Browsing by Subject "Outer membrane vesicles"
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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 Examining the Effect of the Context of Heat-Labile Enterotoxin Presentation on the Host Immune Response(2011) Chutkan, HalimaEnterotoxigenic Escherichia coli (ETEC), the leading cause of traveler's diarrhea and childhood mortality due to diarrhea in the developing world, has been shown to secrete heat-labile enterotoxin (LT) in association with outer membrane vesicles. However, studies on the effect of LT have been performed using soluble LT, which is not its physiologically relevant presentation context. The effect of LT associated with vesicles and its trafficking within human intestinal epithelial cells were compared with soluble LT. Cytokine responses and trafficking of standardized samples of soluble LT and vesicle-associated LT were evaluated in polarized intestinal epithelial cells. Using real-time PCR, immunoblotting, and ELISAs, we found that compared to soluble LT, vesicle-bound LT showed delayed kinetics in the activation of LT. Vesicles containing LT or not also produced cytokines through different signaling pathways than soluble LT. We found that this difference in signaling was due to different trafficking within the cell. Interestingly, not all LT associated with vesicles is active within cells. Vesicle-associated LT must bind to the host receptor GM1 in lipid rafts to be active within cells. This suggests that although vesicles can deliver large amounts of LT to a cell, much of the LT would be inactive and not produce a physiological response. To test this hypothesis, we attempted to develop animal models for ETEC-induced diarrhea. Although the models were largely unsuccessful, the mouse model appears promising for determining the physiological response of a host to LT as fluid accumulation was observed in response to vesicles containing LT. The results in this thesis provide further understanding of the mechanism of LT-induced diarrhea and emphasize the importance of study toxins in their natural context.
Item Open Access Outer Membrane Vesicle Production in Escherichia coli Relieves Envelope Stress and is Modulated by Changes in Peptidoglycan(2014) Schwechheimer, CarmenBacterial outer membrane vesicles (OMVs) are spherical buds of the outer membrane (OM) containing periplasmic lumenal components. OMVs have been demonstrated to play a critical part in the transmission of virulence factors, immunologically active compounds, and bacterial survival, however vesiculation also appears to be a ubiquitous physiological process for Gram-negative bacteria. Despite their characterized biological roles, especially for pathogens, very little is known about their importance for the originating organism as well as regulation and mechanism of production. Only when we have established their biogenesis can we fully uncover their roles in pathogenesis and bacterial physiology. The overall goal of this research was to characterize bacterial mutants which display altered vesiculation phenotypes using genetic and biochemical techniques, and thereby begin to elucidate the mechanism of vesicle production and regulation. One part of this work elucidated a synthetic genetic growth defect for a strain with reduced OMV production (ΔnlpA, inner membrane lipoprotein with a minor role in methionine transport) and envelope stress (ΔdegP, dual function periplasmic chaperone/ protease responsible for managing proteinaceous waste). This research showed that the growth defect of ΔnlpAΔdegP correlated with reduced OMV production with respect to the hyprevesiculator ΔdegP and the accumulation of protein in the periplasm and DegP substrates in the lumen of OMVs. We further demonstrated that OMVs do not solely act as a stress response pathway to rid the periplasm of otherwise damaging misfolded protein but also of accumulated peptidoglycan (PG) fragments and lipopolysaccharide (LPS), elucidating OMVs as a general stress response pathway critical for bacterial well-being. The second part of this work, focused on the role of PG structure, turnover and covalent crosslinks to the OM in vesiculation. We established a direct link between PG degradation and vesiculation: Mutations in the OM lipoprotein nlpI had been previously established as a very strong hypervesiculation phenotype. In the literature NlpI had been associated with another OM lipoprotein, Spr that was recently identified as a PG hydrolase. The data presented here suggest that NlpI acts as a negative regulator of Spr and that the ΔnlpI hypervesiculation phenotype is a result of rampantly degraded PG by Spr. Additionally, we found that changes in PG structure and turnover correlate with altered vesiculation levels, as well as non-canonical D-amino acids, which are secreted by numerous bacteria on the onset of stationary phase, being a natural factor to increase OMV production. Furthermore, we discovered an inverse relationship between the concentration of Lpp-mediated, covalent crosslinks and the level of OMV production under conditions of modulated PG metabolism and structure. In contrast, situations that lead to periplasmic accumulation (protein, PG fragments, and LPS) and consequent hypervesiculation the overall OM-PG crosslink concentration appears to be unchanged. Form this work, we conclude that multiple pathways lead to OMV production: Lpp concentration-dependent and bulk driven, Lpp concentration-independent.
Item Open Access Outer Membrane Vesicles: A New Paradigm of Bacterial Innate Immunity(2013) Manning, AndrewOuter membrane vesicles are an important constitutive product of all Gram-negative bacteria. Bacteria have evolved many responses to alleviate all different types of stress. The primary objective of this dissertation is to investigate the role of outer membrane vesicles (OMVs) as a method by which Gram-negative bacteria can quickly act to protect themselves against particular threats. Generally, we find that stressors whose primary effect is on the outer membrane can be protected against by OMVs. Throughout this study, a variety of different microbiological and biochemical methods are used to answer key questions in the innate ability of OMVs to protect against particular antimicrobials. Using Escherichia coli as well as Pseudomonas aeruginosa as model organisms we tested the ability of purified vesicles from each species to protect themselves and other hosts. Using bacteriophage T4, we investigated the ability of OMVs purified from E. coli to adsorb phage as well as how this interaction affected the efficiency of infection. We found that OMVs are protective against antimicrobial peptides, as well as bacteriophage. In the course of understanding this protection we also observed and characterized the cross species effects of both OMV protection as well as phage infection. Where typically a phage infects a specific species, we found that T4 associated OMVs treating a non-native host P. aeruginosa resulted in the production of a novel prophage. Upon further examination, we determined that this induction was occurring via a novel pathway that we attempted to further characterize by performing a genetic screen to identify genes important to this induction. The work within this dissertation fully supports the hypothesis of a regulated response to outer membrane acting stimuli, resulting in the induction of vesiculation and the adsorption of stressor in the extra-cellular milieu. This model of protection agrees with the idea of a bacterial innate defense system, which acts in the short term before the adaptive response can fully occur, resulting in a bridge between the untreated to the treated and resistant culture.