/JB.02192-12

Stress-Induced Outer Membrane Vesicle Production by Pseudomonas aeruginosa

Authors
MacDonald, IA; Kuehn, MJ
MLA Citation
MacDonald, IA, and Kuehn, MJ. "Stress-Induced Outer Membrane Vesicle Production by Pseudomonas aeruginosa." JOURNAL OF BACTERIOLOGY 195.13 (July 2013): 2971-2981.
Website
http://hdl.handle.net/10161/10655
PMID
23625841
Source
wos-lite
Published In
Journal of bacteriology
Volume
195
Issue
13
Publish Date
2013
Start Page
2971
End Page
2981
DOI
10.1128/JB.02267-12

Envelope control of outer membrane vesicle production in Gram-negative bacteria.

All Gram-negative bacteria studied to date have been shown to produce outer membrane vesicles (OMVs), which are budded, released spheres of outer membrane with periplasmic content. OMVs have been implicated in the delivery of virulence factors in pathogenesis. However, OMVs also benefit nonpathogenic species by delivering degradative enzymes to defend an ecological niche against competing bacterial species, and they can serve as an envelope stress response. Despite these important roles, very little is known about the mechanism of production of OMVs. Here we review the advantage of vesiculation, particularly in a nonpathogenic context, as well as the hurdles that have to be overcome in Gram-negative envelope architecture before a vesicle can form and bud. Lastly, we address the question of whether OMV production is a stochastic or regulated process.

Authors
Schwechheimer, C; Sullivan, CJ; Kuehn, MJ
MLA Citation
Schwechheimer, C, Sullivan, CJ, and Kuehn, MJ. "Envelope control of outer membrane vesicle production in Gram-negative bacteria." Biochemistry 52.18 (May 7, 2013): 3031-3040.
PMID
23521754
Source
pubmed
Published In
Biochemistry
Volume
52
Issue
18
Publish Date
2013
Start Page
3031
End Page
3040
DOI
10.1021/bi400164t

Quantitative and qualitative preparations of bacterial outer membrane vesicles.

Gram-negative bacterial outer membrane vesicle production and function have been studied using a variety of quantitative and qualitative methods. These types of analyses can be hampered by the use of impure vesicle preparations. Here we describe a set of techniques that are useful for the quantitative analysis of vesicle production and for preparative yields of highly purified vesicles for studies of vesicle function or composition. Procedures and advice are also included for the purification of vesicles from encapsulated and low-yield strains.

Authors
Chutkan, H; Macdonald, I; Manning, A; Kuehn, MJ
MLA Citation
Chutkan, H, Macdonald, I, Manning, A, and Kuehn, MJ. "Quantitative and qualitative preparations of bacterial outer membrane vesicles." Methods Mol Biol 966 (2013): 259-272.
PMID
23299740
Source
pubmed
Published In
Methods in molecular biology (Clifton, N.J.)
Volume
966
Publish Date
2013
Start Page
259
End Page
272
DOI
10.1007/978-1-62703-245-2_16

Functional advantages conferred by extracellular prokaryotic membrane vesicles.

The absence of subcellular organelles is a characteristic typically used to distinguish prokaryotic from eukaryotic cells. But recent discoveries do not support this dogma. Over the past 50 years, researchers have begun to appreciate and characterize Gram-negative bacterial outer membrane-derived vesicles and Gram-positive and archaeal membrane vesicles. These extracellular, membrane-bound organelles can perform a variety of functions, including binding and delivery of DNA, transport of virulence factors, protection of the cell from outer membrane targeting antimicrobials and ridding the cell of toxic envelope proteins. Here, we review the contributions of these extracellular organelles to prokaryotic physiology and compare these with the contributions of the bacterial interior membrane-bound organelles responsible for harvesting light energy and for generating magnetic crystals of heavy metals. Understanding the roles of these multifunctional extracellular vesicle organelles as microbial tools will help us to better realize the diverse interactions that occur in our polymicrobial world.

Authors
Manning, AJ; Kuehn, MJ
MLA Citation
Manning, AJ, and Kuehn, MJ. "Functional advantages conferred by extracellular prokaryotic membrane vesicles." J Mol Microbiol Biotechnol 23.1-2 (2013): 131-141. (Review)
PMID
23615201
Source
pubmed
Published In
Journal of molecular microbiology and biotechnology
Volume
23
Issue
1-2
Publish Date
2013
Start Page
131
End Page
141
DOI
10.1159/000346548

Offense and defense: microbial membrane vesicles play both ways.

Microbes have evolved over millennia to become adapted and specialized to the environments that they occupy. These environments may include water or soil, extreme environments such as hydrothermal vents, and can even include a host organism. To become adapted to these locations, microbes have evolved specific tools to mediate interactions with the environment. One such tool that prokaryotes have evolved includes the production of membrane vesicles (MVs). MVs are 10-300 nm spherical blebs derived from the outermost membrane and have known functions in protein secretion, immune activation and suppression, stress response, attachment, internalization and virulence. In this review, we consider the highly conserved role of membrane vesicles derived from Gram-negative, Gram-positive and archaeal species as a mechanism to facilitate intermicrobial and microbe-host interaction. We examine both the offensive and defensive capabilities of MVs in regard to the interaction of MVs with both host and microbial cells in their environment.

Authors
MacDonald, IA; Kuehn, MJ
MLA Citation
MacDonald, IA, and Kuehn, MJ. "Offense and defense: microbial membrane vesicles play both ways." Res Microbiol 163.9-10 (November 2012): 607-618. (Review)
PMID
23123555
Source
pubmed
Published In
Research in Microbiology
Volume
163
Issue
9-10
Publish Date
2012
Start Page
607
End Page
618
DOI
10.1016/j.resmic.2012.10.020

The inoculum effect and band-pass bacterial response to periodic antibiotic treatment.

The inoculum effect (IE) refers to the decreasing efficacy of an antibiotic with increasing bacterial density. It represents a unique strategy of antibiotic tolerance and it can complicate design of effective antibiotic treatment of bacterial infections. To gain insight into this phenomenon, we have analyzed responses of a lab strain of Escherichia coli to antibiotics that target the ribosome. We show that the IE can be explained by bistable inhibition of bacterial growth. A critical requirement for this bistability is sufficiently fast degradation of ribosomes, which can result from antibiotic-induced heat-shock response. Furthermore, antibiotics that elicit the IE can lead to 'band-pass' response of bacterial growth to periodic antibiotic treatment: the treatment efficacy drastically diminishes at intermediate frequencies of treatment. Our proposed mechanism for the IE may be generally applicable to other bacterial species treated with antibiotics targeting the ribosomes.

Authors
Tan, C; Smith, RP; Srimani, JK; Riccione, KA; Prasada, S; Kuehn, M; You, L
MLA Citation
Tan, C, Smith, RP, Srimani, JK, Riccione, KA, Prasada, S, Kuehn, M, and You, L. "The inoculum effect and band-pass bacterial response to periodic antibiotic treatment." Mol Syst Biol 8 (2012): 617-.
Website
http://hdl.handle.net/10161/10659
PMID
23047527
Source
pubmed
Published In
Molecular systems biology
Volume
8
Publish Date
2012
Start Page
617
DOI
10.1038/msb.2012.49

Secreted bacterial vesicles as good samaritans

Bacteriodes fragilis and polysaccharide A capsular antigen (PSA) produced by this commensal bacteria can mediate immune tolerance in the gastrointestinal (GI) tract. When looking for naturally secreted forms of PSA, Shen et al. (2012) suprisingly found that PSA is packaged in outer membrane vesicles (OMVs), bacterial blebs with a disreputable past. © 2012 Elsevier Inc.

Authors
Kuehn, MJ
MLA Citation
Kuehn, MJ. "Secreted bacterial vesicles as good samaritans." Cell Host and Microbe 12.4 (2012): 392-393.
PMID
23084908
Source
scival
Published In
Cell Host & Microbe
Volume
12
Issue
4
Publish Date
2012
Start Page
392
End Page
393
DOI
10.1016/j.chom.2012.10.005

Contribution of bacterial outer membrane vesicles to innate bacterial defense.

BACKGROUND: Outer membrane vesicles (OMVs) are constitutively produced by Gram-negative bacteria throughout growth and have proposed roles in virulence, inflammation, and the response to envelope stress. Here we investigate outer membrane vesiculation as a bacterial mechanism for immediate short-term protection against outer membrane acting stressors. Antimicrobial peptides as well as bacteriophage were used to examine the effectiveness of OMV protection. RESULTS: We found that a hyper-vesiculating mutant of Escherichia coli survived treatment by antimicrobial peptides (AMPs) polymyxin B and colistin better than the wild-type. Supplementation of E. coli cultures with purified outer membrane vesicles provided substantial protection against AMPs, and AMPs significantly induced vesiculation. Vesicle-mediated protection and induction of vesiculation were also observed for a human pathogen, enterotoxigenic E. coli (ETEC), challenged with polymyxin B. When ETEC with was incubated with low concentrations of vesicles concomitant with polymyxin B treatment, bacterial survival increased immediately, and the culture gained resistance to polymyxin B. By contrast, high levels of vesicles also provided immediate protection but prevented acquisition of resistance. Co-incubation of T4 bacteriophage and OMVs showed fast, irreversible binding. The efficiency of T4 infection was significantly reduced by the formation of complexes with the OMVs. CONCLUSIONS: These data reveal a role for OMVs in contributing to innate bacterial defense by adsorption of antimicrobial peptides and bacteriophage. Given the increase in vesiculation in response to the antimicrobial peptides, and loss in efficiency of infection with the T4-OMV complex, we conclude that OMV production may be an important factor in neutralizing environmental agents that target the outer membrane of Gram-negative bacteria.

