Functional Characterization Of T6SS2 DotU And Development Of Multiplex PCR Assay For Detection Of Virulence Genes In Avian Pathogenic E. Coli

Avian pathogenic E. coli can cause parenteral disease of chickens, ducks and other birds, which manifested as diverse clinical symptoms, such as pericarditis, perihepatitis, aerosacculitis, peritonitis, septicemia and even encephalitis, etc. Avian colibacillosis is prevalent all over the world; act as an obstacle against the development of the poultry industry. It can cause meningitis of mice, which undoubtedly threat another mammal, human.Virulence factors are essential for the pathogenicity of APEC. Virulence factors have been reported in APEC including adhesins, invasins, antiserum survival factors, iron uptake system, vacuolating autotransporter toxin and other virulence factors.More proofs indicated that the APEC share virulence genes with human ExPEC strains. Thus, investigation and study of APEC virulence factors is not only necessary for poultry industry, but also for human health.The discovery of new virulence factors can contribute to the explaination of the pathogenesis of APEC. T6SS is a new found secretion system, which have virous roles in different host. It can acts as virulence factors, anti-pathogenesis factors, or regulator in interbacterial interactions. While function of T6SS in APEC remains unknow.In this study, we developed four multiplex PCR for the detection of 18 virulence genes in APEC, and examined the role of T6SS core protein DotU in APEC pathogenicity and protein secretion.1. Development of multiplex PCR assay for detection of virulence genes in avian pathogenic E. coliFor the efficient investigation of the virulence genes’distribution in avian pathogenic E. coli, we developed four multiplex PCR (mPCR) for the detection of adhesin-associated genes, invasin and toxin-associated genes, serum resistance-associated genes and iron acquisition-associated genes.18 pairs of specific primers were designed and synthesized according to gene sequences published in GenBank. Then the sensitivity of mPCRs was determined using diluted bacterial or DNA templates. Finaly, we verified the feasibility of these mPCRs through detecting the virulence genes in 100 APEC isolates with clear background. According to the results, we concluded that each of the 18 genes could be amplified in the mPCRs exactly and effectively.The sensitivities of these four mPCR were 103 colony forming units (CFU),103CFU,105CFU,105CFU bacteria and 1ng, 1ng, 10ng and 10ng DNA, respectively. In addition, the results of virulence genes distribution in 100 APEC isolates detected by mPCRs were same as the single PCR carried out before. In summury, mPCR developed in this study could efficiently detect the virulence genes of APEC, and its characteristics of time and labor saving compared to conventional PCR undoubtedly making it an efficient and applicable method for virulence genes’ identification and epidemiological investigation in APEC.2. Sequence analysis, distribution and expression of T6SS genes of APEC DE719T6SS is a new found secretion system in Gram-negative bacteria, its role in APEC remains unknown. Thus, the sequence of T6SS in DE719 was analyzed according to T6SS in NMEC RS218. Then we investigated the distribution of dotU in 75 APEC isolate. For further study, His-fusion protein DotU, Hcpl and Hcp2 were expressed and purified, then antisera was prepared in rabbits. Consequently, we found the T6SS gene cluster in DE719 is 23,033 bp, and 5 of 75 APEC isolates are positive for dotU. ORF of dotU、hcp1 and hcp2 were cloned into pET system plasmid, then transformed the recombinant plasmid into E. coli BL21 (DE3).The recombinant BL21 expressed fusion proteins after induced by IPTG. DotU-His, Hcpl-His and Hcp2-His is 32 kD,23 kD and 26 kD respectively, then obtained the anti-serum by immunized rabits with purified fusion protein. The results of indirect ELISA and Western Blot indicated the high quality of these anti-sera. Furthermore, we found anti-DotU antibodies in convalescent anti-DE719 serum, suggesting that DotU may play a role in the pathogenicity of DE719.3. Construction of mutant and complementary strains of APEC DE719 T6SS2 or dotU geneTo examine the role of the T6SS2 core component DotU in APEC pathogenesis, we knocked out dotU, hcp1, hcp2 and the whole gene cluster T6SS2 in DE719 respectively, using lambda red recombinase method. And the dotU complementary strain constructed as well. Several assays were carried out to characterize these strains.Consequently, deletion of dotU does not affect growth kinetics and swimming motility of APEC strain DE719, but resulted in the decreased colonization capacity in vitro and in vivo, defective virulence to duck, diminished resistance to environment stress and SPF serum, reduced intramacrophage survival. Furthermore, mutations of DotU, a structural component of T6SSs, also affect the Hcpl secretion by T6SS2, leading to decreased interleukin-6 (IL-6) and IL-8 gene expression in HD-11 cells.