Role Of The Cpx Two-component Regulatory System In Stresss Responses And Virulence Of Edwardsiclla Tarda

The Cpx（conjugative plasmid expression）two-component regulatory system existson the membrane of a variety of gram-negative bacteria. It senses the change ofnatural environment or the systemic infection niches of hosts, and is involved in theresponse to envelope stress and the maintenance of envelope proteins. Function of theCpx in E. tarda is not clarified. Here we studied its role:（1） The impact on bacterial phenotypes. We obtained the2,713bp cpx gene clusterof Edwardsiella tarda LSE40using a homologous cloning strategy. The Cpx iscomposed of a histidine kinase activity protein CpxA, a cognate cytoplasmic responseregulator CpxR and a periplasmic inhibitor CpxP. To investigate the function of Cpx,the cpxR gene and the cpx cluster in-frame deletion mutants were constructed fromLSE40. Neither ΔcpxR or Δcpx mutants showed significant differences with thewild-type strain in the celluar growth, mobility, sensitivities to antibiotics,auto-aggregation and secretion of T3SS component protein EseB, EseC, EseD（P>0.05） in TSA or TSB medium. The Δcpx mutant showed aberrant biofilmformation （P <0.05）, while the ΔcpxR mutant exihibited significant difference（P>0.05）. Mutants didn’t showed significant differences in3.5%NaCl density TSBand Fe2+limited medium （P>0.05）. While and both mutants exhibited increasedsensitivities under1%（w/v） H₂O₂and0.05%（w/v） SDS stresses （P <0.05）. Thesedata indicated that E. tarda Cpx participated in sensing environmental oxidant andsurfactant signal.（2） The impact on bacterial virulence. Bacteria were administrated to blue guoramifish Trichogaster trichopterus by immersing infection and intramuscular injection. Inthe injection route, the Δcpx and ΔcpxR mutants decreased4.58-fold and7.33-fold in virulence compared with the wild-type strain. In the immersion route, the Δcpx andΔcpxR mutants showed1.5-fold and3.0-fold decrease. Both mutants exhibiteddecreased bacterial proliferation ability in host’s liver tissue as well, which indicatedthat Cpx pathway was involved in the virulence of E. tarda.（3） The impact on genes transcription. Expression of LSE40cpxR was dected byreal-time quantitative RT-PCR （qRT-PCR）. The transcriptional level was significantlyaraised in strain under1mM H₂O₂treatment compared with untreated strain, whichindicated H₂O₂could probably activate the expression of cpxR. We further studied theimpact on the expression of the putative CpxR-regulated genes, the functions ofwhich range from antioxidation, protein folding to cell division processes.Transcriptional levels were observed significantly different in deletion mutants andreturned to wild type levels in complementary strain, which indicated the regulationof CpxR to these genes.