Molecular Mechanism Of Expression Regulation Of MSH Gene Cluster Of Vibrio Cholerae

Vibrio cholerae is a Gram-negative bacterium that is the causative agent of epidemic cholera.The infection cycle of V.cholerae is divided into two phases: one is the aquatic environment in vitro and the other is the intestinal environment in the host.The formation of biofilms has played a significant role in the survival of V.cholerae in both the aquatic environment in vitro and the intestinal environment in the body.The type IV pili of V.cholerae(MSHA,Mannose-sensitive Hemagglutinin)is essential for biofilm formation.The study found that the expression of the MSHA gene cluster was inhibited when V.cholerae entered the host gut.However,V.cholerae in the form of a biofilm was observed in the feces of patients infected with cholera.Therefore,we hypothesize that V.cholerae is likely to inhibit the expression of MSHA fimbriae in the early stage of infection in order to escape the host's natural immune system attack.While,in the later stage of infection,a new biofilm is needed to enter the next infection cycle.However,the regulatory mechanism of the MSHA gene cluster in V.cholerae remains unclear.MshH is a homolog of Escherichia coli CsrD,a component of an unusual and complex posttranscriptional regulatory cascade known as Carbon storage regulator(Csr),CsrD enhances the RNase E-dependent degradation of the small csr RNAs that inhibit binding of the regulatory protein CsrA to mRNA.However,its function is unknown in V.cholerae.This study aimed to explore the molecular mechanism of mshH gene expression regulation.Firstly,a V.cholerae gene expression library was constructed.The gene library was screened by a fluorescence reporter system.By screening 25,000 strains,VC1371 was specifically activated to activate mshH transcription in E.coli.In order to further determine whether VC1371 protein directly regulates the expression of mshH gene,VC1371 protein was successfully purified by nickel column purification method.Through EMSA(Electrophoretic Mobility Shift Assay)experiment,VC1371 was found to bind to the promoter sequence of mshH and pass luciferase.The reporter system detected that the expression of mshH in the V.cholerae vc1371 gene deletion mutant(?vc1371)was significantly weaker than that of the wild type.By analyzing the formation of the biofilm of the mutant strain,it was found that the formation of the biofilm was reduced when VC1371 was deleted.This indicates that VC1371 can regulate the transcription of mshH in V.cholerae and affect the formation of V.cholerae biofilm through the regulation of mshH.By preparing MshA antibody and analyzing the MshA protein level of V.cholerae wild type strain and ?vc1371,it was found that the expression level of MshA in ?vc1371 was weaker than that of wild type.In the VC1371 overexpression strain,the expression of MshA was restored,even higher than the wild type.This suggests that V.cholerae VC1371 may regulate the synthesis of MshA by activating the expression of mshH.In order to verify whether the regulation of mshH by VC1371 plays a role in the colonization process of V.cholerae,the gene deletion mutants of ?vc1371 and ?vc0398 were separately competed with wild-type strains for intestinal metabolism of infant mouse and adult mouse.The results showed that the colonization ability of ?vc1371 and ?vc0398 in the intestine of the infant mouse was stronger than that of the wild type strain,while the colonization ability of ?vc1371 and ?vc0398 in the adult mouse was weaker than that of the wild type.Taken together,this study demonstrates that VC1371 specifically activates the expression of mshH in V.cholerae.The loss of VC1371 reduces the expression of PmshH,decreases the expression of MshA protein,and reduces the formation of biofilm,and the VC1371 protein can bind to the promoter sequence of mshH in vitro.Finally,it was found that ?vc1371 and ?vc0398 played a certain role in the initial and late host colonization of V.cholerae infection.The results of this study laid a foundation for studying the expression regulation of MSH gene clusters and the molecular mechanism of biofilm formation.