Preferential Packing of Acidic Glycosidases and Proteases into Bacteroides Outer Membrane Vesicles

Abstract

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.

Prokaryotic membrane vesicles: new insights on biogenesis and biological roles

“…Even though OMV production was documented almost 50 years ago, the subject has been neglected and the OMVs were often regarded as broken cells or artifacts. When the protein composition of the purified OM and OMVs of several Gram-negative bacteria was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by protein staining, a different protein pattern was observed in both fractions (Grenier and Mayrand, 1987;Kadurugamuwa and Beveridge, 1995;Horstman and Kuehn, 2002;Kato et al, 2002;Wai et al, 2003;Sidhu et al, 2008;Frias et al, 2010;Kahnt et al, 2010;Haurat et al, 2011;Lappann et al, 2013;McCaig et al, 2013;Aguilera et al, 2014;Elhenawy et al, 2014;Jang et al, 2014). Certain proteins were more abundant in the OMVs than in the OM fraction and others were missing in one of the fractions.…”

“…Recently, two groups employed totally different approaches, arriving to the same conclusion that Bacteroides OMVs are involved in a complex network dedicated to processing of nutrients in the human gut. Elhenawy et al employed a proteomic approach to demonstrate the selective packing of a large number of carbohydrate and protein hydrolases exclusively in the OMVs of both B. fragilis and B. thetaiotaomicron (Elhenawy et al, 2014). Some of these hydrolases were shown to be active in vitro and were enriched in OMVs in response to external stimuli.…”

“…In addition they could be considered as a defense mechanism by carrying away toxic compounds, phages or unfolded proteins after exposure to stressful conditions (McBroom and Kuehn, 2007;. Recently, an important role has been proposed for outer membrane vesicles (OMVs) in microbiota homeostasis (Elhenawy et al, 2014;Rakoff-Nahoum et al, 2014).…”

Polysaccharide Utilization Loci: Fueling Microbial Communities

“…For example, in some species, CAZymes are selectively packaged into outer membrane vesicles and released into the extracellular environment, where they are thought to mediate the production of free glycan fragments for use by the greater gut community (115,119). Remarkably, certain species, such as B. ovatus, secrete enzymes that are not required for the utilization of glycans such as inulin by the bacterium itself or by its clonemates; rather, this effort appears to benefit other species in the gut community (116).…”

Spheres of Hope, Packets of Doom: the Good and Bad of Outer Membrane Vesicles in Interspecies and Ecological Dynamics

“…OMVs from the cyanobacterium Prochlorococcus contain labile carbon that can be used as the sole carbon source for the heterotrophs Alteromonas and Halomonas (7). Similarly, gut-residing members of the Bacteroides genus distribute hydrolases and polysaccharide lyases via OMVs (45,46); by making these enzymes publicly available, the microbiome can communally break down complex polysaccharides that are immutable to individual species. The exact benefit to the producer of providing these services in each of these circumstances is unclear, but it is assumed to provide a net benefit, such as a higher survival rate (44).…”