Protein secretion pathways in the phylum Bacteroidetes

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Members of the genus Bacteroides are by far the most abundant Gram-negative bacteria in the human gut microbiome. We used mass spectrometry to examine the outer membrane (OM) proteome and secretome of one of the most commonly studied members of the Bacteroides genus, B. fragilis, grown under laboratory conditions. Although we did not identify any novel secretion pathways, we found that this organism uses a very different range of secretion strategies than Proteobacteria such as E. coli. B. fragilis lacks many of the pathways that are widespread among the Protebacteria (e.g., the type II, III and IV pathways) but produces multiple type I secretion systems simultaneously. The substrates of the type I pathways are unknown and cannot be easily predicted based on homology to proteins that are type I substrates in Proteobacteria. B. fragilis also differs dramatically from Proteobacteria in that it produces a large number of lipoproteins that are exposed on the cell surface. We are currently using a variety of methods to investigate the mechanism(s) by which lipoproteins are transported across the OM and to identify targeting signals that earmark specific lipoproteins for export. Interestingly, many of the proteins found in the secretome lack significant homology to proteins that are in the Genbank database and presumably have novel functions. In a recent study we examined the function of fragipain, a poorly characterized clostripain-like protease that is exposed on the cell surface of B. fragilis. Surprisingly, we found that disruption of the gene that encodes this protein (fpn) led to a strong reduction in the level of >100 proteins, many of which are predicted to be lipoproteins, in the secretome. Experiments performed with purified fragipain provided direct evidence that the protease releases at least some of these proteins from the cell surface. The observation that wild-type cells outcompeted an fpn- strain in co-cultivation assays also supported the notion that fragipain plays an important role in cell physiology. Finally, we found that purified fragipain altered the adhesive properties of HT29 intestinal epithelial cells. Our results suggest that fragipain is a broad-spectrum protease that not only catalyzes protein secretion on a wide scale but that also potentially cleaves host cell proteins during colonization.