A Tail Fiber Protein and a Receptor-Binding Protein Mediate ICP2 Bacteriophage Interactions withVibrio choleraeOmpU

AbstractICP2 is a virulent bacteriophage (phage) that preys onVibrio cholerae. ICP2 was first isolated from cholera patient stool samples. Some of these stools also contained ICP2-resistant isogenicV. choleraestrains harboring missense mutations in the trimeric outer membrane porin protein OmpU, identifying it as the ICP2 receptor. In this study, we identify the ICP2 proteins that mediate interactions with OmpU by selecting for ICP2 host-range mutants within infant rabbits infected with a mixture of wild type and OmpU mutant strains. ICP2 host-range mutants had missense mutations in putative tail fiber genegp25and putative adhesingp23. Using site-specific mutagenesis we show that single or double mutations ingp25are sufficient to generate the host-range mutant phenotype. However, at least one additional mutation ingp23is required for robust plaque formation on specific OmpU mutants. Mutations ingp23alone were insufficient to give a host-range mutant phenotype. All ICP2 host-range mutants retained the ability to plaque on wild typeV. choleraecells. The strength of binding of host-range mutants toV. choleraecorrelated with plaque morphology, indicating that the selected mutations ingp25andgp23restore molecular interactions with the receptor. We propose that ICP2 host-range mutants evolve by a two-step process where, first,gp25mutations are selected for their broad host-range, albeit accompanied by low level phage adsorption. Subsequent selection occurs forgp23mutations that further increase productive binding to specific OmpU alleles, allowing for near wild type efficiencies of adsorption and subsequent phage multiplication.ImportanceConcern over multidrug-resistant bacterial pathogens, includingVibrio cholerae, has led to a renewed interest in phage biology and their potential for phage therapy. ICP2 is a genetically unique virulent phage isolated from cholera patient stool samples. It is also one of three phages in a prophylactic cocktail shown to be effective in animal models of infection and the only one of the three that requires a protein receptor (OmpU). This study identifies a ICP2 tail fiber and a receptor binding protein and examines how ICP2 responds to the selective pressures of phage-resistant OmpU mutants. We found that this particular co-evolutionary arms race presents fitness costs to both ICP2 andV. cholerae.