EFFECT OF SALINITY ON THE EXPRESSION OF VIRULENT T3SS GENE OF Vibrio parahaemolyticus

V. parahaemolyticus is a halophilic bacterium of marine origin. Being halophilic, they have a mandatory requirement of sodium chloride for their growth. In this study, the effect of NaCl concentrations on relative expression of T3SS2β effector protein vopA was studied and the gene confirmed to be up regulated in VP22 (tdh^-trh⁺) and VP12 (tdh⁺trh⁺) strains at different time intervals at 1% and 2% NaCl concentrations.

Generally, V. parahaemolyticus isolated from oysters and clams culturing environments were found to contain T3SS (Yu et al., 2013). The expression of   virulence genes of V. parahaemolyticus is influenced and altered by many environmental factors including salinity. The type III secretion system (T3SS) of                             V. parahaemolyticus has been suggested as an important virulence factor (Broberg et al., 2011). T3SS2 is reported to be responsible for enterotoxicity and playing a role in the environmental fitness of strains (Matz et al., 2011).  The present study was undertaken to analyse the impact of different salinity conditions on the virulent T3SS2 gene vopA expression in three different strains of V. parahaemolyticus viz., VP5, VP22 and VP12 at different concentrations of NaCl such as 3.5%, 2% and 1% at different time intervals such as 0 h, 6 h, 12 h and 24 h post infection. RNA polymerase A gene (rpoA) was used as a house keeping gene. The relative expression of virulence genes of                                    V. parahaemolyticus in broth was analyzed by StepOne Plus Real Time PCR using 2X SYBR green super mix. Melt curve analysis was performed immediately after amplification to avoid false positive results. Relative expression ratio (R) was calculated by the formula R=2^-ΔΔC_T.  Pure culture of 10⁷ CFU/ml of                                                V. parahaemolyticus was inoculated in LB broth. The optimum salinity of                                            V. parahaemolyticus such as 35 ppt (3.5%) was used as reference in this study.

It was observed that the virulence gene viz., vopA was expressed at all the salt concentrations whereas the level of expression varied. In post VP22 (tdh^-trh⁺) infected sample, the vopA was significantly up regulated in 1% at 6 h post infection. At 2% NaCl concentration, the gene was significantly up regulated to a level of 8 fold at 24 h post infection. It was noted that there was a significant difference in the expression of gene between time intervals and also between 1% and 2% NaCl concentrations.  Relative expression of vopA gene from VP12 (tdh⁺trh⁺) infected sample was significantly up regulated immediately after 0 h post infection with the value being 12 fold at 1% NaCl and 16 fold at 2% NaCl concentrations which gradually reduced till 24 h post infection. It was found that there was significant difference (p<0.05) between time intervals at 2% NaCl and between 12 h and 24 h in 1% NaCl concentration.