| Actinobacillus pleuropneumoniae(APP)is a gram-negative pathogen causing porcine pleuropneumonia,which has caused great economic losses to the global pig industry.The virulence of the 18 serotypes of APP is different,and the research on its pathogenic mechanism and influencing factors needs to be further studied.Therefore,the study of APP virulence factors is conducive to the comprehensive prevention and control of pleuropneumonia.High temperature requirement A(HtrA)is a highly conserved serine protease,which plays an important role as a protein regulator and protective immune antigen in bacterial periplasm.In the process of invading host cells,app is exposed to a variety of stress conditions,which requires a stress response system to cope with these changing adverse external environment.It is known that HtrA family proteins are closely related to the survival and virulence of some pathogens.At present,little is known about the mechanism of APP stress adaptation and its relationship with bacterial pathogenicity.In this project,we mainly studied the effect of HtrA protein on the survival and virulence of APP under stress:1.Construction of htrA gene deletion mutant and back mutantUsing APP serotype 1 strain SLW01 as the parent strain,a chloramphenicol sensitive and sucrose resistant mutant ΔhtrA was screened by homologous recombination,chloramphenicol mediated positive selection and sucrose mediated negative selection.The complete HtrA gene was amplified and cloned into pJFF224-XN,and the shuttle plasmid was transformed into ΔhtrA strain by electroporation.Through chloramphenicol resistance screening,the revertant ΔhtrA-Compl was obtained.2.Analysis of the high temperature adaptability of htrA mutantThe parental strains SLW01,ΔhtrA and ΔhtrA-Compl were inoculated into TSB,cultured at 37℃ and 42℃ respectively,and the growth curves were drawn.The results showed that the growth levels of the three strains were basically the same at 37℃.Compared with SLW01 and ΔhtrA-Compl,the growth of ΔhtrA was significantly inhibited at 42℃,and the OD600 of ΔhtrA was less than 0.1 within 10 h,indicating that the mutant could hardly grow at 42℃,and the growth ability of ΔhtrA-Compl was restored at 42℃,and its OD600 value was only slightly lower than SLW01(no significant difference).The results of TSA plate counting showed that the viable population of ΔhtrA was significantly lower than that of its parents SLW01 and ΔhtrA-Compl at 42℃.The growth of ΔhtrA-deficient strains under high temperature stress was significantly affected.ΔhtrA is a high temperature(42℃)sensitive strain,indicating that HtrA is an essential protein for APP high temperature stress adaptation.3.Analysis of the oxidative adaptability of htrA mutantThe parent bacteria SLW01,ΔhtrA and ΔhtrA-Compl were cultured to the logarithmic growth phase and then transferred to TSB containing 2.5mM H2O2,and the viable bacteria counted on the plate to calculate the survival rate of the three bacteria.The results show that ΔhtrA has a lower survival rate than SLW01 and ΔhtrA-Compl,and its oxidation sensitivity is higher.The survival rates of ΔhtrA-Compl and SLW01 were basically the same,indicating that supplementing htrA restored the oxidation tolerance ofΔhtrA.The results show that oxidative stress has a more obvious inhibitory effect on the survival of ΔhtrA-deficient strains,which proves that HtrA is more important for APP survival under oxidative stress conditions.4.Study on the pathogenicity of htrA mutant in miceThe LD50 of SLW01,ΔhtrA and ΔhtrA-Compl in mice,the acute toxicity of SLW01,ΔhtrA-Compl in mice and the bacterial load in mice lung tissue were determined to explore the toxicity correlation between HtrA and APP.Compared with SLW01 andΔhtrA-Compl,the mice infected with ΔhtrA had lower mortality and lower virulence.The number of bacteria in lung tissue of mice inoculated with ΔhtrA was significantly lower than that of parents SLW01 and ΔhtrA-Compl.The competitive infection index of ΔhtrA and SLW01 in the lung of mice was far lower than the weakening threshold,indicating that HtrA gene deletion can affect the virulence of APP,and HtrA is the virulence factor of APP.5.Study on the colonization ability of htrA mutant in blood and lung of pigletsThe pathogenicity of HtrA was further verified by piglet infection test.By intranasal injection of the mixture of ΔhtrA and SLW01 with the same amount of bacteria,the infected bacteria in lung tissue and residual blood of thoracic cavity were isolated.The competitive infection index of the two bacteria in piglets was calculated by PCR identification.The results showed that the competitive infection index of ΔhtrA mutant in lung tissue and residual blood of piglets was lower than the weakening threshold,indicating that the pathogenicity of ΔhtrA mutant to pigs decreased,indicating that HtrA is the virulence determinant of APP.In conclusion,this study confirmed that heat stress demand protein HtrA is an important virulence factor of app,and the pathogenic mechanism of HtrA may be related to the immune escape process mediated by bacterial high temperature tolerance and oxidative stress tolerance. |