Authors
Manning, AJ; Kuehn, MJ
MLA Citation
Manning, AJ, and Kuehn, MJ. "Contribution of bacterial outer membrane vesicles to innate bacterial defense. (Published online)" BMC Microbiol 11 (December 1, 2011): 258-.
Website
http://hdl.handle.net/10161/10657
PMID
22133164
Source
pubmed
Published In
BMC Microbiology
Volume
11
Publish Date
2011
Start Page
258
DOI
10.1186/1471-2180-11-258

Recognition of β-strand motifs by RseB is required for σ(E) activity in Escherichia coli.

Gram-negative bacteria react to misfolded proteins in the envelope through a myriad of different stress response pathways. This cohort of pathways allows the bacteria to specifically respond to different types of damage, and many of these have been discovered to have key roles in the virulence of bacterial pathogens. Misfolded outer membrane proteins (OMPs) are typically recognized by the σ(E) pathway, a highly conserved envelope stress response pathway. We examined the features of misfolded OMPs with respect to their ability to generate envelope stress responses. We determined that the secondary structure, particularly the potential to form β strands, is critical to inducing the σ(E) response in an RseB-dependent manner. The sequence of the potential β-strand motif modulates the strength of the σ(E) response generated by the constructs. By understanding the details of how such stress response pathways are activated, we can gain a greater understanding of how bacteria survive in harsh environments.

Authors
Kulp, A; Kuehn, MJ
MLA Citation
Kulp, A, and Kuehn, MJ. "Recognition of β-strand motifs by RseB is required for σ(E) activity in Escherichia coli." J Bacteriol 193.22 (November 2011): 6179-6186.
PMID
21908666
Source
pubmed
Published In
Journal of bacteriology
Volume
193
Issue
22
Publish Date
2011
Start Page
6179
End Page
6186
DOI
10.1128/JB.05657-11

Context-dependent activation kinetics elicited by soluble versus outer membrane vesicle-associated heat-labile enterotoxin.

Enterotoxigenic Escherichia coli (ETEC) is the leading cause of traveler's diarrhea and children's diarrhea worldwide. Among its virulence factors, ETEC produces heat-labile enterotoxin (LT). Most secreted LT is associated with outer membrane vesicles that are rich in lipopolysaccharide. The majority of prior studies have focused on soluble LT purified from ETEC periplasm. We investigated the hypothesis that the extracellular vesicle context of toxin presentation might be important in eliciting immune responses. We compared the polarized epithelial cell responses to apically applied soluble LT and LT-containing vesicles (LT(+) vesicles) as well as controls using a catalytically inactive mutant of LT and vesicles lacking LT. Although vesicle treatments with no or catalytically inactive LT induced a modest amount of interleukin-6 (IL-6), samples containing catalytically active LT elicited higher levels. A combination of soluble LT and LT-deficient vesicles induced significantly higher IL-6 levels than either LT or LT(+) vesicles alone. The responses to LT(+) vesicles were found to be independent of the canonical LT pathway, because the inhibition of cyclic AMP response element (CRE)-binding protein (CREB) phosphorylation did not lead to a decrease in cytokine gene expression levels. Furthermore, soluble LT caused earlier phosphorylation of CREB and activation of CRE compared with LT(+) vesicles. Soluble LT also led to the activation of activator protein 1, whereas LT(+) vesicle IL-6 responses appeared to be mediated by NF-κB. In summary, the results demonstrate that soluble LT and vesicle-bound LT elicit ultimately similar cytokine responses through distinct different activation pathways.

Authors
Chutkan, H; Kuehn, MJ
MLA Citation
Chutkan, H, and Kuehn, MJ. "Context-dependent activation kinetics elicited by soluble versus outer membrane vesicle-associated heat-labile enterotoxin." Infect Immun 79.9 (September 2011): 3760-3769.
PMID
21708992
Source
pubmed
Published In
Infection and immunity
Volume
79
Issue
9
Publish Date
2011
Start Page
3760
End Page
3769
DOI
10.1128/IAI.05336-11

Elicitation of epithelial cell-derived immune effectors by outer membrane vesicles of nontypeable haemophilus influenzae

Outer membrane vesicles (OMVs) are produced by all Gram-negative microorganisms studied to date. The contributions of OMVs to biological processes are diverse and include mediation of bacterial stress responses, selective packaging and secretion of virulence determinants, modulation of the host immune response, and contributions to biofilm formation and stability. First characterized as transformasomes in Haemophilus, these membranous blebs facilitate transfer of DNA among bacteria. Nontypeable Haemophilus influenzae (NTHI), an opportunistic pathogen of the upper and lower respiratory tracts, produces OMVs in vivo, but there is a paucity of information regarding both the composition and role of OMVs during NTHI colonization and pathogenesis. We demonstrated that purified NTHI vesicles are 20 to 200 nm in diameter and contain DNA, adhesin P5, IgA endopeptidase, serine protease, and heme utilization protein, suggesting a multifaceted role in virulence. NTHI OMVs can bind to human pharyngeal epithelial cells, resulting in a time- and temperature-dependent aggregation on the host cell surface, with subsequent internalization. OMVs colocalize with the endocytosis protein caveolin, indicating that internalization is mediated by caveolae, which are cholesterol-rich lipid raft domains. Upon interaction with epithelial cells, NTHI OMVs stimulate significant release of the immunomodulatory cytokine interleukin-8 (IL-8) as well as the antimicrobial peptide LL-37. Thus, we demonstrated that NTHI OMVs contain virulence-associated proteins that dynamically interact with and invade host epithelial cells. Beyond their ability to mediate DNA transfer in Haemophilus, OMV stimulation of host immunomodulatory cytokine and antimicrobial peptide release supports a dynamic role for vesiculation in NTHI pathogenesis and clinically relevant disease progression. © 2011, American Society for Microbiology.

Authors
Sharpe, SW; Kuehn, MJ; Mason, KM
MLA Citation
Sharpe, SW, Kuehn, MJ, and Mason, KM. "Elicitation of epithelial cell-derived immune effectors by outer membrane vesicles of nontypeable haemophilus influenzae." Infection and Immunity 79.11 (2011): 4361-4369.
PMID
21875967
Source
scival
Published In
Infection and immunity
Volume
79
Issue
11
Publish Date
2011
Start Page
4361
End Page
4369
DOI
10.1128/IAI.05332-11

Immunization with Salmonella enterica serovar typhimurium-derived outer membrane vesicles delivering the pneumococcal protein PspA confers protection against challenge with Streptococcus pneumoniae

Gram-negative bacteria produce outer membrane vesicles (OMVs) that serve a variety of functions related to survival and pathogenicity. Periplasmic and outer membrane proteins are naturally captured during vesicle formation. This property has been exploited as a method to derive immunogenic vesicle preparations for use as vaccines. In this work, we constructed a Salmonella enterica serovar Typhimurium strain that synthesized a derivative of the pneumococcal protein PspA engineered to be secreted into the periplasmic space. Vesicles isolated from this strain contained PspA in the lumen. Mice intranasally immunized with the vesicle preparation developed serum antibody responses against vesicle components that included PspA and Salmonella-derived lipopolysaccharide and outer membrane proteins, while no detectable responses developed in mice immunized with an equivalent dose of purified PspA. Mucosal IgA responses developed against the Salmonella components, while the response to PspA was less apparent in most mice. Mice immunized with the vesicle preparation were completely protected against a 10x 50% lethal dose (LD50) challenge of Streptococcus pneumoniae and significantly protected against a 200x LD50 challenge, while control mice immunized with purified PspA or empty vesicles were not protected. These results establish that vesicles can be used to mucosally deliver an antigen from a Gram-positive organism and induce a protective immune response. Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Authors
Muralinath, M; Kuehn, MJ; Roland, KL; III, RC
MLA Citation
Muralinath, M, Kuehn, MJ, Roland, KL, and III, RC. "Immunization with Salmonella enterica serovar typhimurium-derived outer membrane vesicles delivering the pneumococcal protein PspA confers protection against challenge with Streptococcus pneumoniae." Infection and Immunity 79.2 (2011): 887-894.
PMID
21115718
Source
scival
Published In
Infection and immunity
Volume
79
Issue
2
Publish Date
2011
Start Page
887
End Page
894
DOI
10.1128/IAI.00950-10

Naturally produced outer membrane vesicles from Pseudomonas aeruginosa elicit a potent innate immune response via combined sensing of both lipopolysaccharide and protein components.

Pseudomonas aeruginosa is a prevalent opportunistic human pathogen that, like other Gram-negative pathogens, secretes outer membrane vesicles. Vesicles are complex entities composed of a subset of envelope lipid and protein components that have been observed to interact with and be internalized by host cells. This study characterized the inflammatory responses to naturally produced P. aeruginosa vesicles and determined the contribution of vesicle Toll-like receptor (TLR) ligands and vesicle proteins to that response. Analysis of macrophage responses to purified vesicles by real-time PCR and enzyme-linked immunosorbent assay identified proinflammatory cytokines upregulated by vesicles. Intact vesicles were shown to elicit a profoundly greater inflammatory response than the response to purified lipopolysaccharide (LPS). Both TLR ligands LPS and flagellin contributed to specific vesicle cytokine responses, whereas the CpG DNA content of vesicles did not. Neutralization of LPS sensing demonstrated that macrophage responses to the protein composition of vesicles required the adjuvantlike activity of LPS to elicit strain specific responses. Protease treatment to remove proteins from the vesicle surface resulted in decreased interleukin-6 and tumor necrosis factor alpha production, indicating that the production of these specific cytokines may be linked to macrophage recognition of vesicle proteins. Confocal microscopy of vesicle uptake by macrophages revealed that vesicle LPS allows for binding to macrophage surfaces, whereas vesicle protein content is required for internalization. These data demonstrate that macrophage sensing of both LPS and protein components of outer membrane vesicles combine to produce a bacterial strain-specific response that is distinct from those triggered by individual, purified vesicle components.

