Characterization of the Peptide-Binding Domain of FrhA in Vibrio cholerae

Lloyd, Cameron Jacob
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Vibrio cholerae is a highly motile organism that employs a single polar flagellum as a major virulence factor determinant for intestinal colonization and environmental proliferation. Underlying the regulation of the flagella, Vibrio cholerae expresses a large adhesin, Flagellar Regulated Hemagglutinin A (FrhA), which it employs for biofilm formation and intestinal colonization among other characteristic activities. A series of domains from aa 1155 to aa 2016 were predicted to confer the adhesive activities of FrhA. We characterized the domains present in this region and determined that one of the domains, the Peptide-Binding Domain (PBD), was responsible for the major activities ascribed to FrhA. Additionally, work by our collaborators lead to the identification of a small inhibitory peptide, AGYTD, which could associate in the binding pocket of a similar Peptide-Binding Domain found in the organism Marinomonas primoryensis. We characterized the efficacy of this inhibitory peptide in abrogating FrhA-mediated activity and found that it was effective in inhibiting FrhA activity through its Peptide-Binding Domain. Furthermore, we demonstrated that the PBD identified in Marinomonas primoryensis was capable of rescuing FrhA PBD activity in a recombinant mutant. In addition to these results, we also showed that other domains contribute to FrhA-mediated activity with the Sugar-Domain (SBD) facilitating binding to epithelial cells and binding to chitin. We performed additional investigations to design a methodology to transfer mutations in the O395 Classical strain of Vibrio cholerae through the exploitation of horizontal gene transfer machinery. Vibrio cholerae strains are naturally competent when in the presence of chitin, however mutations in key genes in O395 have resulted in an inability to conduct horizontal gene transfer. Strains in the El Tor Biotype express the major quorum-sensing regulatory protein HapR, which integrates signals to cause the transition of Vibrio cholerae to a planktonic state. By expressing the proteins TfoX, QstR, and HapR we were able to reconstitute the ability of O395 to collect free DNA from the environment and undergo recombination. This tool has allowed us to rapidly transform bacteria with existing and synthetic mutations in a rapid manner, which was previously unavailable.

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Adhesin, Biofilm, Inhibitor, Vibrio cholerae
Molecular Microbiology and Immunology