| Summary: | Bacteroides fragilis is a Gram negative, anaerobic colonic commensal that has been implicated in both homeostatic and disease processes ranging from development of the immune system and provision of anti-inflammatory signals, to acute inflammatory diarrhea, inflammatory bowel disease (IBD), colon cancer, sepsis, and abscess formation. Two B. fragilis factors are major contributors to these states. First, B. fragilis capsular polysaccharides in the gut are host-beneficial signals, while in systemic contexts are necessary and sufficient to promote abscess formation. Secondly, the B. fragilis toxin (BFT), produced by Enterotoxigenic B. fragilis (ETBF) strains, is the only known virulence factor in B. fragilis-associated pro-inflammatory colonic disease.
BFT is a zinc metalloprotease that is produced as an inactive zymogen. It has an N-terminal signal sequence and lipobox motif followed by a prodomain and catalytic domain. The prodomain maintains catalytic latency, and its removal derepresses toxin activity. As an active toxin, BFT disrupts the intestinal epithelial barrier and induces cell proliferation, NFkappaB pro-inflammatory signaling, and a Th17 response. BFT is associated with colonic disease in humans and has been shown to cause chronic colitis and progression of colon cancer in susceptible mouse models.
Despite the physiological significance of B. fragilis, the interaction of this microbe with the host remains poorly characterized. There is a paucity of information on processing and export of BFT, and a general mechanism of protein export remains to be identified in B. fragilis . Using a transposon mutagenesis screen, I have identified genes involved in BFT production and shown that post-translational modifications, in particular glycosylation and proteolytic cleavage, are important for toxin release and activity. The protease responsible for BFT cleavage, Fragipain (Fpn), was then characterized further. Fpn is a cysteine protease that undergoes autocatalytic cleavage into two chains and has structural similarity to members of protease clan CD, as seen in a 2.4 A structure of purified Fpn. The enzyme is localized to the external surface of the outer membrane and expressed constitutively in culture. Deletion of fpn results in a reduction of proteins in the supernatant, suggesting that Fpn releases proteins into the environment. While it was expected that colonic infection with an fpn mutant would result in reduced pathology, it had minimal effect in a mouse model of ETBF-mediated colitis. Instead, we found that mouse mucus was sufficient to activate BFT in vitro. In contrast to our findings in the gastrointestinal model, fpn mutants exhibited a substantial reduction in lethality during sepsis and a higher splenic bacterial burden. The spleen is responsible for clearance of encapsulated organisms, and Fpn may allow B. fragilis to avoid splenic clearance. While B. fragilis intra-abdominal abscess formation has been studied extensively, sepsis has not been studied, and, to our knowledge, Fpn is the first identified virulence factor in B. fragilis sepsis. These studies provide a potential mechanism of B. fragilis protein export, insight into mechanisms of pathogenesis of BFT-mediated colitis, and identify Fpn as a novel virulence factor in B. fragilis sepsis, an infection with high mortality in humans.
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