The Type ? Secretory System Mediates VP2 Phage Infection In Vibrio Cholerae

Phages play a unique role in biology,and different phages can specifically infect a group of bacteria.Through bacterium-phage interaction,they also promote the diversity and evolution of bacteria.Moreover,the ability of different phages to lyse target bacteria can be used for bacterial variation analysis and typing.Vibrio cholerae is the host of many phages.Since the 1970s,in the prevention and control of cholera in China,five virulent phages were selected to establish a phage-typing scheme for O1 serogroup E1 Tor biotype of V.cholerae,which was used for etiological and epidemiological monitoring and analysis,and risk assessment of the epidemic caused by the strains in combination with biotyping.It is helpful to understand the variation of Vibrio cholerae and the mechanism of phage typing by investigating the infection and resistance mechanisms of Vibrio cholerae typing phages.Receptors are specific molecules in the first step of bacterial infection by phages.At present,the research on the five phage-typed strains of Vibrio cholerae and their receptors has found that a polyglutamine(polyQ)membrane protein of Vibrio cholerae is the infection receptor of phage VP1,and the receptor of VP3 is composed of core oligosaccharides and outer membrane protein TolC,which is a dual-receptor infection pattern.Additionally,Vibrio cholerae O-antigen is the receptor of phage VP4 and OmpW is the receptor of phage VP5.Furthermore,phage VP2 is a circular double stranded DNA molecule.Observation under an electron microscope shows that VP2 is a short-tailed phage with the full genome length of 39,853 bp(GenBank accession number:NC 005879)and the G+C content of 50.56%.It is predicted that there are 49 open reading frames(ORFs),and the homologous phages include Vibriophage phiCJY,Vibriophage H3 and Vibriophage Rostov 6.In this study,the whole proteome of VP2 mature granules was identified by mass spectrometry,revealing 34 proteins corresponding to the predicted genes.The first step of phage infection is to specifically bind with receptors on the surface of host cells.The lytic phage VP2 can be used for phage typing of V.cholerae biotype El Tor of the O1 serogroup,but its infection receptor is still unknown.In this study,a study on the VP2 receptor for Vibrio cholerae infection was carried out.The mutant library of V.cholerae receptor was established by transposon insertion,and then the VP2 cleavage strategy was added to select the VP2 resistant strains.Firstly,VP2 resistant strains were selected by establishing the mutant library of Vibrio cholerae receptors through transposons and adding VP2 for lysis.In addition,the mutant gene which was identified as membrane protein was further investigated through sequencing and sequence alignment.This gene encoded inner membrane protein EpsM of the type II secretion system(T2SS)of Vibrio cholerae.Subsequently,the epsMgene was deleted and complemented,and deleted epsMstrain(N-?epsM)and complemented strain(N-?epsM+C)were obtained.The phenotypic experiment showed that the N-?epsM was resistance to VP2,but VP2 could lyse the N-? epsM+C strain.We also verified the adsorption of VP2 on the N-?epsM.,The results showed that the N-?epsM could not adsorb VP2,indicating that EpsM played a role in the successful infection of VP2,although it is not related to VP2 adsorption.Moreover,the outermost outer membrane protein epsD gene of the T2SS was deleted and complemented,and deleted epsD strain(N-?epsD)and complemented strain(N-?epsD+C)were obtained.The lysis experiment confirmed that EpsD played a role in VP2 infection of Vibrio cholerae and was the site of VP2 adsorbing Vibrio cholerae.Further in vivo protein-protein interaction and pull-down experiments proved that the VP2 receptor was the outer membrane protein EpsD in the T2SS of Vibrio cholerae.Therefore,it is found that in Vibrio cholerae,the T2SS acts not only as a transport device for secreted cholera toxins and released filamentous phages,but also as a receptor for phage infection,which may be a channel for phage DNA injection.Our study expands the understanding of the role of T2SS in bacteria.