The Molecular Mechnisam Of VPA0041 And VPA1701 Regulate The Motility In Vibrio Parahaemolyticus

The Vibrio parahaemolyticus is a Gram-negative bacterium which could live in all types of aqueous environments,including marine,coastal and freshwater.V parahaemolyticus is an important foodbome pathogen which could cause the vibriosis in the marine animals,and cause the acute gastroenteritis by consumption of raw or undercooked sea-food in humans Vibrio parahaemolyticus is not only threat to the health of humans,but also cause huge economic loss in aquaculture industry.Studies have found that the pathogenicity of Vibrio parahaemolyticus is mainly related to the virulence factors such as adhesion factor,hemolytic toxin,T3SSs,T6SSs,motility,iron uptake system and protease.Transcription regulatory factor(TFs)could directly bound to the promoter region and regulate the expression of downstream genes,including metabolic pathway,quorum sensing system,virulence system and motility.The transposon insertion sequencing(TIS)was used to investigate the virulence genes of V.parahaemolyticus,and 230 genes were identified related to the intestinal colonization.In this study,eight genes transcription factors were selected for the further study,including VPA0041,VPA0076,VPA0240,VPA0692,VPA0806,VPA1365,VPA1562 and VPA1701We successfully constructed the gene deletion mutants by homologous recombination,which are △VPA0041,△VPA0076,△VPA0240,△VPA0692,△VPA0806,△VPAI365,△VPA1562 and △VPA1701.The intestinal colonization of △VPA1365 and △VPA1701 were significantly decreased compared to WT in the infection of infant rabbits.qRT-PCR results showed that the expression of T3SS related genes was downregulated in △VPA1365 compared to WT,indicating that VPA1365 mediated the intestinal colonization of Vibrio parahaemolyticus by regulating the expression of T3SS genes.Motility results showed that swimming motility of the △VPA1562 and △VPA1 701 was lost,and the transmission electron microscopy(TEM)found that no flagellar was observed around the △VPA1562 and △VPA1701 mutant strains cultured in the plates.qRT-PCR and RNA-seq showed that lateral flagellar genes were downregulated in the △VPA1562 and△VPA1701 mutant strains compared to WT.In addition,RNA-seq results also showed that VPA0041 and VPA1701 are global regulators in Vibrio parahaemolyticus,which not only regulate motility,but also participate in the growth and metabolism pathway,quorum sensing system,ABC transport system and two-component system.In order to further investigate the molecular mechanism of VPA0041 and VPA1701 regulation,protein expression strains of VPA0041 and VPA1701 was constructed,and two proteins were purified.EMSA results showed that VPA0041 and VPA1701 directly bound to the promoter region of flagellum gene,and regulate the expression of flagellum system to mediate the motility of Vibrio parahaemolyticus.V parahaemolyticus is an important pathogenic bacterium,which could threat the health of humans.In this study,we constructed eight transcription factors deletion mutant strains,then the infant rabbits and motility was used to valuate the virlence of these mutant strains,these results showed that the the virulence of △VPA0041,△VPA1365 and △VPA1701 significantly decreased compared the WT.Further study indicated that VPA1365,VPA0041 and VPA1701 regulate the expression of T3SSs genes and lateral flagellar genes to mediate the intestinal colonization of Vibrio parahaemolyticus.