The Role Of CpxA/R Two-component System In Stress Response Of Haemophilus Parasuis

Haemophilus parasuis is an opportunistic pathogen in swine upper respiratory tract and the etiological agent of Gl?sser’s disease, which is characterized by fibrinous polyserositis, polyarthritis, and meningitis. The Cpx two-component system is a signal transduction system that respond to environmental signals by modifying the phosphorylation state of the cognate regulatory protein Cpx R.In the present study, we constructed the Cpx mutants in H.parasuis JS0135 and investigated the role of Cpx two-component system in response to the stresses in Haemophilus parasuis JS0135. The main results are as follows: 1. Comparison the biological characteristics of wild strain JS0135 and JS0135 Cpx mutant strainConstruct the recombinant plasmids which were named p K18-cpx A-UKD, p K18-cpx R-UKD and p K18-cpx AR-UKD. The recombinant plasmids were transformed into JS0135 by natural transformation, and the Cpx mutants were screened on kan resistant plate.The results of stress tolerance assays showed that the survival rate of JS0135-Δcpx R and JS0135-Δcpx AR were significantly lower than that of the wild strain JS0135, and the survival rate of JS0135-Δcpx A kept the same with the wild strain JS0135, in response to 0.5M KCl and 0.1M H2O2. The wild type strain JS0135 and JS0135 cpx mutants did not differ in response to 0.02 M Na OH.Moreover, we observed that the survival rate of JS0135-Δcpx A was higher than that of JS0135, JS0135-Δcpx R and JS0135-Δcpx AR on plates containing gentamicin; The survival rates of JS0135 and JS0135-Δcpx A were significantly higher than JS0135-Δcpx R and JS0135-Δcpx AR on plates containing erythromycin. Further, MIC was evaluated by in vitro 96-well microplate. The results showed that the MICs of JS0135, JS0135-Δcpx A were higher than that of JS0135-Δcpx R, JS0135-Δcpx AR to macrolide antibiotics. 2. Cpx two-component system is involved in adhesion ability and resist against phagocytosis of H. parasuisWild type strain JS0135 and the Cpx mutants were cultured with porcine kidney epithelial cells(PK-15). The results showed that the adhesion index of JS0135-Δcpx R、JS0135-Δcpx AR were significantly lower than that of wild type strain. The adhesion index of JS0135-Δcpx A were significantly lower than that of wild strain only at 30 min but showed no difference at 60 min and 90 min.Wild type strain JS0135 and JS0135 cpx mutants were cultured with mouse macrophages(RAW264.7) or porcine alveolar macrophage(PAM). The results showed that the phagocytic resistance ability of JS0135-△cpx R and JS0135-△cpx AR were significantly higher than that of wild strain JS0135, the phagocytic resistance ability of JS0135-△cpx A was significantly lower than that of wild strain JS0135. 3. Identification of differentially expressed genes after cpx deletion by q RT-PCRAccording to the references, we selected 16 genes that are relevant to bacterial virulence and drug resistance to analyze their expression sfter cpx deletion. Results showed that the virulence gene cdt B, drug resistance gene prop and HAPS2067 were differentily expressed. Moreover after adding erythromycin as an inducer, the resules showed that prop, acr A, HAPS1938, HAPS2067 and HAPS2068 were differentily expressed. Taken together, our findings demonstrated that two-component system cpx A/R probably affect the drug resistance of H. parasuis through influencing the expression of efflux pump genes.In conclusion, we demonstrated that the Cpx two-component system is involved in stress tolerance, drug resistance, adhesive ability andt phagocytosis resistance of H.parasuis.