Construction And Characterization Of KpsM Deleted Mutants Of Porcine Extraintestinal Pathogenic Escherichia.Coli

According to the genetic characteristics and clinical manifestations. Escherichia coli are divided into three categories:the symbiotic type Escherichia coli, intestinal pathogenic Escherichia coli and the extraintestinal pathogenic Escherichia coli (ExPEC). ExPEC has become a wide range of pathogens threat to global human and animal health in recent years. It can mainly cause pyelonephritis and neonatal meningitis in the human body, and sepsis, the airbag inflammation, pericarditis and so on in poultry. Due to the fact that serotypes, virulence factors, gene structure and regulation of gene expression of the ExPEC are intricate, the pathogenic mechanisms of ExPEC have not been completely investigated. In2004-2007, we found that0161,08, O11,0138, O101,026are its dominant serotypes and B2and D are its major groups during315pig source parenteral pathogenic E. coli of central China epidemiological studies.Perfringen is important polysaccharide structure covering the bacterial surface, which can protect the bacteria from the removal of the host immune system. kps gene locus from2groups,a conserved structure, is composed of three regions. kpsM in kps gene cluster region3, a highly conserved gene locus, encods the channel protein of. ABC transporter proteins across the inner membrance.We discover that kpsM is preasant in PCN033but not in PCN009, while comparing the Whole sequence alignment of the virulent strain PCN033and attenuated strain PCN009. We treat the virulent strain PCN033separated from pig brain as parental bacteria,whose serotype is011and it belong to D group. On the basis mentioned above, we delet kpsM by homologous recombination and it is carried out to study its function and biological characteristics. The reaserch mainly contents as follows:1Construction and identification of kpsM Deleted Mutants.the upstream and downstream homology arm,amplification of kpsM From PCN033, are connected to pMD-18T vector. Restriction enzyme cuts upstream and downstream homology arm. simultaneously connect the products to the intermediate transfer plasmid pSK with the result that constructing shuttle vector pSK△kpsM. Cut the homology arms with restriction enzyme then connect the product to the reorganizated suicide plasmid pRE112. We then get the recombinant transfer plasmid pRE112△kpsM.After conjugative transfering from the donor strain X7213which is transformed recombinant transfer vector pRE112△kpsM to the recipient strain porcine ExPEC PCN033, we use Method which is SacB sucrose negative screening combining with two-step exchange homologous recombination to build gene deletion strains. The results suggest that the gene deleted mutant is successfully constructed by PCR determine and Southern blot hybridization analysis to verify.2Construction and identification of AkpsM complementary strain.Connect kpsM gene from PCN033genome to the shuttle plasmid pHSG396to get recombinant expression vector pHSG396-kpsM. Recombinant plasmid is transformed into AkpsM strains to get the complementary strain AkpsM-kpsM. The results show that the complementary strain is successfully constructed by using PCR to verify.3Analysis for gene deleted mutants and complementary strain biological characteristics.The complementary strain△kpsM-kpsM and deleted strain AkpsM continuous generat10times on the plate. Each generation is identificated for its genetic stability by PCR, whose results suggesting that the AkpsM and AkpsM-kpsM stably inherited. Measure the strains experiment of growth rate, then draw the growth curve of AkpsM、 PCN033and△kpsM-kpsM. We find that comparing AkpsM and△kpsM-kpsM with the parental strain, the growth rates are not significantly different. Mearing LD50for Kunming mouse, the results show that the poison force of AkpsM reduces14.75times comparing with PCN033, and△kpsM-kpsM is not fully recovered, just restoring the virulence of nearly75%, when comparing with the parent strain virulence.Results of adhesion, engulfing, serum bactericidal experiment show that the adhesion ability of AkpsM decreased by80%, the anti phagocytic ability of AkpsM decreased by nearly40%, and AkpsM resistance to complement-mediated serum decreased55.12%comparing AkpsM with PCN033. The adhesion ability of△kpsM-kpsM recovered73%, the anti phagocytic ability of△kpsM-kpsM recovered nearly80%, and the anti serum bactericidal of△kpsM-kpsM recovered nearly100%comparing△kpsM-kpsM with PCN033.