Identification Of Vibrio Cholerae O1El Tor Biotype Typing Phages Receptor

National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention. State Key Laboratory for Infectious Disease Prevention and Control (China CDC).Cholera is an acute intestinal infection disease caused by bacterium Vibrio cholerae. The ongoing seventh cholera pandemic which began in1961is caused by El Tor biotype of V. cholerae serogroup01. To discriminate El Tor strains, phagetyping is combined with biotyping in China, and five typing phages are used in the scheme, two of which are VP2and VP4. El Tor strains were divided into two class-epidemic strains and non-epidemic strains.long-term observation showed during cholera epidemics the fomer were found and mainly isolated from patients, but the later did not infect people or cause diarrhea. From virulence genes, almost all of epidemic strains have it while non-epidemic strains do not.To study the mechanism of typing phages’ infection on V. cholerae and the difference between the strains sensitive and resistant to VP4helps to understand the genetic diversity of El Tor strains and the mechanism of the phage-biotyping scheme used for many years in China.Receptor identification is the first step in phage infecting bacteria, in this study phage receptors is as the research target to explore differences between the phage sensitive strains and resistant strains. VP4was sequenced on whole genome scale and conducted a whole protein identification with mass spectrum. Under electron microscopy VP4was observed as a short-tailed phage.The genome sequence is 31693bp, with an overall G+C content of46.81%. Preliminary bioinformatics analysis predict there are45encoding genes,3of which were predicted to have function. Spectrum analysis showed that there are26proteins of VP4total proteins were correspond to the predicted genes.Transposon insertion mutant libraries were established to screen VP2and VP4resistant strains, we got9strains of VP4resistant strains and5strains of VP2resistant strains. Transposon insertion cites of VP4resistant strains were in gene manB, manC, gmd, wbeN, wbeG, wbeU and recJ, while the cites of VP2resistant strains were within VC1384, VC0760and VC0761gene.Gene manB, manC, gmd, wbeN wbeG, wbeU are associated with the synthesis of O-antigen and were deleted and complemented in this study. We built manC, wbeN, manB, wbeG, wbeU and recJ expression plasmid Respectively, after complementing them to the corresponding transposon mutant strains, these resistant strains all turned into sensitive strains, prompted that these genes had relationship with vp4resistance; Constructing gene deletion strains by homologous recombination, manB,wbeN, wbeU, and wbeG, gene deletion strains were got and showed resistance to VP4infection, while thes genes were coplemented they restored sensitivity. These results showed these genes were associated with VP4infection. We built manC, wbeN, manB, wbeG, wbeUand recJ expression plasmid Respectively, after complementing them to the corresponding transposon mutant strains, these resistant strains all turned into sensitive strains, prompted that these genes had relationship with vp4resistance; manB,wbeN, wbeU, and wbeG, gene deletion strains were got by homologous recombination and showed resistance to VP4infection, while thes genes were coplemented they restored sensitivity. These results showed these genes were associated with VP4infection.We selected the manB and wbeU deletion strains to analyzes the integrity of O-antigen and found that the strains lack of O-antigen resisted VP4adsorption and infection. When complemented the deletion gene,they all regain complete O-antigen and adsorpted and infected by VP4.And these results proved O-antigen was the receptor of phage VP4. recJ gene deletion strain showed VP4resistance and restored sensitivity when recJ was complemented, but adsorption experiments showed it had nothing to do with the adsorption of VP4. We speculated that it was associated with the replication of VP4afer it entered into the host cells.For the analysis of possible role of O-antigen gene synthesis cluster in the natural VP4resistant strains, five natrual resistant strains’ wbe cluster were sequenced.Mutations were identified in the wbe cluster, which is responsible for the biosynthesis of O-antigen. Mutations in the manB, wbeE and wbell genes caused adsorption failure of VP4to these strains, whereas the observed amino acid residue mutations within wbeW and manC have no effect on VP4infection. Additionally, although mutations in three resistant strains were only found in manB and wbeW, complementing both genes did not restore sensitivity to VP4infection, suggesting that other resistance mechanisms may exist.We also screened VP2phage resistant mutant strains by transposon mutagenesis library. Five VP2resistant mutants were got and the mutant site were identified in VC1384, VC0760and VC0761. The VP2resistant related genes-VC1384, VC0760gene deletion strains were got by knockout experiments.Strain with VC0760gene deletion showed VP2resistance partly; VC1384deletion strain had VP2resistance and restored sensitivity to VP2after being complemented with VC1384gene. Adsorption experiments showed that the resistance of VC1384deletion strain has no relationship with the adsorption. VC1384is predicting to encode a transmembrane protein which belonges to OMPb-brl family which is a transmembrane protein with channel and containes β-folds, and we speculate it may be associated with the DNA injection of VP2.In this study, we conformed that the O-antigen of LPS is the receptor of VP4on the El Tor strains sensitive to VP4. We also analysed the difference of O-antigen synthesis cluster between natural resistant strains and sensitive strains. With this strategy, using this strategy, we can know mechanism of vibrio cholerae typing phage sensitivity and resistance, and genome variation associated with the function difference in these strains. Vibrio cholerae phage existing in natural water can infect and predate sensitive clones, while Phage resistant strains can survive in the nature, and have the possibility to lead the epidemic.So these resistant strains against VP4we identified, at least in the presence of VP4and similar phage environment, have a certain survival advantage.