Construction Of CompT, PompT Knockout Mutants Of In Avian Pathogenic E. Coli E058 And Evaluation Their Pathogenicity

Avian colibacillosis is a complex syndrome characterized by multiple organ lesions with airsacculitis and associated pericarditis, perihepatitis and peritonitis being most typical.Avian pathogenic Escherichia coli (APEC) infection is responsible for great economic losses to the poultry industry worldwide and there is increasing evidence of its potential zoonotic importance.OmpT, located in the outer membrane of Escherichia coli, which belongs to the omptin family of proteases in Gram-negative bacteria. OmpT can be encoded by chromosomal ompT gene (compT) in APEC, and also plasmid pAPEC-O2-ColV ompT gene (pompT). pompT exhibits 79% nucleotide sequence identity with compT. Some studies indicate that both compT and pompT in APEC isolates have high positive rates, were 62.5%and 78.5%, respectively.It is not clear whether ompTs pay a role in pathogenicity of APEC, and whether both of them are involved in invasion and related pathogenesis mechanism of APEC. In order to demonstrate the relationship between compT, pompT and the pathogenicity of APEC and to reveal the pathogenesis mechanism based on cOmpT and pOmpT, compT and pompT knockout mutants of APEC strain E058 were constructed, and their biological characteristics were clarified. This panel may be helpful toward understanding the pathogenic mechanism of APEC and controling avian collibacillosis.Through OmpT known active site, we generated site-directed mutants and with different sizes of ORFs residual in compT or pompT of APEC E058 strain. Using λ Red system to construct E058△50%compT::cat and E058△100%compT::cat, compT was amplified and subcloned into pMD(?)19-T and then the cat, pMD-T-compT fragments was ligated to form pMD-T-compT::cat. And the amplified 50%compT-cat,100%compT-cat fragments was electroporated into E058 to form mutants. Using CRISPR/Cas construction of site-directed mutants we amplified compTD97H, compTS99I, pompTD97H, pompTS99I fragments by OverLap PCR, then electroporated into E058△50%compT and E058A50%pompT, respectively to form E058compTD97H, E058compTS99I, E058pompTD97H and E058pompTS99I site-directed mutants. The growth curves of eight mutants mentioned above were similar to that of their parental strain E058 in LB broth. The protamine inhibition test result indicated that cOmpT, which encoded by compT has the activity of hydrolysis of protamine. RT-PCR result exhibited that the mRNA expression of compT and pompT in E058 were no significant difference. Then, the complete ORF compT fragment with its promoter region and the complete ORF pompT fragment also with compT promoter region were cloned into pACYC184 plasmid, respectively. Plasmids mentioned above were transformed into E058△100%compT::cat mutant to obtain the complementary strain ReE058△100%compT::catl, ReE058△100%compT::cat2, respectively. Protamine inhibition test results showed that two revertant strains were all recovered with protamine inactivation at an equal level compared to their parental strain. In a model of colonization and persistence in 35-day-old specific pathogen free (SPF), the mutants E058△50%pompT::cat, E058△100%compT::cat were attenuated in vivo compared with their parental strain at 24 hours post-challenge. Further, RT-PCR results experienced that mRNA expression of two virulence factors encoding genes iucD and fimC in E058△50%compT::cat were significantly higher than in E058△100%compT::cat. Median lethal dose(LD50)test results showed that E058△50%pompT::cat was 10-times attenuated compared to its parent strain E058, meanwhile, pathogenicity of E058△50%pompT with cat-resistance gene deleted vs the mutant E058A50%pompT::cat was equal as its parent strain E058. To explore the possible reason,50%pompT-FRT gene fragment with pompT promoter was cloned into pACYC184 plasmid, and transformed into E058△50%pompT::cat to construct the complementary strain ReE058△50%pompT, based on LD50 determination, the virulence of the revertant strain was not complemented. Serum bactericidal assay result showed that all mutants mentioned above and wild-type strain are not sensitive to SPF chicken serum. The results of this study suggested that, (1) Outer membrane protein T (OmpT) encoded by the pompT gene located on plasmid ColV of avian pathogenic Escherichia coli (APEC) strain E058 was found unable to inactivate the protamine which is the main substrate of the outer membrane protease encoded by chromosomalompT gene of E. coli. (2) When half of the OmpT encoding gene of ColV plasmid origin (pompT) was knocked out from the APEC strain E058, the virulence of the mutant E058△50%pompT::cat declined at 10-fold level evaluated by 50% lethal dose determination assay compared to the wild-type strain E058. Meanwhile, whole pompT gene was knocked out from the strain E058, the virulence of the mutant E058△100%pompT::cat was as equal as that of the wild-type strain.