Role Of Cpx Two-component Regulatory System In Stresss Responses And Virulence Of Vibrio Anguillarum

Cpx two-component regulatory system exists in a variety of Gram-negative bacteria, mainly involved in the response to envelope stress. The sensor histidine kinase CpxA accepts signals from the environment, and passes them to the response regulator protein CpxR, which then functions as a transcription factor to regulate expression of target genes and helps bacteria respond to environmental changes. In this study,the cpx gene cluster was cloned from the genome DNA of Vibrio anguillarum M3 strain by degenerate PCR and genome walking. The obtained full length of cpx cluster was 2768 bp. The predicted CpxR and CpxA showed 60% and 84%, and 45% and 72% identities with Escherichia coli and Vibrio cholerae.To investigate the function of cpxRA, the cpxR and cpxA in-frame deletion mutants were constructed from M3. The phenotypes of both mutants were comparatively analysed with the wild-type strain. Both cpxR and cpxA mutants showed no difference with the wild-type strain in the mobility, gelatinase activity, growth in TSB containing 3.5% NaCl (P >0.05), while both mutants showed reduced viability in 1% H2O2 and 0.05% SDS, reduced biofilm formation, increased growth ability at pH 9.0 (P <0.05), varied sensitivites to Cephazolin and Cefuroxime. These data indicated that V. anguillarum CpxRA participated in sensing environmental signals and regulating some specific stress responses.Roles of cpxR and cpxA on bacterial virulence were determined. Bacteria were administrated to blue guorami fish Trichogaster trichopterus by intramuscular injection and immersing infection. In the injection route, the cpxR and cpxA mutant decreased 24.6-fold and 13.3-fold in virulence compared with the wild-type strain. In the immersion route, no differences in virulence were observed between both mutants and the wild-type, though, the death time of fish was delayed in the mutant strains. These data indicated that CpxRA was involved in the virulence of Vibrio anguillarum.Then, transcription of ten virulence-related genes were analysed by real-time quantitative RT-PCR (qRT-PCR) in the mutants and the wild-type strains, including transcription regulator genes rpoS and rpoN, metalloproteinase genes empA and prtV, quorum sensing regulator vanT, exopolysaccharide biosythesis and transport genes wza, wbfD, flagellin A gene flaA, and type six secretion sytem genes hcp and icmF. A certain degree of changes in transcription of the ten genes were found between the mutants and the wild-type strains. Bioinformative analysis indicated that none of the ten genes were Cpx regulons. The mechanisms of V. anguillarum Cpx on these genes need further investigate.