Mechanism Of Lower Virulence In Vibrio Parahaomolyticus After Exposure To High Hydrostatic Pressure

In this study, a Vibrio parahaemolyticus strain of C4 was isolated from a gastrointestinal patient’s feces. The C4 strain was treated by 11 rounds of high hydrostatic pressure treatment, and then it became an anti-pressure strain of N11. However, N11 strain has lower virulence than C4 strain based on results of haemolyticus and biofilm-forming ability of bacteria, and the median lethal dose and organ injury of mice after intraperitoneal injection of bacteria. Then we constructed Paired-end library of C4 and N11 strain and Mate-pair library of C4 strain, and found out some mutant nucleotide regions after bioinformatic analysis. Furthermore, the relative expression levels of both virulence genes and genes with mutant flanking region were also analyzed by real time quantitative PCR. Detail conclusions state as follows.The isolate bacterium was identified as V. parahaemolyticus based on the biochemical and PCR sequence results. After 11 rounds of high hydrostatic pressure treatments, the widetype strain of C4 became an anti-pressure strain of N11. Their virulence abilities were difficult to distinguish according to weak hemolysis. However, C4 strain has a high ability to form the biofilm, and a smaller value of 24 h LD50 and higher damage of organs on mice after injection of bacteria than N11 strain, which suggested N11 strain has a lower virulence. Therefore, we suspected that high hydrostatic pressure can change the virulence of V. parahaemolyticus.The whole genome sequence of C4 strain was assembled based on the paired-end and mate-pair library. There are 12 scaffolds containing 4850,110, and 11 of CDS, tRNA, and rRNA, respectively. Phylogenetic tree, constructed by whole-genome protein sequences, suggests that C4 strain is closed to V. parahaemolyticus and its gene prediction is reasonable. Furthermore,21 mutant nucleotides located in 4 frank regions of N11 strain were filtered compared with C4. We suspected that these mutant nucleotides in frank regions may alter the binding ability of RNA polymerase Ⅱ, thereby reducing the expression levels of genes and the toxicity of bacteria.Open reading fram of TDH-S, TDH-A, TLH, type IV pili, mannose-sensitive hemagglutinin genes, and type III secretion system are present in chromosome of C4 strain, while trh gene is absent. Combined with genes having mutant flanking regions, their relative expression levels of mRNA were detected by real-time quantitative PCR in C4 and N11 strians. The expression levels of gene coding hemolysin, mshA gene coding pili, gene coding RNA binding protein and HD-GYP in N11 strain exhibited much lower expression levels than C4 strain, showing N11 strain has a lower binding and invasion ability.