Molecular Mechanism Of Virulenceregulation By VP0057,A Ser/Thr Protein Kinase In Vibrio Parahaemolyticus

Vibrio parahaemolyticus is the leading worldwide cause of food-borne illnesses associated with the consumption of raw sea-food.Although hemolysin,adhesin,type ? and type secretion system ? have been revealed as the major virulence factors of the bacterium,their regulatory mechanism is still unclear.Recently,the phosphorylation modification of proteins triggered by eukaryotic-like Ser / Thr protein kinase has been found to play central roles in bacterial cellular regulation and closely associated with bacterial virulence.By bioinformatics analysis,we found that VP0057 may have a potential phosphorylating activity that may function as Ser / Thr protein kinase.Therefore,we considered that VP0057 may regulate the virulence factors through phosphorylation of proteins.In this study,we firstly constructed the VP0057 gene deletion strains(?VP0057)from wild-type V.parahaemolyticus(VP).Then we verify the correlation of VP0057 and virulence in V.parahaemolyticus through the comparison of VP and ?VP0057in various aspects: the growth rate in different medium,the ability of adhesion to HeLa cells,the formation of biofilm and the effects of T3 SS secretion protein expression.The results showed that the amount of ?VP0057 was much lower than that of VP adhesion to HeLa cells and ?VP0057 was weaker than VP in other three aspects.Based on this results,we concluded that VP0057 can enhance the virulence of V.parahaemolyticus.In order to further determine the regulatory mechanisms,we secondly tested the expression of VP0057 at the level of mRNA and protein in DMEM medium and bile salts,two conditions that can activate T3SS1 and T3SS2,respectively.The increasing expression of the protein was both found at the two conditions.The results suggested that VP0057 may be related to the activation of T3 SSs and secretion of effector proteins.Thirdly,we analyzed the differentiation of phosphorylation caused by the deletion of VP0057.The results showed that phosphorylationat Ser/Thr siteof membrane proteins of?VP0057wasdownregulated,but cytoplasmic proteins had no significant difference.The results suggested that VP0057 may regulate the activation and secretion of T3 SS by phosphorylation.To sum up,VP0057 may regulate the virulence factors of V.parahaemolyticusby phosphorylation of membrane proteins.It not only provide theoretical basis for the in-depth study of the Ser/Thr kinase in regulating virulence in V.parahaemolyticus,but also help us to understand the mechanism of bacterial virulence and infection.