Factors Screening Of TDH Regulation By Pathogenic Vibrio Parahaemolyticus

Pathogenic Vibrio parahaemolyticus(Vp) is one of the most important foodborne pathogenic bacteria. The thermostable direct hemolysin(TDH) is the main pathogenic factor, which coded by tdh gene. However, little information on regulation is known about the hemolysin. In this study, factor, which regulated TDH expression, were evaluated by a 6 factors 5 levels orthogonal experiment. The factors were confirmed by the expression of TDH using the single factor and orthogonal experiment. The results demonstrated that various factors affect the expression of TDH, including different medium, temperature, pH value, the concentration of taurocholic acid and NaCl.In this study, the Mini-Tn5-Km2/Cm was inserted randomly into the genome of pathogenic Vp(ATCC 33846, tdh+) by bioparental mating. The conjugants(KmR/CmRAmpS) were investigated using thiosulfate citrate bile salts sucrose agar medium with appropriate antibiotics(Km/Cm) and ampicillin(Amp) respectively. After detection of the Km/Cm gene by polymerase chain reaction, library of mutation was established, which conjugants with Tn5 in different gene locus. After evaluating hemolytic activity with human blood agar, the phenotype of 5 strains was changed, including 2 up-regulated and 3 down-regulated strains. There was significant difference between the 5 conjugants and parent strain on growth rate, biofilm formation and motility ability. Among 3 down-regulated strains, the biofilm formation ability of 2 conjugants was increased(P<0.05). The residual 3 strains demonstrated the significant difference(P<0.05) on growth rate, biofilm formation ability and motility, comparing with their parent strain. Using the plasmid rescue method, one gene(pst S) that regulated the TDH expression was obtained. The results indicated that pst S gene enhances the hemolytic activity of pathogenic Vp, and reduces the ability of biofilm formation.The conjugants, various factors and up-regulated gene on TDH expression will contribute on investigation of regulation system and molecular mechanism of tdh in further study.