| Vibrio cholerae is the infectious agent which causes the human infectious disease cholera.V.cholerae can cause host acute intestinal infection,watery diarrhea,vomiting,and leads to severe dehydration and even death.Currently,there are still cholera outbreaks in areas with poor hygienic conditions.Studies found that V.cholerae in the aquatic environment usually symbiose with plankton in biofilm form.When V.cholerae entered the animal or human body,the expression of virulence factors would be readjusted to adapt to the host intestinal environment.Then V.cholerae would colonize intestinal epithelial cells and secreted toxins.Disulfide bond oxidoreductase DsbA is located in periplasmic space of V.Cholerae.DsbA can transfer its disulfide bonds to substrate proteins by redox reaction to make substrate proteins in the correct conformation and function.Our studies were designed to investigate the function of VcDsbA.It has been found that VcdsbA-deficient strains can not colonize the small intestine of neonatal rats.Vibrio cholerae toxin has also been found to be a catalytic substrate of VcDsbA.However,which protein can interact with VcDsbA and then regulate life metabolism,how does VcDsbA regulate virulence factors expression,what role VcDsbA plays in Vibrio cholerae survival in the aquatic environment,are still unclear.In this study,the effect of VcDsbA on the regulation of cholera toxin and the formation of biofilm in vitro and in vivo were investigated by capturing substrate proteins of VcDsbA and constructing VcdsbA-deficient Vibrio cholerae strain.The 33 rd amino acid Cysteine which is located in VcDsbA activity center was subjected to site-directed mutagenesis.The possible substrates of VcDsbA were captured by affinity chromatography.The results showed that more than 40 substrate proteins were captured,including outer membrane proteins(OMP),fimbriae synthesis-related proteins,molecular chaperones and transcriptional regulators.We knocked out dsbA(Vc0034)gene and replenished it by homologous recombination and gene cloning.By crystal violet staining assay,we found that biofilm formation ability of dsbA deficient strain(ΔdsbA)decreased significantly compared with wild type strain(WT).The expression levels of Mannose-sensitive hemagglutinin(pili biogenesis protein,MSHA),which is related to biofilm formation,in Vibrio cholerae WT,ΔdsbA and CΔdsbA strains were analyzed by using luciferase gene as a reporter gene and FLAG fusion expression analysis.It was found that the transcription of msh gene in ΔdsbA strain was significantly decreased.Through the MSHA hemagglutination test we found that ΔdsbA could not cause sheep erythrocyte agglutination.The above experiments showed that VcDsbA promoted biofilm formation by enhancing msh gene expression.The expression level of cholera virulence gene in ΔdsbA strain was detected by using luciferase gene as a reporter gene.The results showed that in ΔdsbA strain the expression level of ToxT,a key virulence gene regulator of Vibrio cholerae,was significantly lower than that in wild type.Bacterial two-hybrid experiment results indicated that Vibrio cholerae TcpP protein,which could form intermolecular disulfide bonds to form TcpP dimer,was catalyzed by VcDsbA.AMS trapping assay revealed that TcpP was a substrate of VcDsbA,and VcDsbA catalyzed cysteines located in TcpP periplasmic region to form TcpP dimer.The results showed that VcDsbA catalyzed the oxidation of TcpP to form dimer to activate the expression of downstream virulence factor toxT,which in turn activated the virulence of Vibrio cholerae.In summary,in external aquatic environment,VcDsbA might directly or indirectly enhance the biosynthesis of V.cholerae MSHA by influencing other regulators,thus promoted V.cholerae biofilm formation.In host’s small intestine,VcDsbA not only directly catalyzed the virulence factors,but it was also indispensable in the regulation of cholera toxin regulators expression.Our studies laid the foundation for further research on DsbA based regulation mechanism of biofilm formation and virulence factors expression in V.cholerae. |
