Browsing by Author "Kuehn, Meta J"
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Item Open Access A standardized method to determine the concentration of extracellular vesicles using tunable resistive pulse sensing.(J Extracell Vesicles, 2016) Vogel, Robert; Coumans, Frank AW; Maltesen, Raluca G; Böing, Anita N; Bonnington, Katherine E; Broekman, Marike L; Broom, Murray F; Buzás, Edit I; Christiansen, Gunna; Hajji, Najat; Kristensen, Søren R; Kuehn, Meta J; Lund, Sigrid M; Maas, Sybren LN; Nieuwland, Rienk; Osteikoetxea, Xabier; Schnoor, Rosalie; Scicluna, Benjamin J; Shambrook, Mitch; de Vrij, Jeroen; Mann, Stephen I; Hill, Andrew F; Pedersen, ShonaBACKGROUND: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSION: The results of this study demonstrate the feasibility of this standardized methodology to facilitate comparable and reproducible EV concentration measurements.Item Open Access Aminopeptidase-Dependent Modulation of Bacterial Biofilms by Pseudomonas aeruginosa Outer Membrane Vesicles(2019) Esoda, Caitlin NoelPseudomonas aeruginosa, known as one of the leading causes of morbidity and mortality in cystic fibrosis (CF) patients, secretes a variety of virulence-associated proteases. These enzymes have been shown to contribute significantly to P. aeruginosa pathogenesis and biofilm formation in the chronic colonization of CF patient lungs, as well as playing a role in infections of the cornea, burn wounds and chronic wounds. Our lab has previously characterized a secreted P. aeruginosa peptidase, PaAP, that is highly expressed in chronic CF isolates. This leucine aminopeptidase is also highly expressed during infection and in biofilms, and it associates with bacterial outer membrane vesicles (OMVs), structures known for their contribution to virulence mechanisms in a variety of Gram-negative species and one of the major components of the biofilm matrix. With this in mind, we hypothesized that PaAP may play a role in P. aeruginosa biofilm formation. Using a lung epithelial cell/bacterial biofilm coculture model, we show that PaAP deletion in a clinical P. aeruginosa background alters biofilm microcolony composition to increase cellular density, while decreasing matrix polysaccharide content and resistance to the antibiotic colistin. We recreate this phenotype using a pellicle biofilm model, in which bacteria are grown statically at the culture air-liquid interface, demonstrating that these phenotypes are not dependent on the coculture host cell substrate. We additionally show that OMVs from PaAP expressing strains, but not PaAP alone or in combination with PaAP deletion strain-derived OMVs, could complement this phenotype. Finally, we found that OMVs from PaAP-expressing strains cause protease-mediated biofilm detachment, leading to changes in matrix and colony composition. OMVs mediated the detachment of biofilms formed by both non-self P. aeruginosa strains and K. pneumoniae, another respiratory pathogen, showing that this process may also be relevant in polymicrobial communities and acts on non-P. aeruginosa derived substrates. Our findings represent novel roles for OMVs and the PaAP aminopeptidase in the modulation of bacterial biofilm architecture.
