O antigen is the receptor of Vibrio cholerae serogroup O1 El Tor typing phage VP4

Abstract

Bacteriophage VP4 is a lytic phage of the Vibrio cholerae serogroup O1, and it is used in phage subtyping of V. cholerae biotype El Tor. Studies of phage infection mechanisms will promote the understanding of the basis of phage subtyping as well as the genetic differences between sensitive and resistant strains. In this study, we investigated the receptor that phage VP4 uses to bind to El Tor strains of V. cholerae and found that it infects strains through adsorbing the O antigen of V. cholerae O1. In some natural isolates that are resistant to VP4 infection, mutations were identified in the wb* cluster (O-antigen gene cluster), which is responsible for the biosynthesis of O antigen. Mutations in the manB, wbeE, and wbeU genes caused failure of adsorption of VP4 to these strains, whereas the observed amino acid residue mutations within wbeW and manC have no effect on VP4 infection. Additionally, although mutations in two resistant strains were found only in manB and wbeW, complementing both genes did not restore sensitivity to VP4 infection, suggesting that other resistance mechanisms may exist. Therefore, the mechanism of VP4 infection may provide a basis for subtyping the phage. Elaborate mutations of the O antigen may imbue V. cholerae strains with resistance to phage infection.

Fig 1

Mutations in the transposon insertion and VP4-resistant natural isolates. (A) Strains and transposon insertion sites in the wb* gene cluster and recJ gene. The large white arrows represent the wb* cluster, and the recJ gene and the transposon insertion positions are marked with small black arrows. The gmhD and the rjg junction genes are indicated by gray arrows. (B) Locations of mutations in the wb* gene clusters of VP4-resistant and -sensitive natural isolates. The large white arrows represent the wb* gene cluster, and the mutation sites are marked with small black arrows. The gmhD and the rjg junction genes are indicated by gray arrows.

Fig 2

SDS-PAGE and silver staining of LPS from strain N16961-Sm and its mutants and the naturally VP4-resistant isolates. The top ladder indicates the integrated LPS. LA, lipid A; CO, core oligosaccharide; O-ag, O polysaccharide.

Fig 3

VP4 adsorption to manB and wbeU gene deletion mutants and complemented strains, shown as residual PFU percentages. LB was used as the no-bacterium control, and the phage titer in the control supernatant was set to 100%. Error bars indicate ranges.

Fig 4

Binding of SYBR gold-labeled VP4 on the surfaces of different strains observed by CLSM.