Authors
Ellis, TN; Leiman, SA; Kuehn, MJ
MLA Citation
Ellis, TN, Leiman, SA, and Kuehn, MJ. "Naturally produced outer membrane vesicles from Pseudomonas aeruginosa elicit a potent innate immune response via combined sensing of both lipopolysaccharide and protein components." Infect Immun 78.9 (September 2010): 3822-3831.
PMID
20605984
Source
pubmed
Published In
Infection and immunity
Volume
78
Issue
9
Publish Date
2010
Start Page
3822
End Page
3831
DOI
10.1128/IAI.00433-10

Heat-labile enterotoxin: beyond G(m1) binding.

Enterotoxigenic Escherichia coli (ETEC) is a significant source of morbidity and mortality worldwide. One major virulence factor released by ETEC is the heat-labile enterotoxin LT, which is structurally and functionally similar to cholera toxin. LT consists of five B subunits carrying a single catalytically active A subunit. LTB binds the monosialoganglioside G(M1), the toxin's host receptor, but interactions with A-type blood sugars and E. coli lipopolysaccharide have also been identified within the past decade. Here, we review the regulation, assembly, and binding properties of the LT B-subunit pentamer and discuss the possible roles of its numerous molecular interactions.

Authors
Mudrak, B; Kuehn, MJ
MLA Citation
Mudrak, B, and Kuehn, MJ. "Heat-labile enterotoxin: beyond G(m1) binding." Toxins (Basel) 2.6 (June 2010): 1445-1470. (Review)
Website
http://hdl.handle.net/10161/10656
PMID
22069646
Source
pubmed
Published In
Toxins
Volume
2
Issue
6
Publish Date
2010
Start Page
1445
End Page
1470
DOI
10.3390/toxins2061445

Specificity of the type II secretion systems of enterotoxigenic Escherichia coli and Vibrio cholerae for heat-labile enterotoxin and cholera toxin.

The Gram-negative type II secretion (T2S) system is a multiprotein complex mediating the release of virulence factors from a number of pathogens. While an understanding of the function of T2S components is emerging, little is known about what identifies substrates for export. To investigate T2S substrate recognition, we compared mutations affecting the secretion of two highly homologous substrates: heat-labile enterotoxin (LT) from enterotoxigenic Escherichia coli (ETEC) and cholera toxin (CT) from Vibrio cholerae. Each toxin consists of one enzymatic A subunit and a ring of five B subunits mediating the toxin's secretion. Here, we report two mutations in LT's B subunit (LTB) that reduce its secretion from ETEC without global effects on the toxin. The Q3K mutation reduced levels of secreted LT by half, and as with CT (T. D. Connell, D. J. Metzger, M. Wang, M. G. Jobling, and R. K. Holmes, Infect. Immun. 63:4091-4098, 1995), the E11K mutation impaired LT secretion. Results in vitro and in vivo show that these mutants are not degraded more readily than wild-type LT. The Q3K mutation did not significantly affect CT B subunit (CTB) secretion from V. cholerae, and the E11A mutation altered LT and CTB secretion to various extents, indicating that these toxins are identified as secretion substrates in different ways. The levels of mutant LTB expressed in V. cholerae were low or undetectable, but each CTB mutant expressed and secreted at wild-type levels in ETEC. Therefore, ETEC's T2S system seems to accommodate mutations in CTB that impair the secretion of LTB. Our results highlight the exquisitely fine-tuned relationship between T2S substrates and their coordinate secretion machineries in different bacterial species.

Authors
Mudrak, B; Kuehn, MJ
MLA Citation
Mudrak, B, and Kuehn, MJ. "Specificity of the type II secretion systems of enterotoxigenic Escherichia coli and Vibrio cholerae for heat-labile enterotoxin and cholera toxin." J Bacteriol 192.7 (April 2010): 1902-1911.
PMID
20097854
Source
pubmed
Published In
Journal of bacteriology
Volume
192
Issue
7
Publish Date
2010
Start Page
1902
End Page
1911
DOI
10.1128/JB.01542-09

Virulence and immunomodulatory roles of bacterial outer membrane vesicles.

Outer membrane (OM) vesicles are ubiquitously produced by Gram-negative bacteria during all stages of bacterial growth. OM vesicles are naturally secreted by both pathogenic and nonpathogenic bacteria. Strong experimental evidence exists to categorize OM vesicle production as a type of Gram-negative bacterial virulence factor. A growing body of data demonstrates an association of active virulence factors and toxins with vesicles, suggesting that they play a role in pathogenesis. One of the most popular and best-studied pathogenic functions for membrane vesicles is to serve as natural vehicles for the intercellular transport of virulence factors and other materials directly into host cells. The production of OM vesicles has been identified as an independent bacterial stress response pathway that is activated when bacteria encounter environmental stress, such as what might be experienced during the colonization of host tissues. Their detection in infected human tissues reinforces this theory. Various other virulence factors are also associated with OM vesicles, including adhesins and degradative enzymes. As a result, OM vesicles are heavily laden with pathogen-associated molecular patterns (PAMPs), virulence factors, and other OM components that can impact the course of infection by having toxigenic effects or by the activation of the innate immune response. However, infected hosts can also benefit from OM vesicle production by stimulating their ability to mount an effective defense. Vesicles display antigens and can elicit potent inflammatory and immune responses. In sum, OM vesicles are likely to play a significant role in the virulence of Gram-negative bacterial pathogens.

Authors
Ellis, TN; Kuehn, MJ
MLA Citation
Ellis, TN, and Kuehn, MJ. "Virulence and immunomodulatory roles of bacterial outer membrane vesicles." Microbiol Mol Biol Rev 74.1 (March 2010): 81-94. (Review)
PMID
20197500
Source
pubmed
Published In
Microbiology and molecular biology reviews : MMBR
Volume
74
Issue
1
Publish Date
2010
Start Page
81
End Page
94
DOI
10.1128/MMBR.00031-09

Biological functions and biogenesis of secreted bacterial outer membrane vesicles.

Gram-negative bacteria produce outer membrane vesicles (OMVs) that contain biologically active proteins and perform diverse biological processes. Unlike other secretion mechanisms, OMVs enable bacteria to secrete insoluble molecules in addition to and in complex with soluble material. OMVs allow enzymes to reach distant targets in a concentrated, protected, and targeted form. OMVs also play roles in bacterial survival: Their production is a bacterial stress response and important for nutrient acquisition, biofilm development, and pathogenesis. Key characteristics of OMV biogenesis include outward bulging of areas lacking membrane-peptidoglycan bonds, the capacity to upregulate vesicle production without also losing outer membrane integrity, enrichment or exclusion of certain proteins and lipids, and membrane fission without direct energy from ATP/GTP hydrolysis. Comparisons of similar budding mechanisms from diverse biological domains have provided new insight into evaluating mechanisms for outer membrane vesiculation.

Authors
Kulp, A; Kuehn, MJ
MLA Citation
Kulp, A, and Kuehn, MJ. "Biological functions and biogenesis of secreted bacterial outer membrane vesicles." Annu Rev Microbiol 64 (2010): 163-184. (Review)
PMID
20825345
Source
pubmed
Published In
Annual Review of Microbiology
Volume
64
Publish Date
2010
Start Page
163
End Page
184
DOI
10.1146/annurev.micro.091208.073413

Residues of heat-labile enterotoxin involved in bacterial cell surface binding.

Enterotoxigenic Escherichia coli (ETEC) is a leading cause of traveler's diarrhea worldwide. One major virulence factor released by this pathogen is the heat-labile enterotoxin LT, which upsets the balance of electrolytes in the intestine. After export, LT binds to lipopolysaccharide (LPS) on the bacterial surface. Although the residues responsible for LT's binding to its host receptor are known, the portion of the toxin which mediates LPS binding has not been defined previously. Here, we describe mutations in LT that impair the binding of the toxin to the external surface of E. coli without altering holotoxin assembly. One mutation in particular, T47A, nearly abrogates surface binding without adversely affecting expression or secretion in ETEC. Interestingly, T47A is able to bind mutant E. coli expressing highly truncated forms of LPS, indicating that LT binding to wild-type LPS may be due primarily to association with an outer core sugar. Consequently, we have identified a region of LT distinct from the pocket involved in eukaryotic receptor binding that is responsible for binding to the surface of E. coli.

Authors
Mudrak, B; Rodriguez, DL; Kuehn, MJ
MLA Citation
Mudrak, B, Rodriguez, DL, and Kuehn, MJ. "Residues of heat-labile enterotoxin involved in bacterial cell surface binding." J Bacteriol 191.9 (May 2009): 2917-2925.
PMID
19270095
Source
pubmed
Published In
Journal of bacteriology
Volume
191
Issue
9
Publish Date
2009
Start Page
2917
End Page
2925
DOI
10.1128/JB.01622-08

Pseudomonas aeruginosa vesicles associate with and are internalized by human lung epithelial cells.

