Gluconate-mediated GntR Protein Regulates Intestinal Colonization In Vibrio Parahaemolyticus

Vibrio parahaemolyticus is an important foodborne pathogen and a Gram-negative bacterium,which could colonize to the intestine after infection.Consumption of seafood containing Vibrio parahaemolyticus can cause acute gastroenteritis,and the most common clinical symptoms of Vibrio parahaemolyticus infection are abdominal pain,diarrhea,nausea,vomiting,accompanied with fever and other symptoms.The contamination of Vibrio parahaemolyticus would threat to the health of humans and huge economic losses in the marine aquaculture.Thermophilic direct hemolysin(TDH),thermophilic related hemolysin(TRH),type? secretory system(T3SS),type ? secretory system(T6SS),and adhesives are the main virulence factors of Vibrio parahaemolyticus.Besides,motility is also related to the virulence of Vibrio parahaemolyticus.The type ? secretion system(T3SS)closely related to the intestinal toxicity s,and the effector proteins secreted by T3SS was play an important role in the pathogenicity of Vibrio parahaemolyticu.Infant rabbit models are used to investigate the intestinal pathogencity of V.parahaemolyticus.Study the molecular mechanism of the pathogenicity of V.parahaemolyticus,which could provide a new vision and ideas for the control and prevention of Vibrio parahaemolyticus.The previous studies have identified 230 genes related to the pathogenicity of V.parahaemolyticus in the infant rabbit model by Tn-seq,include the VP0058 gene.In this study,we constructed the ?gntR gene-deficient strain and complementary strain,and the infant rabbit model was used to valuate the infection of mutant strain.The virulence related phonotypes were evaluated through a series of in vitro experiments.Furthermore,the RNA-seq was used to identified the genes regulated by GntR and verify the regulation.1.Construction of ?gntR mutant strain and identification of its biological characteristicsThe gntR gene deletion mutant strain was successfully constructed by homologous recombination,and three-day-old rabbits were used to valuate the pathogenicity of the WT and gntR mutant strain.The diarrhea and the intestinal colonization of ?gntR were significantly weakened.In addition,the growth curve,motility and acid stress were detected,and results showed that the growth curve and motility were no significant difference between WT and ?gntR,indicating that GntR regulate intestinal colonization was not through motility.The acid stress results showed that the?gntR strain showed a significant decrease in acid tolerance compared to the WT,and the complementary strain restored to the level of WT.These results demonstrated that the the survival ability of ?gntR-deficient strains was decreased in the acidic environment,and this may be the reasons for the decrease in colonization of ?gntR-deficient strains in the intestinal tract of infant rabbits.2.gntR regulate gluconate utilization system to mediate intestinal colonization in Vibrio parahaemolyticusGntR is a transcriptional regulatory factor,and the regulon of GntR protein screened by transcriptome sequencing.The results showed that 357 genes were regulated by GntR,61 genes were up-regulated and 296 genes were down-regulated.KEGG pathway analysis revealed that the expression of the gluconate utilization system was regulated by GntR protein,and this gene cluster was high identity with the gluconate utilization system(edd,gntK,gntT and eda)of Vibrio cholerae.qRT-PCR anslysis verified that the transcription levels of edd,gntK,gntT,and eda genes were significantly increased,and the complemented strain restored to the transcript level of WT.Biological information analysis showed that the edd-gntK-gntT-eda gene cluster contains a bidirectional promoter region.EMSA results show that GntR protein could bind to the promoter region to inhibit the expression of the gluconate utiliztion system.Besides,the gluconate could inhibite the binding of GntR to the promoter,and induce the expression of gluconate utilization system.The ?gntR and ?edd?gntK?gntT?eda strains could not survival in M9 medium with gluconate as the sole carbon source.Interestingly,we found that the ?gntR strain could not grow in the M9 medium with glucose and gluconate,and under the low concentration(10 uM)of gluconate,the ?gntR could not grow,while the concentration of gluconate increased to 50 uM,the ?gntR restore grow.At low concentration(10 uM)gluconate,the gluconate utilization system genes overexpressed,and the excessive accumulation of intermediate metabolites inhibit the growth of bacteria,leading to a decrease in intestinal colonization.Under the high concentration(50 uM)gluconate,the expression levels of gluconate utilization gene decreased,and the mutant strain restored the growth ability.In the process of control and prevention Vibrio parahaemolyticus,the bacterial resistence was increased due to the abuse of antibiotics.In this study,the molecular mechanism of GntR to the gluconate utilization system was studied,which provides a new idea and direction for the control and prevention of Vibrio parahaemolyticus.