Construction Of Neuc Mutant Of Avian Pathogenic Escherichia Coli And The Deletion Of Chlormycetin Resistance Gene Introduced By λ Red System From The Mutant And Evaluation Of Their Pathogenicity

Avian colibacillosis is one of the most common infectious diseases caused entirely or partly by avian pathogenic Escherichia coli (APEC) in birds, including E. coli granuloma, peritonitis, salpingitis, umbilical inflammation, synovitis, air sac inflammation, ophthalmia, yolk peritonitis and so on. It is responsible for worldwide economic losses to the poultry industry with the development of intensive poultry industry.Capsular polysaccharide is an important virulence factor of APEC, which can protect the pathogen from the host immune system clearence. The neuC gene is part of region2in capsule coding operon. Cells harboring mutations in neuC are defective in capsule synthesis.In this study, the mutants E058AneuC::cat、E05△neuC were developed, through the technology of allelic recombination. The plasmid pGEX-6P-1-nenc and pGEX-6P-1-neuC’ was electroporated into E058AneuC::cat to generate the revertant of E058△neuC::cat and named as Re-E058neuC\Re-E058△nenC::catAneuC’. Gene neuC was amplificated and subcloned into pMD(?)19-T simple vector, and then the chloramphenicol resistance gene was cloned into neuC to form pMD-neuC-Cat. And the amplificated fragment neuC-Cat was electroporated into E058with pKD46to form mutant E058AneuC::cat. Then the plasmid was electroporated into the mutant E058△neuC::cat through the same technology to form E058AneuC.The growth curves of E058AneuC::cat and E058△neuC were similar to that of their parental strain E058. In the reverse transcription-polymerase chain reaction (RT-PCR), The RT-PCR result of E058△nenC::cat demonstrated that the deletion of the gene neuC of E058disrupted only the transcription of the target gene, while the upstream gene and the downstream gene of the target genes transcribed normally. But in the mutant E058△neuC, an unexpected transcriptome(neuC’) composed of disrupted neuC, primers of the chlormycetin resistance gene and the FLP recognition target (FRT) sites was generated in the mutant E058△neuC while not in the mutant E058AneuC::cat. In12-day old embryonated lethality assay, the lethality rates of E058, E058AneuC::cat and E058△neuC were90.0%,83.3%and86.7%, respectively. The mutants all were slightly attenuated compared with the wild-type. In1-day old chicken lethality assay, the pathogenicity of mutants was also significantly decreased compared to that of the wild-type, the lethality rates of E058△neuC was increased compared to E058△neuC::cat. In the bactericidal assay in SPF chicken serum, the survival of E058△neuC::cat and E058△neuC was significantly decreased compared to that of parental strain E058(P<0.05); The invasion rate of E058AneuC::cat into chicken macrophage cell HD11was significantly higher than that of wild-type strain E058, while the invasion rate of the mutain E058△neuC was equal to that of wild-type strain. The colonization and persistence ability of mutants E058△neuC::cat and E058AneuC to compete for growth in5-week-old female SPF chicken tissues with wild-type strains E058was evaluated, the result showed that the mutants E058AneuC::cat and E058△neuC was highly attenuated in tested tissues (P<0.001or P<0.0001). The results suggested that neuC was crucial to the virulence of APEC E058. At the same time, we put the mutants E058AneuC::cat and E058△neuC togather, the colonies recovered from the liver, spleen, lung and kidney of birds challenged with E058AneuC were increased significantly compared to those from birds challenged with E058AneuC::cat (P<0.05or P<0.01), and the reverant with the unexpected transcriptome (neuC’) of the mutant E058AneuC::cat recovered the ability of colonization and persistence to the mutant E058△neuC.These results suggested that the enhanced colonization and persistence ability of E058△neuC compared to that of E058AneuC::cat was related to the production of the unexpected transcriptome neuC, and we are going to probe the molecular mechanism of the enhanced virulence of E058△neuC.