Function Of Two-component Regulatory System PhoBR On Actinobacillus Pleuropneumoniae

Actinobacillus pleuropneumoniae(APP) is the causative agent of Porcine Contagious pleuropneumonia(PCP), a severe and often fatal respiratory disease of swine, causing serious economic losses worldwide in the swine industry. This organism cause sudden death and colonize the respiratory tracts, tonsils and lungs of pigs, causing chronic and persistent infections, lung lesions, and reduced growth. Phosphate is an essential nutrient for pathogen and has an important role in signal transduction and energy metabolism. Carbohydrate as a carbon source has a vital role in the growth of bacteria. In respiratory tracts and lung are generally thought to be limiting for phosphorus and Carbohydrate. Therefore, bacteria must actively pursue phosphate and Carbohydrate to ensure survival in porcine respiratory tracts and lung.In the process of the infection, the pathogen could sense and respond to extracellular signals through the two component regulatory system(TSC), and then regulate the expression of the various genes to complete its pathogenic process. Five TSC, ArcA/ArcB, CpxA/CpxR, NarP/NarQ, PhoB/PhoR and QseB/QseC, are found in APP based on the analysis of the genome of APP strain JL03. Many researchers study on PhoBR show that PhoBR involved in the regulation of phosphorus, sugar metabolism and the expression of virulence factors, But the function of PhoBR in the APP is still unknown.In view of the above background, this study selects the APP serotype1strain SLW01as the parent strain and use homologous recombination to constructure the double gene deletion mutant strain AphoBR. At the same time, the strain AphoBR of complementary strain CAphoBR is constructured. Biological characteristics and pathogenicity in mice of ΔphoBR is compared with the parent strain. In adition, using Agilent single channel expression profile chip and qRT-PCR, the differentially expressed genes between mutant strains AphoBR and parental strains SLW01are screened. The pathogenic mechanism of phoBR in APP is preliminarily investigated. The main research content are as follows:1. Construction and identification of the mutant strain AphoBR and complementary strain CΔphoBRRefer to the phoBR genes sequence in JL03Genbank genome, the upstream and downstream fragments of phoBR genes are amplified from the genomic DNA of APP serotype1SLW01. The upstream and downstream segments are cloned into the vector pMD-18T. After identification by restriction endonuclease analysis, the segments were further cloned into the vector pEMOC2, creating the recombinant suicide plasmid pEM-phoBR. β2155transferred with recombinant suicide plasmid pEM-phoBR is regarded as the donor strain. Then the donor strain was conjugal transfered with the receptor strain SLW01, obtaining single exchange strain. Through negative and positive selection, the double exchange strain which is Cm-sensitive and sucrose resistant was further verified with PCR, qRT-PCR and sequencing. The mutant strain was constructed successfully, and named it ΔphoBR.The phoBR genes were amplified from the genomic DNA of APP serotype1SLW01, and then cloned into the vector of pEMD-18and shuttle plasmid pJFF224-XN of APP-E. coli in succession. After being verified by PCR, enzyme digestion and sequencing, the recombinant plasmid pJFF-phoBR was then electrotransfered into double mutant strain ΔphoBR. The recombinants were verified by PCR and qRT-PCR to confirm that the complementary strains CΔphoBR was constructed successfully.2. Research on the basic biological characteristics of PhoBRThe research results of biological characteristics of mutant strain show that ΔphoBR could propagate stability in vitro. In normal culture condition, the mutant strain ΔphoBR behave a significantly slower growth rate compared to SLW01. In the condition of phosphorus limited, the growth rate of mutant strains ΔphoBR shows no difference with wild strains SLW01. LD50in mice infection experiment show that the virulence of mutant strains ΔphoBR compared to the parent strain SLW01and complementary strain CΔphoBR rose1.73times and1.73times respectively (Korbor method); Survival rate of mice infected with ΔphoBR was7.14%, while that with parent strain SLW01was42.86%. In early infectin phase, bacteria load in lung and blood of mice infected by AphoBR is siginificantly higher than that infected by SLW01. On120h post infection, the bacteria load in lung of mice infected with the two strains was almost the same while ΔphoBR still maintain a higher level in blood. PMN mediated killing experiments shows that the mutant strains AphoBR exhibits significantly greater resistance than the parent strain SLW01. The above results showed that the virulence of mutant strains ΔphoBR enhanced.3. The regulation mechanism and pathogenesis of PhoBRAnalysis of differentially expressed genes between mutant strains ΔphoBR and parent strain SLW01shows that in normal culture condition, the absence of phoBR led to12genes differently expressed (5upregulated and7down regulated). In phosphorus limited culture condition, the absence of phoBR genes led to up-regulation of119genes fold change more than2and60genes downregulated. These genes are mainly related to energy metabolism, amino acid transport and metabolism, sugar transport and metabolism, coenzyme metabolism, post-translational modifications, protein folding and molecular chaperone, inorganic iron transport and metabolism. Further, the chip results were confirmed by qRT-PCR. In conclusion, PhoB/R is verified to be involved in the pathogensis of APP via regulating virulence factores.