Item Open Access Breaking the bilayer: OMV formation during environmental transitions.(Microb Cell, 2017-02-03) Bonnington, Katherine E; Kuehn, Meta JGram-negative bacteria maintain the barrier properties of the outer membrane (OM) in a wide array of physiological conditions despite their inability to degrade lipopolysaccharide (LPS) and protein material present in the outer leaflet of the OM. Through characterization of the native dynamics of outer membrane LPS change we recently described a mechanism in which these diderm organisms overcome this design flaw. In response to different environmental stimuli Salmonellaenterica modulates the export of specific structural variants of lipid A via outer membrane vesicles (OMVs). We proposed that the polymorphic model for regulation of membrane lipid content could largely account for the structural differences between secreted and retained lipid A species. However, differences in OMV production levels and size observed between environmental conditions remain unexplained. Further exploration into the relationship between OMV production level and content specificity may shed light onto the enigmatic mechanisms of OMV formation.Item Open Access Contribution of bacterial outer membrane vesicles to innate bacterial defense.(BMC Microbiol, 2011-12-01) Manning, Andrew J; Kuehn, Meta JBACKGROUND: 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.Item Open Access Differential Packaging of Outer Membrane Proteins of Enterotoxigenic Escherichia coli into Outer Membrane Vesicles under Oxidative Stress Conditions Reveals a Potential Mechanism for Vesicle Cargo Selectivity(2020) Orench-Rivera, NicholeOuter membrane vesicles (OMVs) are spherical structures that bud from the outer membrane (OM) of bacteria containing OM and periplasmic material. They are known to be produced by all bacteria studied to date and play important roles in inter-bacterial communication, bacterial-host interactions, toxin delivery, survival, nutrient acquisition, and biofilm development. The process of OMV production is known to be genetically regulated and selective cargo packaging into bacterial vesicles has been reported and implicated in many biological processes. While much is known about how cargo gets incorporated into vesicles in eukaryotic systems, the mechanism behind cargo selectivity in bacteria has remained largely unexplored. In this study we aimed to characterize preferential sorting trends in OMV packaging in Escherichia coli under oxidative stress, and investigate the mechanism behind selective sorting into OMVs. Proteomic analysis of outer membrane (OM) and OM vesicle fractions from enterotoxigenic E. coli (ETEC) 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 and we observed a slight increase in periplasmic content. 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. We first inquired whether this sorting was due to the need of the cell to discard or retain OM proteins and tested oxidative stress sensitivity of mutants of lipoproteins and integral proteins. We hypothesized that mutants of lipoproteins would not be more sensitive than integral proteins however both groups showed increased sensitivity. Therefore, this did not explain this preferential sorting. We next wondered if selectivity was dependent on gene expression. By mining gene expression databases and performing qRT-PCR we found the sorting to be independent of transcriptional regulation of the proteins upon oxidative stress. We were also able to validate these preferential sorting trends of lipoproteins vs integral proteins using randomly selected protein candidates from the different cargo classes. We also observed that a shift to oxidative stress conditions improved the fitness of bacteria to a secondary oxidative challenge, suggesting the differential sorting resulted in an OMV-mediated remodeling of the OM during stress. Together, our data showed that oxidative stress induced a differential sorting of proteins into OMVs and OM of E. coli and that OMV production might serve as a disposal mechanism for the cell to rid itself of oxidized proteins. Since our data revealed that the preferentially retained proteins were those known to have ties to other cell envelope components, a hypothetical functional and mechanistic basis for cargo selectivity was tested using OmpA as a model. A full-length and a truncated version of OmpA were used to test whether physical tethering to the cell is a determinant for protein retention in the OM. Quantifying OMV protein packaging of both OmpA constructs revealed a basic mechanism for cargo selectivity into OMVs. We show that the untethered version of OmpA was more likely to be exported than the tethered version and that this preferential selection was exacerbated under oxidative stress. The findings of this study provide insight into the dynamics of bacterial cargo selection and membrane remodeling during stress as well as propose and test a mechanism for cargo incorporation in E. coli.
Item Open Access Effect of LPS remodeling and LPS-binding proteins on outer membrane vesicle production and composition(2017) Bonnington, Katherine BonningtonEnvironmental transitions cause bacteria to simultaneously alter lipopolysaccharide (LPS) structure, release LPS-binding proteins, and increase outer membrane vesicle (OMV) production. However, any relationship between these events is unknown. The research described here aims to fill this gap in knowledge by examining the effect of environmental membrane remodeling factors and LPS-binding proteins on the regulation of OMV production and composition.
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 to remove unfavorable lipopolysaccharide (LPS) glycoforms except by slow dilution through cell growth. As all Gram-negative bacteria constitutively shed outer membrane 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 (TCS). In response to acidic pH, toxic metals, antimicrobial peptides, and lack of divalent cations, these TCS modify the LPS lipid A and core, lengthen the O-antigen, and up-regulate 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, down-regulation 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. A polymorphic model for regulation of OM LPS composition may explain the propensity of different LPS structures, with varied geometries and biophysical properties, to be retained in the OM or to be shed via OMVs.
