| Escherichia coli, which are known to colonize the intestinal tract with no harmful effects or cause of systemic infections in human and animal hosts. While most of these strains are non-pathogenic E.coli, certain few pathogenic strains can cause of intestinal or extraintestinal infection. The pathogenic E.coli can be classified into two groups:intestinal pathogenic E.coli and extraintestinal pathogenic E.coli (ExPEC). Avian pathogenic Escherichia coli (APEC) strains cause one of the most significant extraintestinal infections which take many forms and are collectively termed colibacillosis.Such strains are responsible for serious economic losses to the poultry industry. Today, many pathogenic factors have been found in APEC,and the new virulence factors are constantly being found. Inactivating the target genes is the primary method for studying gene function.Antioxidant defense system is a protective mechanism to protect the bacteria from the oxidative damage caused by metabolic processes, ahpF gene is a member of the system. It works with ahpC to catalyze the endogenous H2O2into the corresponding alcohols and H2O, removing the H2O2 produced in the metabolic process, in order to reduce the damage to the bacteria. Another gene yfgC has a close relationship with the outer membrane protein of bacteria, which can degrade the outer membrane protein, and it plays an important role in the integrity of outer membrane proteins. However, the specific function of yfgC remains unclear. The purpose of this study is to demonstrate the relationship between gene ahpF, yfgC and the pathogenicity of APEC. It will lay the foundations for revealing the pathogenesis mechanism and constructing attenuated vaccine strains.In this study, the mutants E058△ahpF-1, E058△ahpF-2, E058△ahpF-3, E058△yfgC-1, E058△yfgC-2, E058△yfgC-3 were developed by using X Red recombinase system and CRISPR/Cas9 system. The low copy plasmid pACYC184-ahpF and pACYC184-yfgC carried its native promoter was electroporated into mutants to generate the revertant of E058△ahF-1 and E058△yfgC-1. RT-PCR analysis demonstrated that E058AahpF-1, E058△ahpF-2, E058△ahpF-3, E058△yfgC-1, E058△yfgC-2 were not normally transcribed in the mutant strains, while the upstream gene and the downstream gene were not influenced.But in the mutant E058AyfgC-3,an unexpected transcriptome composed of disrupted yfgC.LD50 test showed that E058△yfgC-1, E058△yfgC-2 and E058△yfgC-3 mutants significantly decreased the virulence compared with their parental E058 strain, while E058△ahpF-1, E058△ahpF-2 and E058△ahpF-3 mutants were not obviously attenuated. The colonization and persistence ability of mutants E058△yfgC-1, E058△yfg-2, E058△yfgC-3 to compete for growth in 5-week-old SPF chicken tissues with wild-type strain E058 was significantly reduced. The growth kinetics of all mutants was similar to that of their parental strain E058 in LB broth medium. The growth kinetics of E058△yfg-1, E058△yfgC-2 and E058△yfgC-3 mutant strain were significantly decreased in the medium supplemented with H2O2 compared with the wild-type strain, while E058△ahpF-1, E058△ahpF-2 and E058△ahpF-3 was slightly increased in the medium. Compared with wild-type strain, all the mutants were similar in serum bactericidal test. In vitro competition test showed that all mutants were slightly attenuated in LB broth medium. This phenomenon remains to be explained. These data indicated that deletion of gene yfgC significantly decreased the virulence of the APEC, while gene ahpF was not contributed to the APEC virulence. Comparing of the two methods for APEC mutant construction, λ Red recombinase system and CRISPR/Cas system, the corresponding mutants created with these two systems showed identical biological characteristics, including pathogenity of the same mutant constructed with either of these two systems, except that CRISPR system is more practical and convenient than the X. Red recombinase system during mutant construction. |
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