Regulation Of Ser/thr Protein Kinase VP0057 On Virulence Factors Of Vibrio Parahaemolyticus

Vibrio parahaemolyticus is a typical food borne pathogens from ocean.Patients infected with V.parahaemolyticus usually have symptom like vomiting and diarrhea,moreover,it can lead to gastroenteritis or septicemia.Recent studies have shown that protein phosphorylation exists in bacteria widely and plays an important regulatory role in various life processes of bacteria.However,the detailed regulatory mechanism of protein phosphorylation in V.parahaemolyticus has not been revealed.VP0057 is a Ser/Thr protein kinase in V.parahaemolyticus,which may regulate the virulence and pathogenicity of V.parahaemolyticus.In this study,vp0057 knockout bacteria(?vp0057)and vp0057 complementing bacteria(?vp0057:vp0057)were constructed on the basis of POR1(?tdh A/S),a hemolysin related gene knockout strain of V.parahaemolyticus RIMD 2210633.Firstly,the effect of vp0057 on the virulence of V.parahaemolyticus was analyzed by comparing the growth curve,biofilm formation,adhesion to Caco-2 cells,cytotoxicity to Caco-2 cells and enterotoxicity to rabbit ileum of V.parahaemolyticus.Secondly,the expression of virulence factors of V.parahaemolyticus was analyzed by q PCR.Finally,the phosphorylation difference and differential expression proteome of vp0057 knockout were compared and analyzed by liquid chromatography tandem mass spectrometry(LC-MS/MS)quantitative analysis technology to explore the regulation effect of VP0057 on virulence of V.parahaemolyticus.The results showed that the growth of?vp0057 on LBS medium was weaker than POR1 and ?vp0057:vp0057,and the ability of biofilm formation and adhesion to host cells were also significantly reduced.Knockout of vp0057 gene reduced the enterotoxicity of V.parahaemolyticus,but increased the cytotoxicity to Caco-2;and when type ? secretion systems(T3SSs)were activated,it was found that the transcriptional level of T3SS2 related genes was decreased,while T3SS1 was the opposite,this result was also consistent with the changes of enterotoxicity and cytotoxicity.Further studies showed that vp0057 promoted the transcription of the quorum sensing regulatory gene opa R and transcription regulator cal R of V.parahaemolyticus at low density,and opa R and cal R have been confirmed to inhibit the transcription of T3SS1 secretion system.Therefore,the enhanced cytotoxicity of V.parahaemolyticus after knockout vp0057 may be related to the low density of V.parahaemolyticus during the cytotoxicity test.In addition,the deletion of vp0057 can affect the phosphorylation modification of another Ser/Thr protein kinase VPA1044 in V.parahaemolyticus,indicating that VPA1044 is a potential substrate for VP0057.A total of 197 differentially expressed proteins were identified by proteomic analysis after knockout vp0057,of which 135 were up-regulated and 62 were down-regulated;and the changes of some proteins were consistent with the above-mentioned functional changes.In conclusion,this study revealed the regulation and mechanism of vp0057 gene upon toxicity of V.parahaemolyticus,it lays a foundation for further study on the regulation network of VP0057 protein phosphorylation,and provides a basis for revealing the pathogenic mechanism of Vibrio,and also provides new research ideas for the treatment of foodborne diseases by drugs and vaccines based on phosphorylation site modification.