Dataset Information

The Uptake, Trafficking, and Biodistribution of Bacteroides thetaiotaomicron Generated Outer Membrane Vesicles.

ABSTRACT: Gram-negative bacteria ubiquitously produce and release nano-size, non-replicative outer membrane vesicles (OMVs). In the gastrointestinal (GI-) tract, OMVs generated by members of the intestinal microbiota are believed to contribute to maintaining the intestinal microbial ecosystem and mediating bacteria-host interactions, including the delivery of bacterial effector molecules to host cells to modulate their physiology. Bacterial OMVs have also been found in the bloodstream although their origin and fate are unclear. Here we have investigated the interactions between OMVs produced by the major human gut commensal bacterium, Bacteroides thetaiotaomicron (Bt), with cells of the GI-tract. Using a combination of in vitro culture systems including intestinal epithelial organoids and in vivo imaging we show that intestinal epithelial cells principally acquire Bt OMVs via dynamin-dependent endocytosis followed by intracellular trafficking to LAMP-1 expressing endo-lysosomal vesicles and co-localization with the perinuclear membrane. We observed that Bt OMVs can also transmigrate through epithelial cells via a paracellular route with in vivo imaging demonstrating that within hours of oral administration Bt OMVs can be detected in systemic tissues and in particular, the liver. Our findings raise the intriguing possibility that OMVs may act as a long-distance microbiota-host communication system.

SUBMITTER: Jones EJ

PROVIDER: S-EPMC7015872 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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The Uptake, Trafficking, and Biodistribution of <i>Bacteroides thetaiotaomicron</i> Generated Outer Membrane Vesicles.

Jones Emily J EJ Booth Catherine C Fonseca Sonia S Parker Aimee A Cross Kathryn K Miquel-Clopés Ariadna A Hautefort Isabelle I Mayer Ulrike U Wileman Tom T Stentz Régis R Carding Simon R SR

Frontiers in microbiology 20200206

Gram-negative bacteria ubiquitously produce and release nano-size, non-replicative outer membrane vesicles (OMVs). In the gastrointestinal (GI-) tract, OMVs generated by members of the intestinal microbiota are believed to contribute to maintaining the intestinal microbial ecosystem and mediating bacteria-host interactions, including the delivery of bacterial effector molecules to host cells to modulate their physiology. Bacterial OMVs have also been found in the bloodstream although their origi ...[more]

PMID: 32117106

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Project description:Outer membrane vesicles (OMV) are spherical membranous structures released from the outer membrane (OM) of Gram-negative bacteria. OMV have been proposed to play several different roles during both pathogenesis and symbiosis. Despite the fact that OMV were described several decades ago, their biogenesis is a poorly characterized process. Whether OMV are produced by an active mechanism or by passive disintegration of the OM is a still matter of controversy. Bacteroides fragilis and Bacteroides thetaiotaomicron are important members of the human microbiota. In this work, we determined and compared the protein compositions of OM and OMV from B. fragilis and B. thetaiotaomicron. SDS-PAGE analysis of both fractions revealed dramatically different protein profiles. Proteomic analysis of OM and OMV in B. fragilis identified more than 40 proteins found exclusively in OMV and more than 30 proteins detectable only in the OM. The OMV-specific proteome showed a high prevalence of glycosidases and proteases, some of which were shown to be active in vitro. Similar results were obtained for B. thetaiotaomicron. Most of the OMV-exclusive proteins were acidic. Based on these results, we propose that these species possess machinery devoted to selectively pack acidic proteins into the OMV. These OMV equipped with hydrolytic enzymes could help in securing nutrients for the benefit of the whole bacterial community present in the microbiota, uncovering a novel function for bacterial OMV. IMPORTANCE The members of genus Bacteroides are key players in the symbiosis between the human host and the gut microbiota. It is known for its ability to degrade a wide variety of glycans that are not substrates for human glycosidases. The cleaved glycans can be utilized by Bacteroides and other microbiota members, resulting in the production of short-chain fatty acids that are beneficial for the host. Although members of the genus Bacteroides are known to secrete different hydrolases, their secretion pathways remain uncharacterized. In this article, we show that B. fragilis and B. thetaiotaomicron preferentially pack a large number of hydrolases in outer membrane vesicles (OMV). Most of these hydrolases are acidic and were detected exclusively in OMV. This suggests the presence of a molecular mechanism in Bacteroides responsible for the selection of OMV proteins based on their charge. We propose that OMV contribute to the establishment and balance of the gut microbiota.

Project description:Outer membrane vesicles (OMVs) are spherical structures derived from the outer membranes (OMs) of Gram-negative bacteria. Bacteroides spp. are prominent components of the human gut microbiota, and OMVs produced by these species are proposed to play key roles in gut homeostasis. OMV biogenesis in Bacteroides is a poorly understood process. Here, we revisited the protein composition of Bacteroides thetaiotaomicron OMVs by mass spectrometry. We confirmed that OMVs produced by this organism contain large quantities of glycosidases and proteases, with most of them being lipoproteins. We found that most of these OMV-enriched lipoproteins are encoded by polysaccharide utilization loci (PULs), such as the sus operon. We examined the subcellular locations of the components of the Sus system and found a split localization; the alpha-amylase SusG is highly enriched in OMVs, while the oligosaccharide importer SusC remains mostly in the OM. We found that all OMV-enriched lipoproteins possess a lipoprotein export sequence (LES), and we show that this signal mediates translocation of SusG from the periplasmic face of the OM toward the extracellular milieu. Mutations in the LES motif caused defects in surface exposure and recruitment of SusG into OMVs. These experiments link, for the first time, surface exposure to recruitment of proteins into OMVs. We also show that surface-exposed SusG in OMVs is active and rescues the growth of bacterial cells incapable of growing on starch as the only carbon source. Our results support the role of OMVs as "public goods" that can be utilized by other organisms with different metabolic capabilities.IMPORTANCE Species from the Bacteroides genus are predominant members of the human gut microbiota. OMVs in Bacteroides have been shown to be important for the homeostasis of complex host-commensal relationships, mainly involving immune tolerance and protection from disease. OMVs carry many enzymatic activities involved in the cleavage of complex polysaccharides and have been proposed as public goods that can provide growth to other bacterial species by release of polysaccharide breakdown products into the gut lumen. This work shows that the presence of a negatively charged rich amino acid motif (LES) is required for efficient packing of the surface-exposed alpha-amylase SusG into OMVs. Our findings strongly suggest that surface exposure is coupled to packing of Bacteroides lipoproteins into OMVs. This is the first step in the generation of tailor-made probiotic interventions that can exploit LES-related sequences to generate Bacteroides strains displaying proteins of interest in OMVs.

Dataset Information

The Uptake, Trafficking, and Biodistribution of Bacteroides thetaiotaomicron Generated Outer Membrane Vesicles.

Publications

The Uptake, Trafficking, and Biodistribution of <i>Bacteroides thetaiotaomicron</i> Generated Outer Membrane Vesicles.

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