Receptor And Temporal Analysis Of Transcription Of Vibrio Cholerae Phage VP3

Cholera is an acute intestinal infection caused by ingestion of food or water contaminated with the bacterium Vibrio cholerae.The ongoing seventh cholera pandemic which began in 1961 is caused by E1 Tor biotype of V.cholerae serogroup O1.To discriminate E1 Tor strains,phagetyping is combined with biotyping in China,and five typing phages are used in the scheme,one of which is VP3.To study the mechanism of typing phages' infection on V.cholerae and the difference between the strains sensitive and resistant to VP3 helps to understand the genetic diversity of E1 Tot strains and the mechanism of the phage-biotyping scheme used for many years in China.We sequenced the whole genome of VP3 and did priliminary bioinformatics analysis in previous studies.VP3 is a member of T7 family and predicted to consist of 52 protein encoding open reading frames and 17 conserved T7-like promoters. O1 serogroup El Tor biotype strain N16961 is whole genome sequenced and sensitive to VP3,and its sensitivity is probably correlated to wav gene cluster which is responsible for the synthesis of the core oligosaccharide(OS) region of lipopolysaccharide(LPS).In this study we further determined the relationship between the VP3 sensitivity and the genes in wav cluster.VC0231 in wav cluster ofN16961 was inactivated by deleting most part of the gene,and the deletion mutant get resistant to VP3.The deletion mutant and the transposon insertion mutants constructed in previous studies restored the sensitivity to VP3 when they get complementing plasmids containing corresponding genes.Predicted tail fiber protein of VP3 was expressed in vitro and labeled with FITC.Whether the predicted tail fiber protein can combine to wild type N16961,the mutants and complemeted mutant strains was observed with confocal microscopy,and the results were in accordance with the sensitivity of the strains to VP3.So we concluded that the product of wav gene cluster,OS,is the binding site of VP3,and the predicted tail fiber has the receptor binding function.We found that some wild type El Tor strains can combine VP3 tail fiber protein on the cell surface though they are resistant to the infection of the phage. The nucleotide sequences of their wav cluster are identical or very similar to the strains sensitive to VP3.So there should exist more complex mechanism.The temporal pattern of VP3 transcription was determined by RT-PCR.Total RNA was extracted on 1 minute intervals after N 16961 was infected by phage VP3, and at least one gene downstream each predicted T7-1ike promoter were tested.We found that the genome of VP3 is transcribed from left to right,and the genes were classed into early,middle and late phases according to the time their transcripts could be detected.6 rho-independent terminators were predicted in VP3 genome, and terminator T1 and T6 are presumed to work efficiently to some extent, deduced by analyzing the experiment results and their location in genome.In this study,we conformed that the OS region of LPS is the receptor of VP3 on the El Tor strains sensitive to VP3,and the predicted tail fiber has the receptor-binding function.Some strains could resist VP3 infection due to the mechanism other than OS receptor.VP3 genes were classed into early,middle and late phases according to the time their transcripts could be detected.These studies helped us to understand the mechanism of the phage-biotyping scheme used in China,gave us some preliminary knowledge about the life process of VP3,and posed some new questions we should answer next step. Molecular subtyping is used to distinguish pathogenic bacterial strains during epidemiological surveys of infectious diseases and the discovery of novel pathogens.There is a need to standardize protocols involving Pulse-Field Gel Electrophoresis(PFGE),a crucial method in molecular subtyping,to make inter-laboratory results comparable and information exchange possible.An optimal PFGE protocol produces suitable number of restriction fragments and gives distinct patterns by agarose gel electrophoresis.The number and size of restriction fragments are determined by the restriction enzymes used,while the band distribution in a gel depends on the electrophoretic parameters(EPs) whose discriminatory power can be assessed by similarity coefficients among patterns.The smaller the similarity coefficients,the larger the pattern difference,and the higher the discriminatory power.With a certain restriction enzyme,selection of the electrophoretic parameter with the smallest similarity coefficients will increase the discriminatory power of PFGE.Cholera is an acute communicable disease caused by Vibrio cholerae. PFGE exhibits high discriminatory power and sound epidemiologic concordance in subtyping of V.cholerae.Not I is one of the restriction enzymes most often used for V.cholerae in PFGE analysis.To optimize EPs of PFGE for Vibrio cholerae,we analyzed 24 isolates of V.cholerae O1 biotype E1 Tor and 26 isolates ofO139 by PFGE with Not I.We used four different EPs and compared the similarity coefficients from the four groups by Friedman test.Based on the principle that the electrophoretic parameter producing smaller similarity in the PFGE patterns has higher level of discriminatory power, the one producing the smallest similarity coefficients is considered to be optimal.Here,we established a method of screening and optimizing electrophoretic parameters in bacterial PFGE subtyping.