Study On Hcp Mediated APEC Ce129 Pathogenicity

Avian colibacillosis is one of the most serious infections in poultry and there is no effective therapy of this disease which has caused considerable economic loss. Avian pathogenic E.coli (APEC) has lots of isolates. Some virulence genes have been proved as its major pathogenic factors of APEC causing infection, although the mechanism remains to be elucidated. Hcp tunnel is the main structure of type VI secretion system, which was been discovered recently and it plays an important role in pathogenic processes. This study targets at the major structure subunit hcp1 and hcp2 genes of Hcp, and explores the role of Hcp in pathogenicity of APEC CE129.Based on the original sequences of hcp1 and hcp2 in GeneBank, the wild type Avain Pathogenic Escherichia coli (APEC) strain CE129 was selected as the prototype and constructed the hcp1 mutant and hcp2 mutant by Red recombination system. Firstly, we generated the PCR products by specific primers with homologies extension of hcp1 and hcp2 gene, and then hcp1 and hcp2 gene from strain CE129 were cloned respectively. Secondly, the PCR products for the replacement of hcp1 and hcp2 gene were constructed by employing template plasmid pKD3 and primers which contain a homologies 5’terminal to the target region and a homologies 3’terminal to chloromycin-resistance (cat) gene. With λ Red-mediated recombination system, the PCR products were introduced into wild type strain CE129 carrying a heat-sensitive plasmid pKD46 by electroporation, and hcp1 and hcp2 gene was replaced by cat gene respectively. The successful recombinant bacterial strains were selected by LB plates containing chloramphenicol. Then the chloromycin-resistance gene was eliminated by another heat-sensitive plasmid pCP20, which encoding the Flp recombinase. Using this system, hcp1 and hcp2 gene in chromosome of APEC were deleted. Finally, the isogenic CE129Δhcp1、CE129Δhcp2 and CE129Δhcp1Δhcp2 mutants were confirmed by PCR and subsequent DNA sequencing.The results of competition test in vitro show that CE129Δhcpi grew slower than wild strain,and the virulence of CE129Δhcp1 was slightly weaker. The results of serum bactericidal test show that, compared to the wild strain CE129, the serum complement sterilization resistanceability of CE129Δhcp1、CE129Δhcp2、 CE129Δhcp1Δhcp2 were lower by 32.7%,35.1% and 50.6% respectively (p<0.01). The results of qualitative and quantitative biofilm tests show that,the biofilmforming ability of CE129Δhcp2 decreased by 40% (p<0.01). DF1 cell adhesion test results show that the adhesion ability of deleted mutant strain CE129Δhcp2 decreased by 46% (p<0.01).The results of chicken embryos infection experiment in vivo showed that the proliferation speed of CE129 Δhcp1、CE129Δhcp2 is only about one-third of the wild strain, and the proliferation level of CE129Δhcp1Δhcp2 is about one-second of wild strain. Real Time quantitative PCR test results displayed that hcpi and hcp2 have a collaborative relationship. Compared with wild strain CE129, the expression level of luxS、 lsrR、pfs for quorum sensing of CE129Δhcp1 decreased by 29%,41% and 33% (p<0.05), and the expression level of ompA and iss for serum resistance decreased by 29% and 24% (p<0.05), respectively; the expression level of fimA,fimC and papC in CE129Δhcp2 reduced by 47%,60% and 37%(p<0.01), respectively, and the expression level of ompA and iss for serum resistance reduced by 31% and 41% (p<0.05), respectively. Above all, gene hcp2 plays an important role in bacterial adhesion, biofilm formation and the process of serum sterilization, and gene hcpi plays an important role in bacteria competition in vitro, quorum sensing and complement survival, which proved that the Hcp mediated the pathogenic process of the APEC CE129 directly and indirectly.