BACKGROUND: Pseudomonas aeruginosa is the major pathogen associated with chronic and ultimately fatal lung infections in patients with cystic fibrosis (CF). To investigate how P. aeruginosa-derived vesicles may contribute to lung disease, we explored their ability to associate with human lung cells. RESULTS: Purified vesicles associated with lung cells and were internalized in a time- and dose-dependent manner. Vesicles from a CF isolate exhibited a 3- to 4-fold greater association with lung cells than vesicles from the lab strain PAO1. Vesicle internalization was temperature-dependent and was inhibited by hypertonic sucrose and cyclodextrins. Surface-bound vesicles rarely colocalized with clathrin. Internalized vesicles colocalized with the endoplasmic reticulum (ER) marker, TRAPalpha, as well as with ER-localized pools of cholera toxin and transferrin. CF isolates of P. aeruginosa abundantly secrete PaAP (PA2939), an aminopeptidase that associates with the surface of vesicles. Vesicles from a PaAP knockout strain exhibited a 40% decrease in cell association. Likewise, vesicles from PAO1 overexpressing PaAP displayed a significant increase in cell association. CONCLUSION: These data reveal that PaAP promotes the association of vesicles with lung cells. Taken together, these results suggest that P. aeruginosa vesicles can interact with and be internalized by lung epithelial cells and contribute to the inflammatory response during infection.

Authors
Bauman, SJ; Kuehn, MJ
MLA Citation
Bauman, SJ, and Kuehn, MJ. "Pseudomonas aeruginosa vesicles associate with and are internalized by human lung epithelial cells. (Published online)" BMC Microbiol 9 (February 3, 2009): 26-.
Website
http://hdl.handle.net/10161/10660
PMID
19192306
Source
pubmed
Published In
BMC Microbiology
Volume
9
Publish Date
2009
Start Page
26
DOI
10.1186/1471-2180-9-26

Release of outer membrane vesicles by Gram-negative bacteria is a novel envelope stress response.

Conditions that impair protein folding in the Gram-negative bacterial envelope cause stress. The destabilizing effects of stress in this compartment are recognized and countered by a number of signal transduction mechanisms. Data presented here reveal another facet of the complex bacterial stress response, release of outer membrane vesicles. Native vesicles are composed of outer membrane and periplasmic material, and they are released from the bacterial surface without loss of membrane integrity. Here we demonstrate that the quantity of vesicle release correlates directly with the level of protein accumulation in the cell envelope. Accumulation of material occurs under stress, and is exacerbated upon impairment of the normal housekeeping and stress-responsive mechanisms of the cell. Mutations that cause increased vesiculation enhance bacterial survival upon challenge with stressing agents or accumulation of toxic misfolded proteins. Preferential packaging of a misfolded protein mimic into vesicles for removal indicates that the vesiculation process can act to selectively eliminate unwanted material. Our results demonstrate that production of bacterial outer membrane vesicles is a fully independent, general envelope stress response. In addition to identifying a novel mechanism for alleviating stress, this work provides physiological relevance for vesicle production as a protective mechanism.

Authors
McBroom, AJ; Kuehn, MJ
MLA Citation
McBroom, AJ, and Kuehn, MJ. "Release of outer membrane vesicles by Gram-negative bacteria is a novel envelope stress response." Mol Microbiol 63.2 (January 2007): 545-558.
Website
http://hdl.handle.net/10161/10658
PMID
17163978
Source
pubmed
Published In
Molecular Microbiology
Volume
63
Issue
2
Publish Date
2007
Start Page
545
End Page
558
DOI
10.1111/j.1365-2958.2006.05522.x

Purification of outer membrane vesicles from Pseudomonas aeruginosa and their activation of an IL-8 response.

Considerable lung injury results from the inflammatory response to Pseudomonas aeruginosa infections in patients with cystic fibrosis (CF). The P. aeruginosa laboratory strain PAO1, an environmental isolate, and isolates from CF patients were cultured in vitro and outer membrane vesicles from those cultures were quantitated, purified, and characterized. Vesicles were produced throughout the growth phases of the culture and vesicle yield was strain-independent. Strain-dependent differences in the protein composition of vesicles were quantitated and identified. The aminopeptidase PaAP (PA2939) was highly enriched in vesicles from CF isolates. Vesicles from all strains elicited IL-8 secretion by lung epithelial cells. These results suggest that P. aeruginosa colonizing the CF lung may produce vesicles with a particular composition and that the vesicles could contribute to inflammation.

Authors
Bauman, SJ; Kuehn, MJ
MLA Citation
Bauman, SJ, and Kuehn, MJ. "Purification of outer membrane vesicles from Pseudomonas aeruginosa and their activation of an IL-8 response." Microbes Infect 8.9-10 (August 2006): 2400-2408.
PMID
16807039
Source
pubmed
Published In
Microbes and Infection
Volume
8
Issue
9-10
Publish Date
2006
Start Page
2400
End Page
2408
DOI
10.1016/j.micinf.2006.05.001

Outer membrane vesicle production by Escherichia coli is independent of membrane instability.

It has been long noted that gram-negative bacteria produce outer membrane vesicles, and recent data demonstrate that vesicles released by pathogenic strains can transmit virulence factors to host cells. However, the mechanism of vesicle release has remained undetermined. This genetic study addresses whether these structures are merely a result of membrane instability or are formed by a more directed process. To elucidate the regulatory mechanisms and physiological basis of vesiculation, we conducted a screen in Escherichia coli to identify gene disruptions that caused vesicle over- or underproduction. Only a few low-vesiculation mutants and no null mutants were recovered, suggesting that vesiculation may be a fundamental characteristic of gram-negative bacterial growth. Gene disruptions were identified that caused differences in vesicle production ranging from a 5-fold decrease to a 200-fold increase relative to wild-type levels. These disruptions included loci governing outer membrane components and peptidoglycan synthesis as well as the sigma(E) cell envelope stress response. Mutations causing vesicle overproduction did not result in upregulation of the ompC gene encoding a major outer membrane protein. Detergent sensitivity, leakiness, and growth characteristics of the novel vesiculation mutant strains did not correlate with vesiculation levels, demonstrating that vesicle production is not predictive of envelope instability.

Authors
McBroom, AJ; Johnson, AP; Vemulapalli, S; Kuehn, MJ
MLA Citation
McBroom, AJ, Johnson, AP, Vemulapalli, S, and Kuehn, MJ. "Outer membrane vesicle production by Escherichia coli is independent of membrane instability." J Bacteriol 188.15 (August 2006): 5385-5392.
PMID
16855227
Source
pubmed
Published In
Journal of bacteriology
Volume
188
Issue
15
Publish Date
2006
Start Page
5385
End Page
5392
DOI
10.1128/JB.00498-06

Genetically engineered probiotic competition.

Authors
Kuehn, MJ
MLA Citation
Kuehn, MJ. "Genetically engineered probiotic competition." Gastroenterology 130.6 (May 2006): 1915-1916.
PMID
16697754
Source
pubmed
Published In
Gastroenterology
Volume
130
Issue
6
Publish Date
2006
Start Page
1915
End Page
1916
DOI
10.1053/j.gastro.2006.03.031

Outer Membrane Vesicles

Authors
McBroom, AJ; Kuehn, MJ
MLA Citation
McBroom, AJ, and Kuehn, MJ. "Outer Membrane Vesicles." EcoSal Plus 1.2 (November 23, 2005).
Source
crossref
Published In
EcoSal Plus
Volume
1
Issue
2
Publish Date
2005
DOI
10.1128/ecosal.2.2.4

Bacterial outer membrane vesicles and the host-pathogen interaction.

Extracellular secretion of products is the major mechanism by which Gram-negative pathogens communicate with and intoxicate host cells. Vesicles released from the envelope of growing bacteria serve as secretory vehicles for proteins and lipids of Gram-negative bacteria. Vesicle production occurs in infected tissues and is influenced by environmental factors. Vesicles play roles in establishing a colonization niche, carrying and transmitting virulence factors into host cells, and modulating host defense and response. Vesicle-mediated toxin delivery is a potent virulence mechanism exhibited by diverse Gram-negative pathogens. The biochemical and functional properties of pathogen-derived vesicles reveal their potential to critically impact disease.

Authors
Kuehn, MJ; Kesty, NC
MLA Citation
Kuehn, MJ, and Kesty, NC. "Bacterial outer membrane vesicles and the host-pathogen interaction." Genes Dev 19.22 (November 15, 2005): 2645-2655. (Review)
PMID
16291643
Source
pubmed
Published In
Genes & development
Volume
19
Issue
22
Publish Date
2005
Start Page
2645
End Page
2655
DOI
10.1101/gad.1299905

Enterotoxigenic Escherichia coli vesicles target toxin delivery into mammalian cells.

Enterotoxigenic Escherichia coli (ETEC) is a prevalent cause of traveler's diarrhea and infant mortality in third-world countries. Heat-labile enterotoxin (LT) is secreted from ETEC via vesicles composed of outer membrane and periplasm. We investigated the role of ETEC vesicles in pathogenesis by analyzing vesicle association and entry into eukaryotic cells. Fluorescently labeled vesicles from LT-producing and LT-nonproducing strains were compared in their ability to bind adrenal and intestinal epithelial cells. ETEC-derived vesicles, but not control nonpathogen-derived vesicles, associated with cells in a time-, temperature-, and receptor-dependent manner. Vesicles were visualized on the cell surface at 4 degrees C and detected intracellularly at 37 degrees C. ETEC vesicle endocytosis depended on cholesterol-rich lipid rafts. Entering vesicles partially colocalized with caveolin, and the internalized vesicles accumulated in a nonacidified compartment. We conclude that ETEC vesicles serve as specifically targeted transport vehicles that mediate entry of active enterotoxin and other bacterial envelope components into host cells. These data demonstrate a role in virulence for ETEC vesicles.

