| Vibrio parahaemolyticus is the main foodborne pathogen causing human gastroenteritis.As an important intestinal pathogen,V.parahaemolyticus must overcome numerous challenges in order to successfully colonize and establish infection in the small intestine or colon.Bile is one of the challenges,and a number of enteric pathogens not only could resist the bactericidal conditions of bile but also utilize bile as a signal to regulate virulence gene expression to either colonize or maintain infection in the human gastrointestinal tract.The KP-positive strains posses an pathogenicity island(Vp-PAI)that contains two tdh genes and a set of genes for the type III secretion system(T3SS2).Study discovered that one novel Tox R-like transcriptional regulatory protein Vtr B.Vtr B is induced only in the presence of bile and can induce the expression of TDH and T3SS2 related proteins in a bile-dependent manner,resulting in enhanced cytotoxicity and intestinal toxicity mediated by V.parahaemolyticus tdh and T3SS2.Therefore,Vtr B is considered to be a key regulator for virulence gene expression in the Vp-PAI induced by bile.However,as a horizontally transferred transcriptional regulatory protein,the role of Vtr B in V.parahaemolyticus resistance to bile and the overall regulatory function of virulence genes under bile induction are unclear.Hence,to determine whether Vtr B contributes to bile resistance in tdh~+(KP+)V.parahaemolyticus.At the same time,in response to bile stimulation,Vtr B overall regulation of virulence regulators.In this study,we first screened V.parahaemolyticus containing vtr B gene,and then constructed mutant and complement strains of vtr B gene.The MBC and MIC of WT,Δvtr B,and C-Δvtr B strains were determined by bile to determine whether Vtr B can help paralyze the bile resistance.Subsequently,bile-induced WT andΔvtr B strains transcriptomics analysis was performed to determine the genes regulated by vtr B under bile induction,and the transcriptome sequencing results were verified by q RT-PCR.Finally,the corresponding phenotypic verification is performed.The main results are as follows:1.The primers were designed based on the nucleotide sequence of VPA1348(vtr B)gene in V.parahaemolyticus RIMD2210633.PCR amplification showed that the vtr B gene was selected from 10 strains of tdh~+trh~-V.parahaemolyticus,and the matching rate was obtained after sequencing and comparison up to 99%.Subsequently,suicide vector homologous recombination method was used to successfully construct a mutant of vtr B gene.Using p BAD33 plasmid as a vector,a vtr B recombinant plasmid was constructed and the recombinant plasmid was transferred intoΔvtr B to obtain complemented mutant strain C-Δvtr B.2.To explore a clear role for Vtr B in bile resistance,wild-type V.parahaemolyticus and isogenic vtr B deletion mutant were grown in various concentrations of bile,and MBC and MIC were subsequently determined.The results showed that MBC and MIC of theΔvtr B mutant were 12.5%and 2%,which were lower than those of the WT strain(15%and 2.5%),and C-Δvtr B restored the bile’s MBC and MIC.In order to understand the role of Vtr B in V.parahaemolyticus resistance to bile,a bile-inducedΔvtr B mutant strain motility and biofilm formation phenotype analysis were performed.The results showed that on bile-containing swim,swarm,and twitch plates,the motility of theΔvtr B strain was significantly increased compared with WT and C-Δvtr B(P<0.05);The total amount of biofilms formed byΔvtr B induced by0.04%bile at 24h was approximately twice that of WT,showing a stronger biofilm formation ability than WT(P<0.05),and the supplemented vtr B showed reduced biofilm formation.3.The RNA-seq were performed on RNA extracted from wild-type andΔvtr B strains grown to late exponential-early stationary phase in LB broth supplemented with0.04%bile.701 genes were differentially expressed in the vtr B mutant compared to the WT strain;of these,462 genes were up-regulated and 239 genes were down-regulated by deletion of vtr B.As expected,45 of the 87 Vp-PAI encoded genes(51.7%)were significantly down-regulated in the vtr B mutant.These include genes encoding TDH and T3SS2-related genes.Interestingly,however,RNA-seq results suggested that Vtr B transcriptional activity was not restricted to genes within the Vp-PAI island.Non-T3SS2genes that showed differential expression in the absence of Vtr B belong to several gene classes based on functions such as T6SS,motility,biofilm formation,regulation and stress responses,suggesting that Vtr B has important roles in coordinating global gene regulation during the infectious process.4.Under bile induction,the imp ABCMLKJ and vgr G2 genes involved in T6SS2assembly and the hcp2 gene encoding the T6SS2 effector were significantly up-regulated in theΔvtr B mutant.It showed that Vtr B is a negative regulator of V.parahaemolyticus T6SS2.During intestinal infection,inhibition of Hcp2 and Vgr G2expression by Vtr B under bile conditions could enhance the bile resistance of V.parahaemolyticus,thus favoring infection.Additionally,Vtr B induced the expression of VP1409,VP1410 and vas D genes in a bile-dependent manner,we conclude that Vtr B may contribute to the T6SS1-dependent antibacterial activity in the presence of bile.5.The swimming,swarming and twitching ability inhibited by Vtr B may be caused by increased expression of T3SS.Meanwhile,Vtr B not only influence the expression of flagellar biosynthesis pathway related proteins but also the biofilm formation related proteins,GGDEF family proteins.Vtr B may mediate the dispersion of biofilm in the presence of bile,thus enhancing tne colonization of V.parahaemolyticus in the intestine.6.At the same time,the analysis results showed that in response to bile,the expression levels of cad A and cad B genes related to V.parahaemolyticus acid tolerance inΔvtr B mutant were down-regulated,and the expression levels of heat shock protein and Csp A were also down-regulated,indicating that Vtr B was induced by it may also induce the adaptability of V.parahaemolyticus to other environments,such as acid,heat and cold stress.The auxiliary colonization factor Acf A also showed significant down-regulation.This indicates that it may help Vtr B to colonize the intestinal tract of the host under the induction of Vtr B.In summary,this study indicated that Vtr B contributes to bile resistance of V.parahaemolyticus and can exercise transcriptional control outside Vp-PAI islands to coordinate virulence gene expression to help V.parahaemolyticus successfully infect the gastrointestinal tract. |
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