Study Of Regulation Mechanism Of Virulence Genes In ToxR Regulon In Vibrio Cholerae

Vibrio cholerae is the causative agent of cholera. The two major virulence determinants of Vibrio cholerae are encoded by two separate genetic elements: cholera toxin(CT), which causes the diarrhea characteristic of cholera, and the toxin-coregulated pilus(TCP), which is essential for attachment and colonization of intestinal epithelia. Extensive studies reveal that complicated regulatory cascades regulate expression of virulence genes. The primary direct transcriptional activator of Vibrio cholerae virulence genes, including ctxAB and tcpA, is ToxT, a member of the AraC/Xyls family. Besides, ToxT is able to repress the transcription of the msh operon. ToxR acts in conjunction with another transcriptional activator, TcpP, to regulate expression of toxT. Moreover, ToxR activates the transcription of ompU and represses the transcription of ompT, outer membrane porins of Vibrio cholerae. In this study, we investigated the expression of the key virulence regulator ToxR under different conditions. We found that compared to that of wild type grown to stationary phase, the toxR expression was lower in an aphB mutant strain. AphB has been previously shown that it can activate tcpP requiring interaction with AphA. EMSA indicates that AphB binds toxR promoter region directly and activate toxR promoter. Furthermore, we demonstrated that AphB effects on toxR expression affect toxT expression. We also analyzed the function that AphB regulate outer membrane porins, OmpT and OmpU. Our data indicated that Vibrio cholerae possesses an additional regulatory loop that use AphB to activate the expression of two virulence regulators, ToxR and TcpP, which together control the expression of the master virulence regulator ToxT.ToxT plays critical roles in pathogenesis, including the regulation of two type IV pili, the anticolonization factor mannose-sensitive hemagglutinin(MSHA) and the toxin-coregulated pilus(TCP). The presence of MSHA pili, which act as potent anticolonization factors, on the other hand, is actively detrimental to survival in the host. Previously, it was thought ToxT required dimerization in order to effect transcriptional regulation at its cognate promoters. Here, we presented evidence that ToxT directly represses transcription of the msh operon by binding to three promoters within this operon and that dimerization may not be required for transcriptional repression of target promoters by ToxT, suggesting that this regulator uses different mechanisms to modulate the transcriptional repertoire of Vibrio cholerae. Vibrio cholerae is the aetiologic agent of cholera that inhabits in aquatic environment. Upon exposure to an unfavourable environment, the microorganism can survive by entering a dormant state, that is, the viable but nonculturable (VBNC) state. VBNC cells cannot be detected by standard culture methods but retaining virulence, and they can be resuscitated and cause disease through access to an animal host. In this study, we successfully induced Vibrio cholerae into the VBNC state in lab environment. During this process, we characterized the tendency of the number of culturable bacteria through plate count and the percentage of viable cells through LIVE/DEAD fluorescence staining. Furthermore, we compared the global transcription pattern of the cells(incubate 4 hours and 24 hours under low temperature and low nutrition culture condition) with that of stationary-phase cells grown in rich medium and described the gene expression profile at this two time points. We found that more than 300 genes changed, and we selected a pair of toxin-antitoxin genes(VCA0391/2) to investigate whether they can help Vibrio cholerae to escape from death under the bad condition...