Authors
Kesty, NC; Mason, KM; Reedy, M; Miller, SE; Kuehn, MJ
MLA Citation
Kesty, NC, Mason, KM, Reedy, M, Miller, SE, and Kuehn, MJ. "Enterotoxigenic Escherichia coli vesicles target toxin delivery into mammalian cells." EMBO J 23.23 (November 24, 2004): 4538-4549.
PMID
15549136
Source
pubmed
Published In
EMBO Journal
Volume
23
Issue
23
Publish Date
2004
Start Page
4538
End Page
4549
DOI
10.1038/sj.emboj.7600471

Lipopolysaccharide 3-deoxy-D-manno-octulosonic acid (Kdo) core determines bacterial association of secreted toxins.

In contrast to cholera toxin (CT), which is secreted solubly by Vibrio cholerae across the outer membrane, heat-labile enterotoxin (LT) is retained on the surface of enterotoxigenic Escherichia coli (ETEC) via an interaction with lipopolysaccharide (LPS). We examined the nature of the association between LT and LPS. Soluble LT binds to the surface of LPS deep-rough biosynthesis mutants but not to lipid A, indicating that only the Kdo (3-deoxy-d-manno-octulosonic acid) core is required for binding. Although capable of binding truncated LPS and Kdo, LT has a higher affinity for longer, more complete LPS species. A putative LPS binding pocket is proposed based on the crystal structure of the toxin. The ability to bind LPS and remain associated with the bacterial surface is not unique to LT, as CT also binds to E. coli LPS. However, neither LT nor CT is capable of binding to the surface of Vibrio. The core structures of Vibrio and E. coli LPS differ in that Vibrio contains a phosphorylated single Kdo-lipid A, and E. coli LPS contains unphosphorylated Kdo2-lipid A. We determined that the phosphate group on the Kdo core of Vibrio LPS prevents CT from binding, resulting in the secretion of soluble toxin. Because LT binds E. coli LPS, it remains associated with the extracellular bacterial surface and is released in association with outer membrane vesicles. We propose that difference in the extracellular fates of LT and CT contribute to the differences in disease caused by ETEC and Vibrio cholerae.

Authors
Horstman, AL; Bauman, SJ; Kuehn, MJ
MLA Citation
Horstman, AL, Bauman, SJ, and Kuehn, MJ. "Lipopolysaccharide 3-deoxy-D-manno-octulosonic acid (Kdo) core determines bacterial association of secreted toxins." J Biol Chem 279.9 (February 27, 2004): 8070-8075.
PMID
14660669
Source
pubmed
Published In
The Journal of biological chemistry
Volume
279
Issue
9
Publish Date
2004
Start Page
8070
End Page
8075
DOI
10.1074/jbc.M308633200

Incorporation of heterologous outer membrane and periplasmic proteins into Escherichia coli outer membrane vesicles.

Gram-negative bacteria shed outer membrane vesicles composed of outer membrane and periplasmic components. Since vesicles from pathogenic bacteria contain virulence factors and have been shown to interact with eukaryotic cells, it has been proposed that vesicles behave as delivery vehicles. We wanted to determine whether heterologously expressed proteins would be incorporated into the membrane and lumen of vesicles and whether these altered vesicles would associate with host cells. Ail, an outer membrane adhesin/invasin from Yersinia enterocolitica, was detected in purified outer membrane and in vesicles from Escherichia coli strains DH5alpha, HB101, and MC4100 transformed with plasmid-encoded Ail. In vesicle-host cell co-incubation assays we found that vesicles containing Ail were internalized by eukaryotic cells, unlike vesicles without Ail. To determine whether lumenal vesicle contents could be modified and delivered to host cells, we used periplasmically expressed green fluorescent protein (GFP). GFP fused with the Tat signal sequence was secreted into the periplasm via the twin arginine transporter (Tat) in both the laboratory E. coli strain DH5alpha and the pathogenic enterotoxigenic E. coli ATCC strain 43886. Pronase-resistant fluorescence was detectable in vesicles from Tat-GFP-transformed strains, demonstrating that GFP was inside intact vesicles. Inclusion of GFP cargo increased vesicle density but did not result in morphological changes in vesicles. These studies are the first to demonstrate the incorporation of heterologously expressed outer membrane and periplasmic proteins into bacterial vesicles.

Authors
Kesty, NC; Kuehn, MJ
MLA Citation
Kesty, NC, and Kuehn, MJ. "Incorporation of heterologous outer membrane and periplasmic proteins into Escherichia coli outer membrane vesicles." J Biol Chem 279.3 (January 16, 2004): 2069-2076.
PMID
14578354
Source
pubmed
Published In
The Journal of biological chemistry
Volume
279
Issue
3
Publish Date
2004
Start Page
2069
End Page
2076
DOI
10.1074/jbc.M307628200

Bacterial surface association of heat-labile enterotoxin through lipopolysaccharide after secretion via the general secretory pathway.

Heat-labile enterotoxin (LT) is an important virulence factor expressed by enterotoxigenic Escherichia coli. The route of LT secretion through the outer membrane and the cellular and extracellular localization of secreted LT were examined. Using a fluorescently labeled receptor, LT was found to be specifically secreted onto the surface of wild type enterotoxigenic Escherichia coli. The main terminal branch of the general secretory pathway (GSP) was necessary and sufficient to localize LT to the bacterial surface in a K-12 strain. LT is a heteromeric toxin, and we determined that its cell surface localization was mediated by the its B subunit independent of an intact G(M1) ganglioside binding site and that LT binds lipopolysaccharide and G(M1) concurrently. The majority of LT secreted into the culture supernatant by the GSP in E. coli associated with vesicles. Only a mutation in hns, not overexpression of the GSP or LT, caused an increase in vesicle yield, supporting a specific vesicle formation machinery regulated by the nucleoid-associated protein HNS. We propose a model in which LT is secreted by the GSP across the outer membrane, secreted LT binds lipopolysaccharide via a G(M1)-independent binding region on its B subunit, and LT on the surface of released outer membrane vesicles interacts with host cell receptors, leading to intoxication. These data explain a novel mechanism of vesicle-mediated receptor-dependent delivery of a bacterial toxin into a host cell.

Authors
Horstman, AL; Kuehn, MJ
MLA Citation
Horstman, AL, and Kuehn, MJ. "Bacterial surface association of heat-labile enterotoxin through lipopolysaccharide after secretion via the general secretory pathway." J Biol Chem 277.36 (September 6, 2002): 32538-32545.
PMID
12087095
Source
pubmed
Published In
The Journal of biological chemistry
Volume
277
Issue
36
Publish Date
2002
Start Page
32538
End Page
32545
DOI
10.1074/jbc.M203740200

Polarized secretion.

Authors
Kuehn, MJ
MLA Citation
Kuehn, MJ. "Polarized secretion." Trends Microbiol 10.3 (March 2002): 116-.
PMID
11864814
Source
pubmed
Published In
Trends in Microbiology
Volume
10
Issue
3
Publish Date
2002
Start Page
116

The E. coli BaeSR two-component regulatory system

Authors
Kuehn, M
MLA Citation
Kuehn, M. "The E. coli BaeSR two-component regulatory system." Trends in Microbiology 10.12 (2002): 553--.
Source
scival
Published In
Trends in Microbiology
Volume
10
Issue
12
Publish Date
2002
Start Page
553-

Bacterial density dictates virulence in cholera

Authors
Kuehn, MJ
MLA Citation
Kuehn, MJ. "Bacterial density dictates virulence in cholera." Trends in Microbiology 10.10 (2002): 449--.
Source
scival
Published In
Trends in Microbiology
Volume
10
Issue
10
Publish Date
2002
Start Page
449-

FRET probes toxin activity in situ

Authors
Kuehn, MJ
MLA Citation
Kuehn, MJ. "FRET probes toxin activity in situ." Trends in Microbiology 10.8 (2002): 355--.
Source
scival
Published In
Trends in Microbiology
Volume
10
Issue
8
Publish Date
2002
Start Page
355-
DOI
10.1016/S0966-842X(02)02414-9

Surrogate host succumbs to virulent Pseudomonas

Authors
Kuehn, MJ
MLA Citation
Kuehn, MJ. "Surrogate host succumbs to virulent Pseudomonas." Trends in Microbiology 10.5 (2002): 215--.
Source
scival
Published In
Trends in Microbiology
Volume
10
Issue
5
Publish Date
2002
Start Page
215-

Bacterial-host-cell tethers.

