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The ℇ34 Phage Tailspike Protein : An In vitro Characterization, Structure Prediction, Potential Interaction withS. newingtonLPS and Cytotoxicity Assessment to Animal Cell Line

Joseph A Ayariga, Logan Gildea, Hongzhuan Wu, Robert Villafane

The ℇ34 phage is a member of the podoviridae family of phages, (short non-contractile tailed bacteriophages) that uses Salmonella newington as its host. This phage initiates the infection of its host via a specific interaction between the lipopolysaccharides (LPS) of the bacterial host and its tailspike protein (TSP). The ℇ34 TSP is structurally similar and functionally equivalent to the P22 phage whose TSP has been well characterized and electron micrographs of both phages appear indistinguishable. The crystal structure of P22 phage TSP in complex with the O-antigen of S. typhimurium has been determined; and the active site of the TSP demonstrated to be the residues Asp392, Asp395 and Glu359 of the receptor binding domain. In another phage called E15, a phylogenetic relative of ℇ34 phage, a short polysaccharide consisting of α-Gal-Man-Rha repeating units is responsible for the interaction between the E15 phage and Salmonella anatum’s LPS leading to the adsorption of the phage to the bacteria. Studies on ℇ34 phage shows that it interacts with Salmonella newington’s O antigen polysaccharide component of the LPS, this polysaccharide consists of mannosyl-rhamnosyl-galactose repeating units joined together by β-galactosyl linkages. However, no data exist regarding the specific residues of ℇ34 TSP that are responsible for LPS binding and hydrolysis.

In this study, the tailspike gene was cloned onto vector pET30a-LIC and expressed as a fusion protein termed the extended ℇ34 TSP (Eℇ34 TSP). We characterized the protein base on resistance to heat, SDS, and proteases; showing that the protein is heat resistant, shows aberrant electrophoretic mobility in the presence of SDS gradient, and actively binds to P22 phage heads to form hybrid phages that cannot infect P22 host. We also demonstrate via in silico study that the ℇ34 TSP binds to and hydrolyses the O-antigen of its host via the ALA250, SER279 and ASP280 residues.