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Characterization of colonization factors from enteric pathogens Vibrio cholerae and enterotoxigenic Escherichia coli

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Thesis type
((Thesis)/(Dissertation)) Ph.D.
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Vibrio cholerae and enterotoxigenic Escherichia coli (ETEC) cause severe gastrointestinal diseases and are a significant cause of mortality in developing countries. These bacteria colonize the gut mucosa and secrete exotoxins resulting in severe diarrhea. Colonization of the small intestine by V. cholerae requires the toxin coregulated pilus (TCP), a type IV pilus that self-associates to hold the bacteria in microcolonies. The TCP assembly apparatus is responsible for secreting a soluble colonization factor, TcpF, which is encoded on the tcp operon along with all genes necessary for TCP assembly. Its function is unknown, but is critical for V. cholerae colonization in the infant mouse model and antibodies against this protein are protective. The ETEC colonization process is less well-characterized, but some ETEC express CFA/III, a type IV pilus that is homologous to TCP, encoded by the cof operon. This operon has a gene cofJ, at a position syntenic to tcpF in the tcp operon, which encodes a putative soluble protein, CofJ, that has similar size to TcpF. We showed that CofJ, like TcpF, is secreted by its type IV pilus system. Although CofJ and TcpF share no amino acid sequence homology with each other or with any other known protein, we hypothesized that they may nonetheless have similar structures and roles in pathogenesis. We solved the TcpF crystal structure to 2.4 Å resolution, revealing a novel bilobed protein with two domains joined by a flexible linker. The N-terminal domain resembles a C-type lectin-like domain and the C-terminal domain has a fibronectin type III fold. We solved the CofJ structure at 2.55 Å resolution. CofJ is very different from TcpF, composed primarily of β-strands forming a large β-sandwich. Structural homology searches revealed CofJ has very limited similarity to the C-terminal domain of perfringolysin O, a pore-forming protein secreted by Clostridium perfringens. As both CofJ and TcpF have patches of surface-exposed hydrophobic residues, we hypothesized that they may interact with epithelial cell membranes. Both proteins associated with synthetic lipid vesicles and CofJ associates with cultured epithelial cells and oligomerizes in their presence. Thus, our preliminary data suggest that CofJ and TcpF bind to epithelial cell membranes.
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Supervisor or Senior Supervisor
Thesis advisor: Craig, Lisa
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