Construction And Characterization Of Enterobactin Associated Genes EntE, EntS, TolC Mutants Of Avian Pathogenic Escherichia Coli

Avian pathogenic Escherichia coli (APEC), a subgroup of extraintestinal pathogenic E. coli (ExPEC), enters through different routes including respiratory and genital tracts and causes various extraintestinal diseases collectively termed as colibacillosis in chickens, which are responsible for significant economic losses in the chicken industry, especially in countries such as Brazil, China, and the United States, where the poultry industry is highly developed.Iron is an essential component for Escherichia coli, but iron availability is very limited. Escherichia coli have to evolve various iron uptake mechanism to compete for iron from host. Enterobactin iron acquisition system is one of those. As iron is critical for survival and full virulence of pathogens in the host tissues, iron acquisition systems such as siderophores could constitute a potential target for the development of vaccines. The purpose of this study is to demonstrate the relationship between enterobactin associated gene entE, entS, tolC and the pathogenicity of APEC. It will lay the foundations for revealing the pathogenic mechanism and constructing attenuated vaccine strains.In this study, the mutants E058â–³entE, E05â–³entS, E058â–³talC, E058â–³entEAentS, and E058â–³entEâ–³entSAtolC were developed by using X Red recombinase system. The low copy plasmid pACYC184-talC carried its native promoter was electroporated into mutants to generate the revertant of E058â–³tolC and E058â–³entEâ–³entSâ–³tolC. RT-PCR analysis demonstrated that entE and tolC genes were not normally transcribed in the mutant strains, while the upstream gene and the downstream gene were not influenced.1-day-old SPF chicken lethal test showed that E058â–³tolC and E058â–³entEâ–³entSAtolC mutants significantly decreased the virulence compared with the E058 strain, while E058AentE, E058â–³entS, E058â–³entEâ–³entS mutants were not obviously. The colonization and persistence ability of mutants E058â–³tolC and E058AentEâ–³entSâ–³tolC to compete for growth in 5-week-old SPF chicken tissues with wild-type strains E058 was significantly reduced, meanwhile, the E058â–³entEAentSAtolC mutant significantly decreased the virulence compared with the E058â–³tolC strain. The growth kinetics of all mutants was similar to that of their parental strain E058 in LB broth medium. The growth kinetics of E058AentEAentSâ–³tolC mutant strain was significantly decreased in iron-restricted medium compared with the wild-type strain, while the other strains exhibited no growth defect in iron-restricted medium. But 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 except for E058â–³entS mutant in LB broth medium. But E058AentEAentSâ–³tolC mutant strain was moderate attenuated. HD-11 test showed that there was a significant increase of an invasion rate in the strain E058â–³entEAentSAtolC compared to that in the strain E058â–³talC. This phenomenon remains to be explained. These data indicated that gene tolC involved in enterobactin efflux across the outer membrane significantly decreased the virulence of the APEC, while enterobactin synthesis gene entE and gene entS involved in enterobactin efflux across the inner membrane were unable to contribute to APEC virulence. Since E058â–³entEAentSâ–³tolC mutant revealed growth defection, it may cause pathogenicity decrease in vivo.