Development of an assay for the identification of campylobacter bacteriophage receptor binding proteins

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  • Bacterial viruses (phages) are ubiquitous in the environment and are believed to target every bacterial species. Phages utilize receptor binding proteins (RBPs) to bind specifically to their host cell surface. The Campylobacter jejuni phage NCTC 12673 RBP and its capsular polysaccharide (CPS) receptor have been studied in our laboratory. Using this model system, we have demonstrated that phage RBPs can be used as diagnostics against bacterial pathogens, and have developed different techniques for RBP immobilization onto surface plasmon resonance (SPR) chips and magnetic microbeads. The possibility of using NCTC 12673 RBPs as therapeutics against Campylobacter infections is promising, as these proteins are capable of agglutinating Campylobacter cells and reducing their motility in vitro. As well, NCTC 12673 RBP antibodies cross-react with Salmonella enterica serovar Typhimurium phage P22 RBPs, which have been demonstrated to reduce salmonella colonization in chickens. Although phage RBPs show great promise in applications using antibody platforms, these RBPs are generally difficult to identify through homology. Also, in spite of the rapid progress in DNA sequencing technologies, phage genome sequencing continues to remain a challenge. Thus, a high-throughput method for the identification of RBPs from any phage is necessary in order to further exploit RBP-based technologies. We are developing a random whole-phage genome expression library method to screen for putative phage RBPs. In proof-of-principal studies, an RBP from the C. jejuni phage NCTC 12673 was expressed in Escherichia coli. Then, lysates from E. coli colonies capable or incapable of RBP expression were probed with intact Campylobacter cells and examined through a bacterial growth assay. Our preliminary results indicate that it is possible to determine which E. coli colonies are producing recombinant NCTC 12673 RBPs. We are now extending this methodology to examine E. coli total expression libraries of phage genomic DNA from NCTC 12673 and other uncharacterized Campylobacter phages.

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    Conference/Workshop Presentation
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    CC0 1.0 Universal