A Tail Fiber Protein and a Receptor-Binding Protein Mediate ICP2 Bacteriophage Interactions with Vibrio cholerae OmpU

Abstract

ICP2 is a virulent bacteriophage (phage) that preys on Vibrio cholerae. ICP2 was first isolated from cholera patient stool samples. Some of these stools also contained ICP2-resistant isogenic V. cholerae strains 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, that can now infect OmpU mutant strains, had missense mutations in putative tail fiber gene gp25 and putative adhesin gp23. Using site-specific mutagenesis we show that single or double mutations in gp25 are sufficient to generate the host-range mutant phenotype. However, at least one additional mutation in gp23 is required for robust plaque formation on specific OmpU mutants. Mutations in gp23 alone were insufficient to give a host-range mutant phenotype. All ICP2 host-range mutants retained the ability to plaque on wild type V. cholerae cells. The strength of binding of host-range mutants to V. cholerae correlated with plaque morphology, indicating that the selected mutations in gp25 and gp23 restore molecular interactions with the receptor. We propose that ICP2 host-range mutants evolve by a two-step process where, first, gp25 mutations are selected for their broad host-range, albeit accompanied by low level phage adsorption. Subsequent selection occurs for gp23 mutations that further increase productive binding to specific OmpU alleles, allowing for near wild type efficiencies of adsorption and subsequent phage multiplication. Importance Concern over multidrug-resistant bacterial pathogens, including Vibrio 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 an 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 and V. cholerae.

Vibrio choleraephage ICP3 requires O1 antigen for infection

“…However, the requirement of the O1 antigen for infection by ICP2 has not been reported. OmpU was previously identified as a receptor for ICP2 (16), and it was recently determined that the ICP2 tail protein Gp23 interacts with OmpU (28, Fig. 8).…”

Allelic diversity uncovers protein domains contributing to the emergence of antimicrobial resistance

“…This vast diversity of allelic variants suggests a scenario in which variations in this protein are strongly driven by environmental pressures and likely confer a set of phenotypes much wider than anticipated. It is known that OmpU acts as the receptor of phage ICP2, which directly interacts with domains that are present on extracellular loops of the porin (L4 and L8) [ 56 , 57 ]. Nonetheless, the extensive variability of OmpU goes beyond the region of the porin that acts as the phage receptor.…”

Allelic diversity uncovers protein domains contributing to the emergence of antimicrobial resistance

“…This vast diversity of allelic variants suggests a scenario in which variations in this protein are strongly driven by environmental pressures and likely confer a set of phenotypes much wider than anticipated. It is known that OmpU acts as the receptor of phage ICP2, which directly interacts with domains that are present on extracellular loops of the porin (L4 and L8) [55, 56]. Nonetheless, the extensive variability of OmpU goes beyond the region of the porin that acts as the phage receptor.…”