Characterization of a Novel Metalloprotease Secreted by the Type II Secretion System in Vibrio cholerae Public

http://ir.library.oregonstate.edu/concern/honors_college_theses/df65v9739

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  • Vibrio cholerae, a Gram-negative bacterium, is the etiological agent of cholera. V. cholerae shuttles between the human host and the aquatic reservoir, where it associates with marine organisms such as copepods and vertebrate fish. The bacteria use the Type II Secretion System (T2SS) to release proteins that facilitate V. cholerae survival. Here, we describe a novel T2SS-dependent protein, collagenase (Clg). The analysis of the predicted amino acid sequence of Clg revealed a signal peptide, pro-peptide, peptidase M9 domain with a zinc-metalloprotease HEXXH consensus, and two pre-peptidase Cterminal (PPC) domains. Clg was purified to apparent homogeneity from the culture supernatant of V. cholerae and its activity was examined using synthetic and putative natural substrates. The protease showed activity in zymogram assays, against DQ gelatin and FALGPA, as well as a purified fish collagen but not against tested human host derived proteins. The enzymatic activity of Clg was blocked in the presence of metalloprotease inhibitors. Additionally, site-directed mutagenesis of the Clg predicted catalytic residues followed by enzymatic assays and SDS-PAGE analyses demonstrated that the zinc-binding motif is crucial for protein activity, but not for protease secretion. The maturation of Clg, which leads to disassembly of the two PPC domains, was also demonstrated.
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