Many pathogenic bacteria secrete toxins which are also lectins, and an increasing number of these proteins have been found to bind to LPS in addition to the host receptor. To investigate the effect of LPS-binding proteins on OM dynamics, we first defined the binding affinity and specificity of heat-labile enterotoxin-LPS binding. We then characterized the ability of the toxin to modulate membrane properties and stimulate OMV production in enterotoxigenic E. coli. Finally, we present a model wherein external toxin binds to LPS and crowds upon the OM to stimulate OMV formation.
Item Open Access Environmentally controlled bacterial vesicle-mediated export.(Cell Microbiol, 2016-11) Orench-Rivera, Nichole; Kuehn, Meta JOver 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.Item Open Access Genome-Wide Assessment of Outer Membrane Vesicle Production in Escherichia coli.(PLoS One, 2015) Kulp, Adam J; Sun, Bo; Ai, Teresa; Manning, Andrew J; Orench-Rivera, Nichole; Schmid, Amy K; Kuehn, Meta JThe 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.Item Open Access Heat-labile enterotoxin: beyond G(m1) binding.(Toxins (Basel), 2010-06) Mudrak, Benjamin; Kuehn, Meta JEnterotoxigenic 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.Item Open Access Inflammasome Activation by Bacterial Outer Membrane Vesicles Requires Guanylate Binding Proteins.(MBio, 2017-10-03) Finethy, Ryan; Luoma, Sarah; Orench-Rivera, Nichole; Feeley, Eric M; Haldar, Arun K; Yamamoto, Masahiro; Kanneganti, Thirumala-Devi; Kuehn, Meta J; Coers, JörnThe Gram-negative bacterial cell wall component lipopolysaccharide (LPS) is recognized by the noncanonical inflammasome protein caspase-11 in the cytosol of infected host cells and thereby prompts an inflammatory immune response linked to sepsis. Host guanylate binding proteins (GBPs) promote infection-induced caspase-11 activation in tissue culture models, and yet their in vivo role in LPS-mediated sepsis has remained unexplored. LPS can be released from lysed bacteria as "free" LPS aggregates or actively secreted by live bacteria as a component of outer membrane vesicles (OMVs). Here, we report that GBPs control inflammation and sepsis in mice injected with either free LPS or purified OMVs derived from Gram-negative Escherichia coli In agreement with our observations from in vivo experiments, we demonstrate that macrophages lacking GBP2 expression fail to induce pyroptotic cell death and proinflammatory interleukin-1β (IL-1β) and IL-18 secretion when exposed to OMVs. We propose that in order to activate caspase-11 in vivo, GBPs control the processing of bacterium-derived OMVs by macrophages as well as the processing of circulating free LPS by as-yet-undetermined cell types.IMPORTANCE The bacterial cell wall component LPS is a strong inducer of inflammation and is responsible for much of the toxicity of Gram-negative bacteria. Bacteria shed some of their cell wall and its associated LPS in the form of outer membrane vesicles (OMVs). Recent work demonstrated that secreted OMVs deliver LPS into the host cell cytosol by an unknown mechanism, resulting in the activation of the proinflammatory LPS sensor caspase-11. Here, we show that activation of cytosolic caspase-11 by OMVs requires additional host factors, the so-called guanylate binding proteins (GBPs). The discovery of GBPs as regulators of OMV-mediated inflammation paves the way toward a mechanistic understanding of the host response toward bacterial OMVs and may lead to effective strategies to ameliorate inflammation induced by bacterial infections.Item Open Access Interactions of a secreted Pseudomonas aeruginosa aminopeptidase with bacterial outer membrane molecules: characterization and implications for bacterial pathogenic phenotypes.