Construction And Characterization Of OmpA、ompT Mutants Of Avian Pathogenic Escherichia Coli And OmpA、pgtE Mutants Of Salmonella Enteritidis

Avian colibacillosis is one of the most common infectious diseases caused entirely or partly by avian pathogenic Escherichia coli (APEC) in birds, and is often infected with other pathogens such as bacteria or viruses. So far, this disease has been an important bacteriosis and it is responsible for worldwide economic losses to the poultry industry.Salmonella enteritidis is a kind of pantropic salmonella and also important pathogens of humans and animals. It not only cause poultry diseases, resulting in systemic infections, diarrhea and gastroenteritis, moreover it is one of the main foodborne pathogenic bacteria, especially for eggs and poultry food contamination and it has great significance in public health.OmpA is a major protein of the gram-negative bacterial OMPs with important founction in the stability of the bacterial adventitia and receptors of phage Tu Ⅱ, and also act as adhesin or invasin associated with the virulence of the bacteria. OmpT (outer-membrane proteases T) is an important defense factor of gam-negative bacteria, especially for E. coli and Salmonella enteritidis. OmpT belongs to Omptin outer membrane protein family including Salmonella enteritidis PgtE protein, which has been proved to be an important virulence factor of Salmonella enteritidis. ompT encodes OmpT protease and ompA encodes OmpA protein. With high detection rate of urinary tract infections and meningitis in clinical isolates, both of ompA and ompT are putative virulence factor for pathogenic E. coli and Salmonella enteritidis. In order to demonstrate the relationship between ompA, ompT, pgtE gene and the pathogenicity of APEC strain E058and SE strain CVCC3378, and also to reveal the pathogenic mechanism and the biological characteristics of the mutants, the ompA, ompT and pgtE gene were chosen and mutated.In this study, the mutants E058ΔompA, E058ΔompT, CVCC3378ΔompA and CV CC3378ΔpgtE were developed, through the technology of allelic recombination. The plasmid pGEX-6P-1-ompA was electroporated into E058ΔompA to generate the revertant of E058ΔompA. Gene ompA was amplificated and subcloned into pMD18-T simple vector, and then the Zeor gene was cloned into ompA to form pT-ompA-Zeo. And the amplificated fragment ompA-Zeo was electroporated into E058with pKD46to form mutant E058ΔompA. Mutant E058ΔompT, CVCC3378ΔompA and CVCC3378ΔpgtE were conducted and selected through the same technology of allelic recombination.The results of RT-PCR demonstrated that the deletion of the gene ompA, ompT of E058and ompA, pgtE gene of CVCC3378was disrupted in the transcription of the target genes, while the upstream gene and the downstream gene of the target genes were expressed normally. The growth curves of E058AompA, Re-E058ΔompA, E058AompT, Re-E058ΔompT, CVCC3378AompA, Re-CVCC3378ΔompA, CVCC3378ApgtE and Re-CVCC3378ΔpgtE were similar to that of their parental strain E058and CVCC3378in LB broth. The result of ingestion assays of HD-11cell showed an inconspicuous difference of mutant strains compared with that of the parent strain E058and CVCC3378. The mutants were slightly out-competed by the wild-type strain in vitro. After24h post-challenge in vivo competition assay, the mutant E058ΔompA, E058ΔompT, CVCC3378ΔompA and CVCC3378ApgtE showed significant attenuation growth in blood, liver, spleen, lung and kidney. The capacity of the wild-type strain and its mutants to colonize internal organs was studied by determining colony numbers in selected organs24h after infection. Compared to those of wild-type strain E058and CVCC3378, the mutant E058ΔompA, E058ΔompT, CVCC3378ΔompA and CVCC3378ApgtE colony numbers were significantly decreased. While the complemented strain Re-E058ΔompA, Re-E058ΔompT, Re-CVCC3378ΔompA and Re-CVCC3378ApgtE showed significantly rescued virulence compared to the responding mutants.