Margarethe Kuehn

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

University of Washington

Ph.D. 1993

Washington University in St. Louis

Postdoctoral Fellow, Molecular Microbiology

Washington University in St. Louis

Howard Hughes Postdoctoral Research Fellow, Biochemistry And Molecular Biolovy

University of California - Berkeley

Grants:

Enterotoxin targeting and delivery mechanisms

Administered By
Biochemistry
Awarded By
National Institutes of Health
Role
Principal Investigator
Start Date
End Date

Detection of in vivo ETEC vesicle production

Administered By
Biochemistry
Awarded By
National Institutes of Health
Role
Principal Investigator
Start Date
End Date

Triggers and Consequences of Bacterial Envelope Stress

Administered By
Biochemistry
Awarded By
National Institutes of Health
Role
Principal Investigator
Start Date
End Date

Regulation of Host Defense by IRG Proteins

Administered By
Medicine, Geriatrics
Awarded By
National Institutes of Health
Role
Advisor
Start Date
End Date

Heat-Labile Enterotoxin Secretion

Administered By
Biochemistry
Awarded By
National Institutes of Health
Role
Principal Investigator
Start Date
End Date

Publications:

The extracellular vesicle generation paradox: a bacterial point of view.

All bacteria produce secreted vesicles that carry out a variety of important biological functions. These extracellular vesicles can improve adaptation and survival by relieving bacterial stress and eliminating toxic compounds, as well as by facilitating membrane remodeling and ameliorating inhospitable environments. However, vesicle production comes with a price. It is energetically costly and, in the case of colonizing pathogens, it elicits host immune responses, which reduce bacterial viability. This raises an interesting paradox regarding why bacteria produce vesicles and begs the question as to whether the benefits of producing vesicles outweigh their costs. In this review, we discuss the various advantages and disadvantages associated with Gram-negative and Gram-positive bacterial vesicle production and offer perspective on the ultimate score. We also highlight questions needed to advance the field in determining the role for vesicles in bacterial survival, interkingdom communication, and virulence.
Authors
McMillan, HM; Kuehn, MJ
MLA Citation
McMillan, Hannah M., and Meta J. Kuehn. “The extracellular vesicle generation paradox: a bacterial point of view.Embo J, vol. 40, no. 21, Nov. 2021, p. e108174. Pubmed, doi:10.15252/embj.2021108174.
URI
https://scholars.duke.edu/individual/pub1498808
PMID
34636061
Source
pubmed
Published In
The Embo Journal
Volume
40
Published Date
Start Page
e108174
DOI
10.15252/embj.2021108174

Microbial vesicle-mediated communication: convergence to understand interactions within and between domains of life.

All cells produce extracellular vesicles (EVs). These biological packages contain complex mixtures of molecular cargo and have a variety of functions, including interkingdom communication. Recent discoveries highlight the roles microbial EVs may play in the environment with respect to interactions with plants as well as nutrient cycling. These studies have also identified molecules present within EVs and associated with EV surfaces that contribute to these functions. In parallel, studies of engineered nanomaterials have developed methods to track and model small particle behavior in complex systems and measure the relative importance of various surface features on transport and function. While studies of EV behavior in complex environmental conditions have not yet employed transdisciplinary approaches, it is increasingly clear that expertise from disparate fields will be critical to understand the role of EVs in these systems. Here, we outline how the convergence of biology, soil geochemistry, and colloid science can both develop and address questions surrounding the basic principles governing EV-mediated interkingdom interactions.
Authors
McMillan, HM; Rogers, N; Wadle, A; Hsu-Kim, H; Wiesner, MR; Kuehn, MJ; Hendren, CO
MLA Citation
McMillan, Hannah M., et al. “Microbial vesicle-mediated communication: convergence to understand interactions within and between domains of life.Environ Sci Process Impacts, vol. 23, no. 5, May 2021, pp. 664–77. Pubmed, doi:10.1039/d1em00022e.
URI
https://scholars.duke.edu/individual/pub1480629
PMID
33899070
Source
pubmed
Published In
Environ Sci Process Impacts
Volume
23
Published Date
Start Page
664
End Page
677
DOI
10.1039/d1em00022e

Differential Packaging Into Outer Membrane Vesicles Upon Oxidative Stress Reveals a General Mechanism for Cargo Selectivity.