(2017-05-08) Campbell, TimothyThe protein Pseudomonas aeruginosa aminopeptidase (PaAP) is secreted by the opportunistic, Gram-negative bacterial pathogen Pseudomonas aeruginosa. PaAP expression appears to be upregulated in clinical isolates of P. aeruginosa, especially in strains found in the lungs of cystic fibrosis patients, likely indicating that it serves a role in the development of pathogenic phenotypes. However, the function(s) that PaAP serves in pathogenic contexts are as yet unknown, as are the mechanisms by which PaAP accomplishes them. This work seeks to identify molecules of the Gram negative outer membrane with which PaAP interacts in order to understand the role that PaAP plays in the establishment and maintenance of P. aeruginosa infections. Particular attention is given to determining whether PaAP interacts with lipopolysaccharide (LPS), which is the major constituent of the Gram negative outer membrane and a potent toxin and immune system agonist. To accomplish this goal, a range of biochemical analyses are used, particularly immunochemistry techniques. While results from these assays are preliminary, our findings suggest that PaAP does not interact with P. aeruginosa LPS, suggesting that proteins or non-LPS saccharides are the primary interaction partners of PaAP in the outer membrane.Item Open Access Modulation of bacterial outer membrane vesicle production by envelope structure and content.(BMC Microbiol, 2014-12-21) Schwechheimer, Carmen; Kulp, Adam; Kuehn, Meta JBACKGROUND: 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. RESULTS: 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. CONCLUSIONS: From this work, we conclude that OMV production can be driven by distinct Lpp concentration-dependent and Lpp concentration-independent pathways.Item Open Access NlpI-mediated modulation of outer membrane vesicle production through peptidoglycan dynamics in Escherichia coli.(Microbiologyopen, 2015-06) Schwechheimer, Carmen; Rodriguez, Daniel L; Kuehn, Meta JOuter 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.Item Open Access Novel Protein Regulators of Heat Shock Transcription Factor 1 During Stress and Disease(2019) Burchfiel, Eileen Therese MalloyHeat Shock Transcription Factor 1 (HSF1) is a critical regulator of transcription that facilitates cellular stress protection in response to protein misfolding, rapid cell proliferation, and other stressful conditions. Defective HSF1 regulation is observed in cellular and animal models of cancer, where hyperactive and dysregulated HSF1 supports cancer survival, and in neurodegenerative disease, where HSF1 function is compromised, further exacerbating protein misfolding. HSF1 is tightly regulated through intramolecular interactions, post-translational modifications, and protein-protein interactions; however, little is known about ho HSF1 regulation differs in response to stresses such as acute or chronic protein misfolding.
We identified one mechanism that contributes to the diminution of HSF1 in chronic protein misfolding in the context of Huntington’s Disease involving inappropriate interactions of HSF1 with CK2α’ and FBXW7 E3 ligase. We found these protein-protein interactions coordinate the abnormal phosphorylation-dependent degradation of HSF1. Importantly, inhibition of this aberrant HSF1 degradation attenuates the biochemical defects and protein misfolding in Huntington’s Disease. To further elucidate how HSF1-interacting proteins regulate HSF1 in acute and chronic stress, we carried out quantitative proteomics studies of the HSF1 interactome under control, acute heat shock, and in a cell model of Huntington’s Disease. We recapitulated many previously described interaction partners of HSF1 and identified several novel HSF1-interacting proteins that encompass a wide variety of cellular functions, including roles in DNA repair, mRNA processing, and regulation of RNA polymerase II. We further report on the interaction of HSF1 with CCCTC binding factor (CTCF), which modulates target gene activation and repression function of HSF1 by facilitating DNA binding at CTCF and HSF1 co-regulated loci. Given the role and elevated expression of both pro-inflammatory proteins and Tau in Huntington’s Disease, and their defective repression by HSF1, understanding the mechanisms of HSF1 repression is of great interest. The studies presented in this thesis expand our understanding of HSF1-mediated gene activation and repression, and the regulation of HSF1 via protein-protein interactions.
