Construction And Characterization Of OmpT And CvaC Mutants Of Avian Pathogenic Escherichia Coli

Colibacillosis refers to any localized or systemic infection caused entirely or partly by avian pathogenic Escherichia coli (APEC), including septicaemia, granuloma (Hjarre’s disease), synovitis, air sac disease/chronic respiratory disease (CRD), peritonitis, salpingitis, avian cellulites, swollen head syndrome, panophthalmitis, and omphalitis/yolk sac infection. Collectively, infections caused by Escherichia coli (E. coli) are responsible for significant economic losses to the poultry industry in many parts of the world. ompT encodes OmpT protease, and cvaC is the structural gene for colicin V. Epidemiologic survey shows that ompT and cvaC are of high ratio in the virulence strains. In this study, the relationship between ompT gene or cvaC gene and the pathogenicity of APEC strain E058was studied. All the actual roles of the known virulence factors of APEC are not fully elucidated and certain steps of the infection process have not been related to previously identified factors. Furthermore, the mechanisms by which APEC cause infection are largely unknown.In this study, genes in APEC serogroup02strain E058chosen for deletion were ompT and cvaC. The fragments were amplified and subcloned to pMD(?)18-T simple vector. The vector T-ompT was inserted with a Zeor gene to form T-ompT-Zeo and T-cvaC was inserted with a Zeor gene to form T-cvaC-Zeo. The disrupted ompT and cvaC genes with Zeor gene inserted in T-ompT-Zeo and T-cvaC-Zeo were amplified by PCR and purified for electroporation to E058competent cells, the resulting Zeor colonies were selected and named as E058AompT, E058AcvaC, respectively.Southern blot showed single copy of Zeor gene was inserted into the plasmid DNA of APEC E058; RT-PCR showed that the insertion didn’t have a polar effect on the upstream or downstream gene of ompT or cvaC. None of the mutants deviated from the growth pattern of the wide-type strain E058in LB medium. It is not surprising that there are no differences in the virulence of mutants E058AompT and E058AcvaC compared to their parent strain E058in the embryo lethality together with the50%lethal doses (LD50), meanwhile, in the colonization and persistence test, the recovery colonies of E058AompT were significantly reduced in the spleen (p＜0.05), lung, heart, kidney (P＜0.01), and very significantly reduced in the liver (p＜0.001) of the challenged birds compared with those birds challenged with the wild-type strain E058. While compared to the parental strain E058, there was no difference in the tested tissues for the mutant E058△cvaC. ompT showed a strong contribution to the pathogenicity of APEC strain E058, while cvaC did not. The actual relationship between the cvaC gene and the pathogenicity of APEC E058needs further study.