Authors
Kuehn, MJ
MLA Citation
Kuehn, MJ. "Bacterial-host-cell tethers." Trends Microbiol 9.7 (July 2001): 310-.
PMID
11435085
Source
pubmed
Published In
Trends in Microbiology
Volume
9
Issue
7
Publish Date
2001
Start Page
310

Engineering a biosensor from a bacterial periplasmic protein

Authors
Kuehn, MJ
MLA Citation
Kuehn, MJ. "Engineering a biosensor from a bacterial periplasmic protein." Trends in Microbiology 9.11 (2001): 527--.
Source
scival
Published In
Trends in Microbiology
Volume
9
Issue
11
Publish Date
2001
Start Page
527-

Bacterial cave dwellers

Authors
Kuehn, MJ
MLA Citation
Kuehn, MJ. "Bacterial cave dwellers." Trends Microbiol 8.10 (October 2000): 450-451.
PMID
11044675
Source
pubmed
Published In
Trends in Microbiology
Volume
8
Issue
10
Publish Date
2000
Start Page
450
End Page
451

Enterotoxigenic Escherichia coli secretes active heat-labile enterotoxin via outer membrane vesicles.

Escherichia coli and other Gram-negative bacteria produce outer membrane vesicles during normal growth. Vesicles may contribute to bacterial pathogenicity by serving as vehicles for toxins to encounter host cells. Enterotoxigenic E. coli (ETEC) vesicles were isolated from culture supernatants and purified on velocity gradients, thereby removing any soluble proteins and contaminants from the crude preparation. Vesicle protein profiles were similar but not identical to outer membranes and differed between strains. Most vesicle proteins were resistant to dissociation, suggesting they were integral or internal. Thin layer chromatography revealed that major outer membrane lipid components are present in vesicles. Cytoplasmic membranes and cytosol were absent in vesicles; however, alkaline phosphatase and AcrA, periplasmic residents, were localized to vesicles. In addition, physiologically active heat-labile enterotoxin (LT) was associated with ETEC vesicles. LT activity correlated directly with the gradient peak of vesicles, suggesting specific association, but could be removed from vesicles under dissociating conditions. Further analysis revealed that LT is enriched in vesicles and is located both inside and on the exterior of vesicles. The distinct protein composition of ETEC vesicles and their ability to carry toxin may contribute to the pathogenicity of ETEC strains.

Authors
Horstman, AL; Kuehn, MJ
MLA Citation
Horstman, AL, and Kuehn, MJ. "Enterotoxigenic Escherichia coli secretes active heat-labile enterotoxin via outer membrane vesicles." J Biol Chem 275.17 (April 28, 2000): 12489-12496.
PMID
10777535
Source
pubmed
Published In
The Journal of biological chemistry
Volume
275
Issue
17
Publish Date
2000
Start Page
12489
End Page
12496

Definitive typing of LPS core structures

Authors
Kuehn, M
MLA Citation
Kuehn, M. "Definitive typing of LPS core structures." Trends in Microbiology 8.5 (2000): 212--.
Source
scival
Published In
Trends in Microbiology
Volume
8
Issue
5
Publish Date
2000
Start Page
212-

Lighting the path to virulence

Authors
Kuehn, M
MLA Citation
Kuehn, M. "Lighting the path to virulence." Trends in Microbiology 8.1 (2000): 16--.
Source
scival
Published In
Trends in Microbiology
Volume
8
Issue
1
Publish Date
2000
Start Page
16-

Foreign travel

Authors
Kuehn, M
MLA Citation
Kuehn, M. "Foreign travel." Trends in Microbiology 7.3 (1999): 102--.
Source
scival
Published In
Trends in Microbiology
Volume
7
Issue
3
Publish Date
1999
Start Page
102-

Expressed by stress

Authors
Kuehn, M
MLA Citation
Kuehn, M. "Expressed by stress." Trends in Microbiology 7.6 (1999): 231--.
Source
scival
Published In
Trends in Microbiology
Volume
7
Issue
6
Publish Date
1999
Start Page
231-

A divisive role for lipoproteins

Authors
Kuehn, M
MLA Citation
Kuehn, M. "A divisive role for lipoproteins." Trends in Microbiology 7.10 (1999): 400--.
Source
scival
Published In
Trends in Microbiology
Volume
7
Issue
10
Publish Date
1999
Start Page
400-

COPII-cargo interactions direct protein sorting into ER-derived transport vesicles.

Vesicles coated with coat protein complex II (COPII) selectively transport molecules (cargo) and vesicle fusion proteins from the endoplasmic reticulum (ER) to the Golgi complex. We have investigated the role of coat proteins in cargo selection and recruitment. We isolated integral membrane and soluble cargo proteins destined for transport from the ER in complexes formed in the presence of Sar1 and Sec23/24, a subset of the COPII components, and GTP or GMP-PNP. Vesicle fusion proteins of the vSNARE family and Emp24, a member of a putative cargo carrier family, were also found in COPII complexes. The inclusion of amino-acid permease molecules into the complex depended on the presence of Shr3, a protein required for the permease to leave the ER. Resident ER proteins Sec61, BiP (Kar2) and Shr3 were not included in the complexes, indicating that the COPII components bound specifically to vesicle cargo. COPII-cargo complexes and putative cargo adaptor-cargo complexes were also isolated from COPII vesicles. Our results indicate that cargo packaging signals and soluble cargo adaptors are recognized by a recruitment complex comprising Sar1-GTP and Sec23/24.

Authors
Kuehn, MJ; Herrmann, JM; Schekman, R
MLA Citation
Kuehn, MJ, Herrmann, JM, and Schekman, R. "COPII-cargo interactions direct protein sorting into ER-derived transport vesicles." Nature 391.6663 (January 8, 1998): 187-190.
PMID
9428766
Source
pubmed
Published In
Nature
Volume
391
Issue
6663
Publish Date
1998
Start Page
187
End Page
190
DOI
10.1038/34438

New transporter family

Authors
Kuehn, M
MLA Citation
Kuehn, M. "New transporter family." Trends in Microbiology 6.9 (1998): 351--.
Source
scival
Published In
Trends in Microbiology
Volume
6
Issue
9
Publish Date
1998
Start Page
351-
DOI
10.1016/S0966-842X(98)01335-3

COPII and secretory cargo capture into transport vesicles.

Yeast cylosolic coat proteins (COPII) direct the formation of vesicles from the endoplasmic reticulum. The vesicles selectively capture both cargo molecules and the secretory machinery that is necessary for the fusion of the vesicle with the recipient compartment, the Golgi apparatus. Recent efforts have aimed to understand how proteins are selected for inclusion into these vesicles. A variety of cargo adaptors may concentrate and sort secretory and membrane proteins by direct or indirect interaction with a subset of coat protein subunits.

Authors
Kuehn, MJ; Schekman, R
MLA Citation
Kuehn, MJ, and Schekman, R. "COPII and secretory cargo capture into transport vesicles." Curr Opin Cell Biol 9.4 (August 1997): 477-483. (Review)
PMID
9261052
Source
pubmed
Published In
Current Opinion in Cell Biology
Volume
9
Issue
4
Publish Date
1997
Start Page
477
End Page
483

Establishing communication via gram-negative bacterial pili.

Authors
Kuehn, MJ
MLA Citation
Kuehn, MJ. "Establishing communication via gram-negative bacterial pili." Trends Microbiol 5.4 (April 1997): 130-132. (Review)
PMID
9141184
Source
pubmed
Published In
Trends in Microbiology
Volume
5
Issue
4
Publish Date
1997
Start Page
130
End Page
132
DOI
10.1016/S0966-842X(96)30045-0

Leaping into the outer membrane

Authors
Kuehn, M
MLA Citation
Kuehn, M. "Leaping into the outer membrane." Trends in Microbiology 5.10 (1997): 387--.
Source
scival
Published In
Trends in Microbiology
Volume
5
Issue
10
Publish Date
1997
Start Page
387-

Cargo selection and vesicle budding

The cytosolic yeast proteins Secl3-31p, Sec23/24p, and the small GTP binding protein Sarlp form the membrane coat termed COPII. Purified COPII components and GTP form anterograde transport vesicles directly from the endoplasmic reticulum. Cargo capture by COPII proteins is highly selective. Only properly folded forms of secretory and plasma membrane proteins are collected whereas resident ER proteins are ignored. Binding and chemical crosslinking experiments indicate that Sarlp-GTP and Sec23/24p form a non-covalent recruit-me: complex with membrane and lumenal cargo molecules. The recovery of soluble secretory proteins in such a complex re-quires the intervention of a putative membrane receptor or adaptor protein. Coated buds and vesicles form when Sec13/31p is added to membranes that have bound SarI-GTP and Sec23/24p. Sec13-31p may serve to cluster cargo recruitment complexes to produce a membrane patch depleted of ER resident protein:s. No special membrane proteins are required to anchor the formation of a COPII coat. Synthetic liposomes containing PE or PC and a mixture of acidic phospholipids are just as active as native ER membranes in binding COPII proteins in the presence of a non-hydrolyzeble analog of GTP, GMP-PNP.

Authors
Schekman, R; Hamamoto, S; Kuehn, M; Matsuoka, K; Yeung, T; Orci, L
MLA Citation
Schekman, R, Hamamoto, S, Kuehn, M, Matsuoka, K, Yeung, T, and Orci, L. "Cargo selection and vesicle budding." FASEB Journal 11.9 (1997): A1011-.
Source
scival
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
11
Issue
9
Publish Date
1997
Start Page
A1011

Cryptococcus and calcineurin

Authors
Kuehn, M
MLA Citation
Kuehn, M. "Cryptococcus and calcineurin." Trends in Microbiology 5.8 (1997): 307--.
Source
scival
Published In
Trends in Microbiology
Volume
5
Issue
8
Publish Date
1997
Start Page
307-

Amino acid permeases require COPII components and the ER resident membrane protein Shr3p for packaging into transport vesicles in vitro.

