Screening And Studying On Gluconate-Related Genes Of Vibrio Parahaemolyticus

Vibrio parahaemolyticus is an important foodborne pathogen in aquaculture industries in China and worldwide,and widely distributed in marine environments.Consuming raw seafood contaminated with V.parahaemolyticus can cause symptoms such as abdominal pain,diarrhea,vomiting,and fever,and in severe cases,even death.Gluconate is a commonly used food additive that can improve the taste,nutritional value,and shelf life of food.In aquaculture,gluconate,as a feed additive,can promote the growth of probiotics in the intestines,improve the growth performance of cultured species,and intestinal health.Studies have shown that V.parahaemolyticus can use gluconate as the sole carbon source to survive under nutrient-deficient conditions,but the effects of gluconate,as a feed additive,on intestinal pathogenic microorganisms remains unknown.To investigate the effect of gluconate on the gene expression profile of V.parahaemolyticus,we used RNA-seq to analysis the different expression genes of V.parahaemolyticus under gluconate conditions.The results showed that a total of 623 genes had significant differences expressed,with 287 genes significantly upregulated and 336 genes significantly downregulated.Notably,the upregulated genes included signal pathwayrelated to glycolysis,the tricarboxylic acid cycle,and the pentose phosphate.The downregulated genes mainly involved virulence-related pathway genes such as type Ⅲsecretion system 1(T3SS1)and type Ⅵ secretion system 1(T6SS1),which was further confirmed by qRT-PCR.Additionally,both RNA-seq and qRT-PCR results showed that the gluconate could significantly induced the expression of the gluconate utilization system(gntR-edd-gntK-gntT-eda).Therefore,we constructed the ΔgntR and Δgnt deletion mutant strains using homologous recombination and determined their growth curves and biofilm formation abilities under different gluconate conditions.The results showed that ΔgntR andΔgnt could not grow when gluconate was used as the sole carbon source,further confirming that gntR-edd-gntK-gntT-eda is the gluconate utilization system of V.parahaemolyticus.The growth curve results showed that with the increase of gluconate concentration,the growth ability of WT also increased,indicating that gluconate can promote the growth of Vibrio parahaemolyticus.However,ΔgntR could not grow under low-concentrations gluconate conditions,but could grow under 50 mM and 100 mM gluconate conditions,which may be due to the accumulation of intermediate metabolites under low-concentration gluconate conditions,causing Vibrio parahaemolyticus to be unable to grow.With the increase of gluconate concentration,the accumulation of intermediate metabolites formed feedback inhibition,allowing the pathogen to grow again.The detection of biofilm formation showed that GntR protein could sensor gluconate signals and inhibit the biofilm formation ability of Vibrio parahaemolyticus.To further investigate the molecular mechanism of GntR protein sensor gluconate to regulate the expression of the utilization of gluconate genes in Vibrio parahaemolyticus,we expressed and purified GntR protein,and EMSA results showed that GntR protein can directly bind to the bidirectional promoter region of gnt and two gntR protein binding sites were found in the gnt promoter.Based on the bioinformatics,we predicted the structure of GntR protein,and found that GntR protein had an AF-2 pocket for pairing with sugar molecules.The molecular docking simulation to identify the binding mode of GntR protein to gluconate and screen the key amino acid residues and constructed the point mutant strains of key amino acids.The qRT-PCR results showed that Tyr141,Arg231,and Arg235 are key amino acid residues for the binding of GntR protein to gluconate.Finally,we determined the effect of gluconate on the virulence of Vibrio parahaemolyticus in cell and animal models.The cytotoxicity results showed that gluconate can significantly enhance the cytotoxicity of Vibrio parahaemolyticus to HeLa cells,while the cytotoxicity of AgntR was significantly downregulated and was not affected by changes in gluconate concentration,indicating that GntR protein can sensor gluconate signals to regulate the cytotoxicity of Vibrio parahaemolyticus.Zebrafish virulence experiments also showed that gluconate can induce the virulence of Vibrio parahaemolyticus under in vivo conditions.In summary,as a feed additive,gluconate can enhance the pathogenicity of V.parahaemolyticus and has a potential risk.