Item Open Access Outer Membrane Vesicle Production Facilitates LPS Remodeling and Outer Membrane Maintenance in Salmonella during Environmental Transitions.(MBio, 2016-10-18) Bonnington, Katherine E; Kuehn, Meta JThe 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. IMPORTANCE: 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.Item Open Access Outer Membrane Vesiculation Facilitates Surface Exchange and In Vivo Adaptation of Vibrio cholerae.(Cell host & microbe, 2019-12-23) Zingl, Franz G; Kohl, Paul; Cakar, Fatih; Leitner, Deborah R; Mitterer, Fabian; Bonnington, Katherine E; Rechberger, Gerald N; Kuehn, Meta J; Guan, Ziqiang; Reidl, Joachim; Schild, StefanGram-negative bacteria release outer membrane vesicles into the external milieu to deliver effector molecules that alter the host and facilitate virulence. Vesicle formation is driven by phospholipid accumulation in the outer membrane and regulated by the phospholipid transporter VacJ/Yrb. We use the facultative human pathogen Vibrio cholerae to show that VacJ/Yrb is silenced early during mammalian infection, which stimulates vesiculation that expedites bacterial surface exchange and adaptation to the host environment. Hypervesiculating strains rapidly alter their bacterial membrane composition and exhibit enhanced intestinal colonization fitness. This adaptation is exemplified by faster accumulation of glycine-modified lipopolysaccharide (LPS) and depletion of outer membrane porin OmpT, which confers resistance to host-derived antimicrobial peptides and bile, respectively. The competitive advantage of hypervesiculation is lost upon pre-adaptation to bile and antimicrobial peptides, indicating the importance of these adaptive processes. Thus, bacteria use outer membrane vesiculation to exchange cell surface components, thereby increasing survival during mammalian infection.Item Open Access Protective plant immune responses are elicited by bacterial outer membrane vesicles.(Cell reports, 2021-01) McMillan, Hannah M; Zebell, Sophia G; Ristaino, Jean B; Dong, Xinnian; Kuehn, Meta JBacterial 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.Item Open Access Pseudomonas aeruginosa vesicles associate with and are internalized by human lung epithelial cells.(BMC Microbiol, 2009-02-03) Bauman, Susanne J; Kuehn, Meta JBACKGROUND: 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.Item Open Access Release of outer membrane vesicles by Gram-negative bacteria is a novel envelope stress response.(Mol Microbiol, 2007-01) McBroom, Amanda J; Kuehn, Meta JConditions 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.Item Open Access Secretion and delivery of bacteria-derived immunomodulatory RNA and DNA via extracellular membrane vesicles(2021) Rodriguez, Blanca VictoriaNucleic acids produced by the human pathogen, Staphylococcus aureus, have become increasingly recognized as important pathogen-associated molecular patterns capable of eliciting Type I Interferon (IFN) signaling in host cells. Intracellular Toll-like receptors (TLRs) have been identified as sensors of S. aureus-derived RNA and DNA. However, there is a lack of mechanistic insight into how S. aureus secretes nucleic acids and how nucleic acids derived from S. aureus—which is classified as an extracellular pathogen—are transported into host cells to activate TLRs to modulate host cell functions. Recent studies have uncovered extracellular membrane vesicles (MVs) produced by bacteria as a novel secretion mechanism for biologically active RNA and DNA. These findings raised the intriguing possibility of a new paradigm in bacterial secretion whereby MVs act as a vehicle for both local and distant delivery of regulatory and immunomodulatory RNA and DNA to target cells. As such, we hypothesized that MVs secreted by S. aureus could represent an important source of functional and structurally relevant bacterial RNA and DNA molecules that modulate the host immune response. Through biochemical analysis of S. aureus MVs and their nucleic acid cargo, we found that subpopulations of MV-associated RNA and DNA can withstand nuclease degradation. We report that in cultured mouse macrophages, IFN-β mRNA is induced by S. aureus MVs and that MV-induced IFN-β expression is specifically promoted by activation of endosomal TLR3, TLR7, and TLR9 receptors—pointing to the likelihood that MV-associated, protected RNA and DNA are inducing such a response. By metabolically labeling S. aureus RNA in culture and imaging the resultant MVs using super-resolution microscopy, we provide visual evidence that MVs and their stably associated RNA cargo are internalized by macrophages. Our work suggests that both unprotected and protected S. aureus MV-associated RNA and DNA molecules induce significant responses in host cells, but do so by different pathways, and that these differences could have important functional consequences. Altogether, we provide the first piece of evidence that S. aureus releases MVs laden with RNA and DNA molecules that are internalized by host immune cells to induce IFN-β via TLR-dependent signaling. These novel insights could promote the development of novel therapeutic strategies for the treatment of staphylococcal disease.