In S. cerevisiae lacking SHR3, amino acid permeases specifically accumulate in membranes of the endoplasmic reticulum (ER) and fail to be transported to the plasma membrane. We examined the requirements of transport of the permeases from the ER to the Golgi in vitro. Addition of soluble COPII components (Sec23/24p, Sec13/31p, and Sar1p) to yeast membrane preparations generated vesicles containing the general amino acid permease. Gap1p, and the histidine permease, Hip1p. Shr3p was required for the packaging of Gap1p and Hip1p but was not itself incorporated into transport vesicles. In contrast, the packaging of the plasma membrane ATPase, Pma1p, and the soluble yeast pheromone precursor, glycosylated pro alpha factor, was independent of Shr3p. In addition, we show that integral membrane and soluble cargo colocalize in transport vesicles, indicating that different types of cargo are not segregated at an early step in secretion. Our data suggest that specific ancillary proteins in the ER membrane recruit subsets of integral membrane protein cargo into COPII transport vesicles.

Authors
Kuehn, MJ; Schekman, R; Ljungdahl, PO
MLA Citation
Kuehn, MJ, Schekman, R, and Ljungdahl, PO. "Amino acid permeases require COPII components and the ER resident membrane protein Shr3p for packaging into transport vesicles in vitro." J Cell Biol 135.3 (November 1996): 585-595.
Website
http://hdl.handle.net/10161/10661
PMID
8909535
Source
pubmed
Published In
The Journal of Cell Biology
Volume
135
Issue
3
Publish Date
1996
Start Page
585
End Page
595

Coat proteins and selective protein packaging into transport vesicles

Authors
Schekman, R; Barlowe, C; Bednarek, S; Campbell, J; Doering, T; Duden, R; Kuehn, M; Rexach, M; Yeung, T; Orci, L
MLA Citation
Schekman, R, Barlowe, C, Bednarek, S, Campbell, J, Doering, T, Duden, R, Kuehn, M, Rexach, M, Yeung, T, and Orci, L. "Coat proteins and selective protein packaging into transport vesicles." Cold Spring Harbor Symposia on Quantitative Biology 60 (1995): 11-22.
PMID
8824373
Source
scival
Published In
Cold Spring Harbor Laboratory: Symposia on Quantitative Biology
Volume
60
Publish Date
1995
Start Page
11
End Page
22

Genetic, biochemical, and structural studies of biogenesis of adhesive pili in bacteria.

Authors
Kuehn, MJ; Jacob-Dubuisson, F; Dodson, K; Slonim, L; Striker, R; Hultgren, SJ
MLA Citation
Kuehn, MJ, Jacob-Dubuisson, F, Dodson, K, Slonim, L, Striker, R, and Hultgren, SJ. "Genetic, biochemical, and structural studies of biogenesis of adhesive pili in bacteria." Methods Enzymol 236 (1994): 282-306.
PMID
7968616
Source
pubmed
Published In
Methods in Enzymology
Volume
236
Publish Date
1994
Start Page
282
End Page
306

Structural basis of pilus subunit recognition by the PapD chaperone.

The assembly of different types of virulence-associated surface fibers called pili in Gram-negative bacteria requires periplasmic chaperones. PapD is the prototype member of the periplasmic chaperone family, and the structural basis of its interactions with pilus subunits was investigated. Peptides corresponding to the carboxyl terminus of pilus subunits bound PapD and blocked the ability of PapD to bind to the pilus adhesin PapG in vitro. The crystal structure of PapD complexed to the PapG carboxyl-terminal peptide was determined to 3.0 A resolution. The peptide bound in an extended conformation with its carboxyl terminus anchored in the interdomain cleft of the chaperone via hydrogen bonds to invariant chaperone residues Arg8 and Lys112. Main chain hydrogen bonds and contacts between hydrophobic residues in the peptide and the chaperone stabilized the complex and may play a role in determining binding specificity. Site-directed mutations in Arg8 and Lys112 abolished the ability of PapD to bind pilus subunits and mediate pilus assembly in vivo, an indication that the PapD-peptide crystal structure is a reflection of at least part of the PapD-subunit interaction.

Authors
Kuehn, MJ; Ogg, DJ; Kihlberg, J; Slonim, LN; Flemmer, K; Bergfors, T; Hultgren, SJ
MLA Citation
Kuehn, MJ, Ogg, DJ, Kihlberg, J, Slonim, LN, Flemmer, K, Bergfors, T, and Hultgren, SJ. "Structural basis of pilus subunit recognition by the PapD chaperone." Science 262.5137 (November 19, 1993): 1234-1241.
PMID
7901913
Source
pubmed
Published In
Science
Volume
262
Issue
5137
Publish Date
1993
Start Page
1234
End Page
1241

A novel secretion apparatus for the assembly of adhesive bacterial pili

The biogenesis of most types of bacterial pili requires two specialized proteins: a chaperone that caps the pilus subunits in the periplasm, and an outer membrane usher that receives the subunits and serves as an assembly platform. This secretion and assembly machinery is proposed to be a novel export apparatus found widely in Gram-negative pathogens.

Authors
Jacob-Dubuisson, F; Kuehn, M; Hultgren, SJ
MLA Citation
Jacob-Dubuisson, F, Kuehn, M, and Hultgren, SJ. "A novel secretion apparatus for the assembly of adhesive bacterial pili." Trends in Microbiology 1.2 (1993): 50-55.
PMID
7913856
Source
scival
Published In
Trends in Microbiology
Volume
1
Issue
2
Publish Date
1993
Start Page
50
End Page
55
DOI
10.1016/0966-842X(93)90032-M

Conserved immunoglobulin-like features in a family of periplasmic pilus chaperones in bacteria.

Detailed structural analyses revealed a family of periplasmic chaperones in Gram-negative prokaryotes which are structurally and possibly evolutionarily related to the immunoglobulin superfamily and assist in the assembly of adhesive pili. The members of this family have similar structures consistent with the overall topology of an immunoglobulin fold. Seven pilus chaperone sequences from Escherichia coli, Haemophilus influenzae and Klebsiella pneumoniae were aligned and their consensus sequence was superimposed onto the known three-dimensional structure of PapD, a representative member of the family. The molecular details of the conserved and variable structural motifs in this family of periplasmic chaperones give important insight into their structure, function, mechanism of action and evolutionary relationship with the immunoglobulin superfamily.

Authors
Holmgren, A; Kuehn, MJ; Brändén, CI; Hultgren, SJ
MLA Citation
Holmgren, A, Kuehn, MJ, Brändén, CI, and Hultgren, SJ. "Conserved immunoglobulin-like features in a family of periplasmic pilus chaperones in bacteria." EMBO J 11.4 (April 1992): 1617-1622.
PMID
1348692
Source
pubmed
Published In
EMBO Journal
Volume
11
Issue
4
Publish Date
1992
Start Page
1617
End Page
1622

P pili in uropathogenic E. coli are composite fibres with distinct fibrillar adhesive tips.

Escherichia coli is a frequent cause of several common bacterial infections in humans and animals, including urinary tract infections, bacteraemia and bacteria-related diarrhoea and is also the main cause of neonatal meningitis. Microbial attachment to surfaces is a key event in colonization and infection and results mainly from a stereochemical fit between microbial adhesins and complementary receptors on host cells. Bacterial adhesins required for extracellular colonization by Gram-negative bacteria are often minor components of heteropolymeric fibres called pili which must be oriented in an accessible manner in these structures to be able to bind to specific receptor architectures. P pili mediate the binding of uropathogenic E. coli to a digalactoside receptor determinant present in the urinary tract epithelium. We report here that the adhesin is a component of distinct fibrillar structures present at the tips of the pili. These virulence-associated tip fibrillae are thin, flexible polymers composed mostly of repeating subunits of PapE that frequently terminate with the alpha-D-galactopyranosyl-(1-4)-beta-D-galactopyranose or Gal alpha (1-4)Gal binding PapG adhesin.

Authors
Kuehn, MJ; Heuser, J; Normark, S; Hultgren, SJ
MLA Citation
Kuehn, MJ, Heuser, J, Normark, S, and Hultgren, SJ. "P pili in uropathogenic E. coli are composite fibres with distinct fibrillar adhesive tips." Nature 356.6366 (March 19, 1992): 252-255.
PMID
1348107
Source
pubmed
Published In
Nature
Volume
356
Issue
6366
Publish Date
1992
Start Page
252
End Page
255
DOI
10.1038/356252a0

Fimbriation of Pseudomonas cepacia

Fimbriae (pili) on the surface of bacteria have been suggested to facilitate adherence to mucosal epithelial surfaces. Three Pseudomonas cepacia cystic fibrosis isolates were screened for their ability to agglutinate erythrocytes (HA), a characteristic of some fimbrial types. One strain, designated PC103, was HA+, while another, PC109, was HA-. A fimbriated (f+) HA+ derivative of PC109 (PCE213) was selected by repeated erythrocyte adsorption. The two HA+ strains were shown by transmission electron microscopy to possess fimbriae which averaged 4.8 ± 1.36 nm in width and 200 to >2,100 nm in length (PCE213) and 3.4 to 11.4 nm in diameter and 280 to 720 nm in length (PC103). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of outer membrane proteins prepared from PC103, PC109, and PCE213 indicated that the putative fimbrial subunit had a mass of 16 kDa. Western blot (immunoblot) analysis of sheared cell supernatants indicated that the 16-kDa subunit from PC103 and PCE213 reacted with antibody to the P. aeruginosa PAK pilin subunit. Southern blot analysis of a SalI digest of PC103 DNA showed DNA fragments which hybridized to P. aeruginosa PAK probes containing either the pilin structural gene (pilA) or the pilin accessory genes (pilB, -C, and -D) but not the conserved N-terminal region of pilA. A 15-kb band was common to both hybridizations, indicating that this fragment contains the PC103 fimbrial gene cluster. These results indicated the presence of homology between P. aeruginosa PAK and PC103 fimbriae but also suggested that the P. cepacia fimbriae are not type IV- like. The importance of fimbriae in adherence to A549 cells (type II pneumocytes) was assessed with PC109 (f-) and PCE213 (f+). PCE213 had an approximately 20-fold-higher level of adherence to A549 cells than PC109. This suggested that fimbriation of P. cepacia is associated with increased adherence in vitro.