Selective cargo packaging into bacterial extracellular vesicles has been reported and implicated in many biological processes, however, the mechanism behind the selectivity has remained largely unexplored. In this study, proteomic analysis of outer membrane (OM) and OM vesicle (OMV) fractions from enterotoxigenic E. coli revealed significant differences in protein abundance in the OMV and OM fractions for cultures shifted to oxidative stress conditions. Analysis of sequences of proteins preferentially packaged into OMVs showed that proteins with oxidizable residues were more packaged into OMVs in comparison with those retained in the membrane. In addition, the results indicated two distinct classes of OM-associated proteins were differentially packaged into OMVs as a function of peroxide treatment. Implementing a Bayesian hierarchical model, OM lipoproteins were determined to be preferentially exported during stress whereas integral OM proteins were preferentially retained in the cell. Selectivity was determined to be independent of transcriptional regulation of the proteins upon oxidative stress and was validated using randomly selected protein candidates from the different cargo classes. Based on these data, a hypothetical functional and mechanistic basis for cargo selectivity was tested using OmpA constructs. Our study reveals a basic mechanism for cargo selectivity into OMVs that may be useful for the engineering of OMVs for future biotechnological applications.
Authors
Orench-Rivera, N; Kuehn, MJ
MLA Citation
Orench-Rivera, Nichole, and Meta J. Kuehn. “Differential Packaging Into Outer Membrane Vesicles Upon Oxidative Stress Reveals a General Mechanism for Cargo Selectivity.Front Microbiol, vol. 12, 2021, p. 561863. Pubmed, doi:10.3389/fmicb.2021.561863.
URI
https://scholars.duke.edu/individual/pub1488964
PMID
34276573
Source
pubmed
Published In
Frontiers in Microbiology
Volume
12
Published Date
Start Page
561863
DOI
10.3389/fmicb.2021.561863

Protective plant immune responses are elicited by bacterial outer membrane vesicles.

Bacterial outer membrane vesicles (OMVs) perform a variety of functions in bacterial survival and virulence. In mammalian systems, OMVs activate immune responses and are exploited as vaccines. However, little work has focused on the interactions of OMVs with plant hosts. Here, we report that OMVs from Pseudomonas syringae and P. fluorescens activate plant immune responses that protect against bacterial and oomycete pathogens. OMV-mediated immunomodulatory activity from these species displayed different sensitivity to biochemical stressors, reflecting differences in OMV content. Importantly, OMV-mediated plant responses are distinct from those triggered by conserved bacterial epitopes or effector molecules alone. Our study shows that OMV-induced protective immune responses are independent of the T3SS and protein, but that OMV-mediated seedling growth inhibition largely depends on proteinaceous components. OMVs provide a unique opportunity to understand the interplay between virulence and host response strategies and add a new dimension to consider in host-microbe interactions.
Authors
McMillan, HM; Zebell, SG; Ristaino, JB; Dong, X; Kuehn, MJ
MLA Citation
McMillan, Hannah M., et al. “Protective plant immune responses are elicited by bacterial outer membrane vesicles.Cell Rep, vol. 34, no. 3, Jan. 2021, p. 108645. Pubmed, doi:10.1016/j.celrep.2020.108645.
URI
https://scholars.duke.edu/individual/pub1472544
PMID
33472073
Source
pubmed
Published In
Cell Reports
Volume
34
Published Date
Start Page
108645
DOI
10.1016/j.celrep.2020.108645

Dynamin-related Irgm proteins modulate LPS-induced caspase-11 activation and septic shock.

Inflammation associated with gram-negative bacterial infections is often instigated by the bacterial cell wall component lipopolysaccharide (LPS). LPS-induced inflammation and resulting life-threatening sepsis are mediated by the two distinct LPS receptors TLR4 and caspase-11 (caspase-4/-5 in humans). Whereas the regulation of TLR4 activation by extracellular and phago-endosomal LPS has been studied in great detail, auxiliary host factors that specifically modulate recognition of cytosolic LPS by caspase-11 are largely unknown. This study identifies autophagy-related and dynamin-related membrane remodeling proteins belonging to the family of Immunity-related GTPases M clade (IRGM) as negative regulators of caspase-11 activation in macrophages. Phagocytes lacking expression of mouse isoform Irgm2 aberrantly activate caspase-11-dependent inflammatory responses when exposed to extracellular LPS, bacterial outer membrane vesicles, or gram-negative bacteria. Consequently, Irgm2-deficient mice display increased susceptibility to caspase-11-mediated septic shock in vivo. This Irgm2 phenotype is partly reversed by the simultaneous genetic deletion of the two additional Irgm paralogs Irgm1 and Irgm3, indicating that dysregulated Irgm isoform expression disrupts intracellular LPS processing pathways that limit LPS availability for caspase-11 activation.
Authors
Finethy, R; Dockterman, J; Kutsch, M; Orench-Rivera, N; Wallace, GD; Piro, AS; Luoma, S; Haldar, AK; Hwang, S; Martinez, J; Kuehn, MJ; Taylor, GA; Coers, J
MLA Citation
Finethy, Ryan, et al. “Dynamin-related Irgm proteins modulate LPS-induced caspase-11 activation and septic shock.Embo Rep, vol. 21, no. 11, Nov. 2020, p. e50830. Pubmed, doi:10.15252/embr.202050830.
URI
https://scholars.duke.edu/individual/pub1464242
PMID
33124745
Source
pubmed
Published In
Embo Reports
Volume
21
Published Date
Start Page
e50830
DOI
10.15252/embr.202050830

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