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Surface exposure and packing of lipoproteins into Outer Membrane Vesicles are coupled processes in Bacteroides.

ABSTRACT: Outer membrane vesicles (OMV) are spherical structures derived from the outer membrane (OM) of Gram-negative bacteria. OMVs from the predominant gut microbiota members, Bacteroides, 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 B. theta 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 many of these OMV-enriched lipoproteins are encoded by polysaccharide utilization loci (PULs), such as the sus operon. We examined the subcellular localization of the components of the sus operon and found that the alpha-amylase SusG is highly enriched in OMVs while the oligosaccharide importer SusC remains mostly in the OM. We show that all OMV-enriched lipoproteins possess a lipoprotein export sequence (LES) that mediates translocation of SusG from the periplasmic face of the OM towards the extracellular milieu and is required for SusG to localize preferentially to OMVs. We also show that surface-exposed SusG in OMVs is active and can rescue growth of bacterial cells incapable of growing on starch as only carbon source. Our results support the role of OMVs as "public goods" that can be utilized by other organisms with different metabolic capabilities.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Bacteroides Fragilis

SUBMITTER: Nichollas Scott

LAB HEAD: Mario Feldman

PROVIDER: PXD011378 | Pride | 2018-11-04

REPOSITORIES: Pride

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Surface Exposure and Packing of Lipoproteins into Outer Membrane Vesicles Are Coupled Processes in Bacteroides.

Valguarnera Ezequiel E Scott Nichollas E NE Azimzadeh Philippe P Feldman Mario F MF

mSphere 20181107 6

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 t ...[more]

PMID: 30404931

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Project description:In Gram-negative bacteria, lipoproteins are major components of the outer membrane (OM) where they play a variety of roles, from the involvement in β-barrel assembly to virulence. Bacteroidetes, a widespread phylum of Gram-negative bacteria, including free-living organisms, commensals and pathogens, encode an exceptionally high number of outer membrane lipoproteins. These proteins are crucial in this phylum mainly because they are key components of SUS-like nutrient acquisition systems as well as of the Type IX secretion and gliding motility machineries. The transport of lipoproteins to the OM has mainly been studied in E. coli and relies on the Lol system, composed of the inner membrane extraction machinery LolCDE, the periplasmic carrier LolA and the OM lipoprotein LolB. While most Lol proteins are essential and conserved across Gram-negative bacteria, to date, no LolB homologs have been identified outside of γ- and β-proteobacteria. How lipoproteins reach and are inserted in the OM of Bacteroidetes is not known. Here we identified LolB homologs in Bacteroidetes and disclosed the co-existence of several LolA and LolB homologs in several species. We provide evidence that LolA1 and LolB1 of F. johnsoniae are devoted to targeting gliding and T9SS lipoproteins to the OM. A proteomic analysis of the OM composition of the lolA1 and lolB1 deletion strain supports this evidence. Furthermore, we show that, while LolB1 and LolA1 have conserved functions in Bacteroidetes, they are partially functionally different from their E. coli counterparts. We also show that surface lipoprotein transport is LolA and LolB independent. Finally, the finding that, in the absence of LolA and LolB homologs, lipoproteins still localize to the OM, strongly suggests the presence in Bacteroidetes of a yet unidentified Lol-alternative transport pathway. In conclusion, Bacteroidetes seem to have evolved different and probably more complex lipoprotein transport pathways than other Gram-negative bacteria and further research is required to uncover their complexity.