Browsing by Author "Orench-Rivera, Nichole"
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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 Dynamin-related Irgm proteins modulate LPS-induced caspase-4 activation and septic shock(2020) Finethy, Ryan; Dockterman, Jacob; Kutsch, Miriam; Orench-Rivera, Nichole; Wallace, Graham; Piro, Anthony; Luoma, Sarah; Haldar, Arun; Hwang, Seungmin; Martinez, Jennifer; Kuehn, Meta; Taylor, Gregory; Coers, JörnABSTRACT
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-4. 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-4 are largely unknown. This study identifies dynamin-related membrane remodeling proteins belonging to the family of Immunity related GTPases M clade (IRGM) as negative regulators of caspase-4 activation in macrophages. Phagocytes lacking expression of mouse isoform Irgm2 aberrantly activate caspase-4-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-4-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-4 activation.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 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.