| Cholera is an acute bacterial gastrointestinal infectious disease caused by ingestion of water or food containing Gram-negative bacteria Vibrio cholerae(a member of the Homyx family).Once infected,it can easily cause severe dehydration diarrhea,hypovolemia and even death.Cholera is classified as a Class A infectious disease in China,and it is also one of the most serious infectious diseases recognized internationally.In the aquatic environment V.cholerae is found in its highly mobile planktonic form as well as in biofilms.They generally promote the attachment of Vibrio cholerae to the surface of phytoplankton,zooplankton and marine crustaceans through a type Ⅳ pili mannose-sensitive hemagglutinin(MSHA),which mediates biofilm formation.At the same time biofilm plays a pivotal role in the entire life cycle of Vibrio cholerae.Three genes,mshH,mshI and mshB,are contained upstream of the msh operon.Existing studies suggest that ToxT,a key virulence regulator of Vibrio cholerae,may regulate the expression of the msh operon by directly binding to the promoters of these genes.However,there have been no reports of enhanced msh expression.To find the genes that regulate the msh operon,we use the Vibrio cholerae genomic library to screen for genes that promote mshH expression.Through screening of 25,000 candidate strains,we obtained a strain capable of activating mshH expression.By sequencing we determined that it is able to activate mshH expression and the gene is rfaH(it will be called Vc rfaH later).The expression of mshH can be promoted by inducing expression of Vc RfaH in E.coli using an arabinose promoter.To further determine whether this gene also plays a role in Vibrio cholerae,an in-frame deletion strain of Vc rfaH was constructed,and Vc RfaH was able to activate mshH expression in Vibrio cholerae by PmshH-lux reporter plasmid.Studies on the transcriptional regulation of E.coli rfaH revealed that the gene activated by RfaH generally has a conserved JUMPstart(just upstream of many polysaccharide associated gene starts)sequence containing two ops elements.Subsequently,by analyzing the promoter of mshH,it was found that there is also a conserved ops element in the promoter region of the gene mshH activated by Vc RfaH in Vibrio cholerae.In order to explore the function of the ops acting element.a deletion and a mutant strain of the ops element sequence were constructed.The ops domain was confirmed to play an important role in the activation of mshH gene by Vc RfaH through LUX reports and RT-PCR experiments.To investigate whether Vc RfaH interacts with the mshH promoter sequence,The Vc RfaH protein was purified and the Electroprotective mobility shift assay(EMSA)of the Vc RfaH and mshH promoters was performed.It was found by EMSA experimental analysis that the Vc RfaH activate mshH by direct interaction.But the binding of Vc RfaH to PmshH was not due to the ops element in the mshH promoter region.Phenotypic analysis of the Vc rfaH in-frame deletion mutant showed that Vc RfaH promoted mshA expression at the protein level.Biofilm experiments have demonstrated that Vc RfaH can promote the formation of Vibrio cholerae biofilm.The LUX reporters gene detected that Vc RfaH promote the expression of virulence genes slightly.Through the competitive colonization experiments of WT and ΔVc rfaH in infant mouse,it was found that Vc rfaH plays an important role in the early stage of Vibrio cholerae infection.However,The competitive colonization experiments of adult mouse showed that the role of Vc rfaH in the late infection of Vibrio cholerae is not significant due to the complex regulatory relationship.In summary,the paper discovered the gene Vc rfaH that can activate mshH expression in Vibrio cholerae.The molecular mechanism of activating mshH gene expression by Vc RfaH is elucidated.That is,Vc RfaH activates the expression of mshH by binding to the promoter of mshH.It can affect the expression of MshA and the formation of Vibrio cholerae biofilm,and ultimately affect Vibrio cholerae in infant mouse. |
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