A dual motif mediates outer-membrane translocation and packing of glycosidases into Bacteroides Outer Membrane Vesicles.

Abstract

Outer membrane vesicles (OMV) are spherical structures derived from the outer membrane (OM) of Gram-negative bacteria. Bacteroides spp. are prominent components of the human gut microbiota, and OMV produced with 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 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 most 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 system, 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.\n\nIMPORTANCESpecies 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. Nevertheless, the mechanistic nature of OMV biogenesis is unclear for Bacteroides spp. This works shows the presence of a negatively-charged rich amino acid dual motif that is required for efficient packing of the surface-exposed alpha-amylase SusG into OMVs. Discovery of this motif (LES) 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.