Construction Of Mutant Strains And Biological Characteristics Of Two-Component Regulatory System CpxA/R On Actinobacillus Pleuropneumoniae

Porcine contagious pleuropneumonia (PCP),which is caused by Actinobacillus pleuropneumoniae (APP), is an infectious porcine respiratory tract disease and a major economic problem in the swine industry worldwide. Treatment of the disease, characterized by hemorrhagic, fibrinous, and necrotic lesions, is increasingly difficult due to the occurrence of antibiotic-resistant strains. Although many virulence factors involved in pathogenesis of APP have been reported, as its regulation system complex,what roles they play in the whole infection and pathogenesis is still not completely clear.Two-component regulatory systems(TCS) are widely used signal transduction mechanisms that bacterium use to mediate the response and adaptation of organisms to environmental changes. Besides, TCSs play an important role in the process of bacterial pathogens infection,such as growth and metabolism,, drug resistance and the expression of virulence factors,so in order to demonstrate pathogenesis of APP, we should make a further step on the research of the TCSs.By analysis the APP JL03strain genome sequencing information, there are five pairs of TCSs exists in APP, are CpxA/CpxR, NarP/NarQ, ArcA/ArcB, PhoB/PhoR and QseB/QseC.The CpxA/R pathway is a prototypical two-component signal transduction system related to envelope stress response, supervising and maintaining the integrity of cell membrane, involving in the Fimbriae synthesis and the expression of virulence factors. it plays important role in the pathogenesis of APP.In view of the above background, in the currently popular APP serotype1(SLW01) as parent strains, based on whole-genome sequencing of serotype3strain (JL03) of APP construct the deleted CpxA/R mutant strain by negative selection and homologous recombination, further research on the biological characteristics and pathogenicity in mice. The main research as follows:1. Construction and identification of the double gene deletion mutant ACpxA/R and complementation strain C ACpxA/Rwith reference to the CpxA CpxR of JLO3Genbank gene sequence, the APP SLW01genomic DNA as template, amplify the upstream and downstream fragments of CpxA/R genes, connected to the PMD-18T by T/A cloning. After digested and retrieved, connected successively to the suicide plasmid pEMOC2. then the recombinant suicide plasmid pEM-CpxA/R that missing2300bp was constructed successfully. Transfer recombinant suicide plasmid pEM-CpxA/R into β2155as the donor strain, then conjugal transfer with the receptor strain SLW01,obtain single exchange strain.Based on negative selection and two-step homologous method,select the clones that Cm-sensitive and sucrose resistant, PCR identification, determine the occurrence of a second single exchange. And the deletion mutant is further identified by DNA sequencing and Southern blot, then validated that the double gene deletion mutant△CpxA/R was successfully construct.with reference to the JL03Genbank gene sequence,design primes, CpxA/R gene from SLW01genomic DNA was amplified,connected to E. coli-APP shuttle vector pJFF224-XN to construct the complementation plasmid PJFF-CpxA/R. Then electrotransform PJFF-CpxA/R into△CpxA/R mutant strains. PCR identification, the complementation strain was successfully constructed.2. The biological characteristics of gene deletion mutants and explore the virulence in miceIn order to confirm whether the deletion in CpxA/R gene affected the growth ability of A. pleuropneumoniae, SLW01、△CpxA/R、C△CpxA/R were cultured at37℃overnight, The next day, strains were transfered into fresh TSB at a1:1000dilution, The OD600was recorded every1h,viable count and draw the growth curves, The result suggested the mutant strain was slower slightly than parental strain during log phase, there is little difference between parent strain and component strain. The△CpxA/R strains was cultured in TSA for10generations,identification of genetic stability by PCR, the results showed that the deletion mutant strain can grow stably. To test the hemolytic activity of the deletion mutant strain,inoculate the strains in10%(v/v) fresh sheep blood NAD plate at37℃with24h, the result showed that there is no significant difference between mutant strains and parental strain. The mutant LD50is tested with six weeks Balb/C mice, virulence of△CpxA/R was5.36times lower than parental strain, while virulence of C△CpxA/R is almost no difference with the parental strain. Electric mirror revealed that the thickness of capsule didn’t change obviously,but the bacterial surface morphology change to a certain extent.the edge line becomes loose. Adhesion and invasion assay demonstrated that the adhesion of mutant dropped markly.Phagocytosis test found the mutant resistance to Phagocytosis was dropped.the mutant was easier to be killed. The above results show that:the delect of CpxA/R cause reduce of bacterial virulence.3. Screening of differentially expressed genes regulated by qRT-PCR With reference to documentation, selected11genes that relevant to material transport, outer membrane protein,fimbriae synthesis, expression differencnce were analyzed by qRT-PCR.There were8genes significant differences in expression, inside6genes up were regulated,and2gens were down regulated.Through the above experiment,we found that CpxA/R regulate system probably affect pathogenicity of APP through regulate the expression of outer membrane protein, fimbriae synthesis.4. Expression and purification of response regulator CpxRwith reference to the CpxR of JL03Genbank gene sequence, CpxR gene from SLW01genomic DNA was amplified, connected to the PMD-18T by T/A cloning, digested and retrieved,then connected to expression vector PET-28a(+), the PET-CpxR plasmid was constructed successfully. Then transformed it into E.coli BL21strain. Its expression was induced by IPTG with3h. obtain24.24KDa response regulator.