Characterisation of proteins secreted in the outer membrane vesicles of Bacteroides fragilis
Kowal, Maria Theresa
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Bacteroides fragilis is an important, anaerobic commensal of the human gastro-intestinal tract. As a Gram-negative bacterium, B. fragilis produces a large number of outer membrane vesicles (OMV), spherical globules consisting of outer membrane and periplasmic material, which have a range of potential functions and which are known to be able to deliver their cargo to host dendritic cells (DCs). One of the proteins believed to be packaged into the OMV of B. fragilis is BfUbb (encoded by the ubb gene) which shares 63% homology with human ubiquitin. Ubiquitin is a small, common, eukaryotic protein modifier, which is conjugated to target proteins via a series of activating, conjugating and ligating enzymes, and which has known roles in a wide range of eukaryotic cell processes. Due to key differences between the two proteins, BfUbb has the potential to act as a suicide substrate mimic of ubiquitin. BfUbb was therefore assayed for its ability to interact with ubiquitin E2 conjugating enzymes of the ubiquitylation cascade in vitro, and was found to covalently bind the majority of available enzymes in a DTT-sensitive manner. BfUbb showed a preference for three specific E2 enzymes, all of which are involved in the degradation of mitotic check point proteins, suggesting a role for BfUbb in the inhibition of cell cycle progression and, consequently, tumorigenesis. No binding partners of BfUbb were identified outside of the ubiquitylation cascade, however BfUbb was found to form spontaneous multimers in vitro, the biological function of which is unknown. This study also describes the construction of two sets of plasmids. The first set will allow the expression of untagged and fluorescently tagged forms of BfUbb for purification and use in biochemical assays. The second set will allow the expression of his-tagged and fluorescently tagged forms of BfUbb in mammalian cells, so that the effects of BfUbb on the host epithelial cells may be studied. The proteome of the OMV of B. fragilis was solved using LTQ-Orbitrap mass spectrometry. The identified proteins indicated several putative roles for B. fragilis OMV, including nutrient acquisition and protease inhibition. The suitability of techniques used during the isolation and proteomic analysis of OMV in different studies is discussed. BfUbb-carrying B. fragilis OMV were able to inhibit growth of Salmonella enterica Typhimurium, thus indicating a role for BfUbb in the inhibition of competing, pathogenic bacteria in the gastro-intestinal tract. The conclusions of this study are that the putative roles of both BfUbb and the OMV of B. fragilis may promote both survival of the bacterium and the gastro-intestinal health of the host.