The Functions Of RpoN For Virulence Regulation In Vibrio Parahaemolyticus

Vibrio parahaemolyticus is an important food-borne pathogen in the marine aquaculture industry,which could cause the acute gastroenteritis including vomiting and diarrhea in human beings.V.parhaemolytics contains a variety of virulence factors,and the expressions of these virulence factors are strictly regulated by transcriptional factors.RpoN is an important sigma factor,which is belongs to the ?54 family,and widely involved in the regulation of virulence-related genes in bacteria.However,the function of RpoN protein is less studied in V.parahaemolyticus,and the molecular mechanism of RpoN in the regulation of virulence is still poorly understood.Bile salt has been reported as an important signal molecule for inducing the expression of virulence in V.parahaemolyticus.In this study,the transposon insertion sequencing(Tn-seq)was used to screen the essential genes under the bile salt conditions and verify the function of the candidate genes in the virulence.We successfully constructed 7 deletion mutant strains by the principle of homologous recombination and screen the virulence-associated genes by the cytotoxicity.These results showed that the cytotoxicity of ?rpoN was significantly reduced compared to the WT.Furthermore,our studies found that rpoN deletion mutant strain could cause the lost of swimming and swarming ability,decrease the biofilm formation,hemolytic activity and the adhension of V.parahaemolyticus.Taken together,these results demonstrated that RpoN protein is an important regulatory factor of virulence-associated genes in V.parahaemolyticus.To systematically explore the molecular mechanism of RpoN protein regulate virulence factor,RNA-seq was used to compare the different transcription levels of WT and ?rpoN.The results showed that 394 genes were regulated by RpoN protein,including flagella system,type? secretion system,type ? secretion system and iron uptake system.Furthermore,the conservative binding sites TTGGCA(-24 box)and TTGC(-12 box)of RpoN protein was predicted by MEME.qRT-PCR and EMSA verified that the RpoN protein could directly bind to the promoter of Vp2250 and Vp0775 to regulate the expression of polar flagella genes in V.parahaemolyticus.These results indicated that the RpoN protein could directly regulate the expression of polar flagellar genes to mediate the motility of V parahaemolyticus.In addition,the infant rabbit's intestinal colonization of ?rpoN was appreciably increased compared to the WT,and the ?rpoN could use the cellobiose to maintain the growth while the WT could not use cellobiose.