Authors
Kuehn, M; Lent, K; Haas, J; Hagenzieker, J; Cervin, M; Smith, AL
MLA Citation
Kuehn, M, Lent, K, Haas, J, Hagenzieker, J, Cervin, M, and Smith, AL. "Fimbriation of Pseudomonas cepacia." Infection and Immunity 60.5 (1992): 2002-2007.
PMID
1373402
Source
scival
Published In
Infection and Immunity
Volume
60
Issue
5
Publish Date
1992
Start Page
2002
End Page
2007

Adhesin presentation in bacteria requires molecular chaperones and ushers

Authors
Jones, CH; Jacob-Dubuisson, F; Dodson, K; Kuehn, M; Slonim, L; Striker, R; Hultgren, SJ
MLA Citation
Jones, CH, Jacob-Dubuisson, F, Dodson, K, Kuehn, M, Slonim, L, Striker, R, and Hultgren, SJ. "Adhesin presentation in bacteria requires molecular chaperones and ushers." Infection and Immunity 60.11 (1992): 4445-4451.
PMID
1356928
Source
scival
Published In
Infection and Immunity
Volume
60
Issue
11
Publish Date
1992
Start Page
4445
End Page
4451

Immunoglobulin-like PapD chaperone caps and uncaps interactive surfaces of nascently translocated pilus subunits.

Molecular chaperones are found in the cytoplasm of bacteria and in various cellular compartments in eukaryotes to maintain proteins in nonnative conformations that permit their secretion across membranes or assembly into oligomeric structures. Virtually nothing, however, has been reported about a similar requirement for molecular chaperones in the periplasm of Gram-negative bacteria. We used the well-characterized P pilus biogenesis system in Escherichia coli as a model to elucidate the mechanism of action of a periplasmic chaperone, PapD, which is specifically required for P pilus biogenesis. PapD probably associates with at least six P pilus subunits after their secretion across the cytoplasmic membrane, but PapD is not incorporated into the pilus. We used purified periplasmic complex that PapD forms with the PapG adhesin to investigate the function of interactions between the chaperone and its targets. We demonstrated that PapD binds to PapG to form a stable, discrete bimolecular complex and that, unlike cytoplasmic chaperones, the periplasmic PapD chaperone maintained PapG in a native-like conformation. Bound PapD in the complex was displaced by free PapD in vitro; however, the in vivo release of subunits to the nascent pilus is probably driven by an ATP-independent mechanism involving the outer membrane protein PapC. In addition, the binding of PapD to PapG in vitro prevented aggregation of PapG. We propose that the function of PapD and other periplasmic pilus chaperones is to partition newly translocated pilus subunits into assembly-competent complexes and thereby prevent nonproductive aggregation of the subunits in the periplasm. These data provide important information for understanding the mechanism of action of this general class of chaperones that function in the periplasmic space.

Authors
Kuehn, MJ; Normark, S; Hultgren, SJ
MLA Citation
Kuehn, MJ, Normark, S, and Hultgren, SJ. "Immunoglobulin-like PapD chaperone caps and uncaps interactive surfaces of nascently translocated pilus subunits." Proc Natl Acad Sci U S A 88.23 (December 1, 1991): 10586-10590.
PMID
1683704
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
88
Issue
23
Publish Date
1991
Start Page
10586
End Page
10590

Trimethoprim resistance in Haemophilus influenzae is due to altered dihydrofolate reductase(s)

We characterized a highly purified preparation of the chromosomally encoded dihydrofolate reductase (DHFR) from a trimethoprim-susceptible (Tmp(S); strain MAP) and two trimethoprim-resistant (Tmp(R)) strains (MAP/47 and MAP/42) of Haemophilus influenzae. The enzymes were purified between 650- and 3000-fold by gel-filtration and dye-ligand chromatography. The apparent molecular mass of the three proteins was 18400 Da by PAGE under denaturing and nondenaturing conditions. Total enzyme activity was greater in all fractions from the Tmp(R) strains compared with the Tmp(S) isolate. The three enzymes had a similar K(m) for dihydrofolate (7, 9 and 5 μM) and NADPH (2, 5 and 6 μM). However, the Tmp IC 50 (the concentration necessary for 50% inhibition of DHFR activity) for the Tmp(S) strain MAP was 0.001 μM, whereas DHFR from the Tmp(R) strains MAP/47 and MAP/42 had values of 0.1 μM and 0.3 μM respectively. The methotrexate IC 50 of the MAP/42 DHFR was 0.06 μM in comparison with the enzyme from MAP (0.008 μM) and MAP/47 (0.007 μM). Isoelectric focusing indicated that the DHFR from MAP/42 had a different isoelectric point (pI 7.6) compared with the enzymes from MAP and MAP/47 (pI 7.3). Peptide mapping after digestion with trypsin revealed one major peptide fragment (7.9 kDa) in the DHFR of MAP and MAP/47 and three major tryptic fragments (7.9, 9.6 and 12.5 kDa) in DHFR from MAP/42. We conclude that trimethoprim resistance in H. influenzae results from overproduction of structurally altered DHFR(s).

Authors
Groot, RD; Chaffin, DO; Kuehn, M; Smith, AL
MLA Citation
Groot, RD, Chaffin, DO, Kuehn, M, and Smith, AL. "Trimethoprim resistance in Haemophilus influenzae is due to altered dihydrofolate reductase(s)." Biochemical Journal 274.3 (1991): 657-662.
PMID
2012595
Source
scival
Published In
The Biochemical journal
Volume
274
Issue
3
Publish Date
1991
Start Page
657
End Page
662

Contribution of a 28-kilodalton membrane protein to the virulence of Haemophilus influenzae

A Haemophilus influenzae b (Hib) membrane protein with a molecular mass of 28 kDa bound polyclonal antisera raised against a highly purified Hib fimbrial subunit. We cloned the gene encoding this protein and found that the gene was expressed in Escherichia coli. DNA sequence analysis identified an 843-bp open reading frame which predicted a 26.78-kDa protein with an amino-terminal signal sequence and a mature protein with 70% similarity to the 28-kDa lipoprotein of E. coli (F. Yu, S. Inouye, and M. Inouye, J. Biol. Chem. 261:2284, 1986). Colony blot hybridization analysis with an intergenic probe of the cloned gene demonstrated that 29 of 32 H. influenzae strains hybridize with this gene. Insertion of a chloramphenicol acetyltransferase gene into the open reading frame inactivated expression of the 28-kDa protein in E. coli. Isogenic Hib strains were derived by marker exchange mutagenesis to generate mutants which no longer expressed the 28-kDa protein as recognized with Western immunoblot analysis. There was no difference in the rate of nasopharyngeal colonization of infant rats or monkeys by the isogenic mutants which lacked the 28-kDa protein compared with colonization by the wild-type strain. In contrast, the frequency of invasion and density of bacteremia in infant rats caused by the isogenic mutants were reduced relative to those caused by the wild-type Hib strain. We conclude that this 28-kDa outer membrane protein aids transepithelial invasion of type b strains but is not essential.

Authors
Chanyangam, M; Smith, AL; Moseley, SL; Kuehn, M; Jenny, P
MLA Citation
Chanyangam, M, Smith, AL, Moseley, SL, Kuehn, M, and Jenny, P. "Contribution of a 28-kilodalton membrane protein to the virulence of Haemophilus influenzae." Infection and Immunity 59.2 (1991): 600-608.
PMID
1987077
Source
scival
Published In
Infection and Immunity
Volume
59
Issue
2
Publish Date
1991
Start Page
600
End Page
608
Show More

Research Areas:

  • Bacteria
  • Bacterial Outer Membrane Proteins
  • Bacterial Proteins
  • Bacterial Toxins
  • Bacteriophage T4
  • Biofilms
  • Biological Transport
  • Cell Fractionation
  • Cell Membrane
  • Cystic Fibrosis
  • Enterotoxigenic Escherichia coli
  • Epithelial Cells
  • Epithelium
  • Escherichia coli
  • Escherichia coli Proteins
  • Exosomes
  • Fimbriae, Bacterial
  • Gram-Negative Bacteria
  • Gram-Negative Bacterial Infections
  • HT29 Cells
  • Lipopolysaccharides
  • Lipoproteins
  • Macrophages
  • Membrane Fusion
  • Membrane Microdomains
  • Membrane Proteins
  • Membrane Transport Proteins
  • Microbial Interactions
  • Microbial Viability
  • Molecular Chaperones
  • Periplasm
  • Protein Sorting Signals
  • Protein Transport
  • Pseudomonas Infections
  • Pseudomonas aeruginosa
  • Secretory Vesicles
  • Sigma Factor
  • Transport Vesicles
  • Vesicular Transport Proteins
  • Virulence