Construction of enterhemorrhagic Escherichia coli strain deleted for espO gene and analysis of its biological functions
10.3760/cma.j.cn112309-20201006-00461
- VernacularTitle:肠出血性大肠埃希菌 espO基因敲除菌株的构建及其生物学功能初步研究
- Author:
Qiaoling LEI
;
Juan XUE
;
Xing PAN
;
Jun LYU
;
Jin YANG
;
Ping ZHU
;
Kun MENG
;
Shan LI
- From:
Chinese Journal of Microbiology and Immunology
2021;41(2):88-96
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To analyze the effects of espO gene knockout on the biological characteristics of enterhemorrhagic Escherichia coli (EHEC). Methods:Two-step methods mediated by the suicide plasmid pCVD442-Δ espO and plasmid pTrc99a were used to construct the espO gene-deleted strain (Δ espO) and the complemented mutant (CΔ espO), respectively. HeLa cells were infected with different EHEC strains to analyze the biological functions and lethal effects of espO gene during infection. Results:PCR, electrophoresis and gene sequencing showed that the Δ espO and CΔ espO mutants were successfully constructed. Compared with the wild-type strain, neither the Δ espO nor CΔ espO mutant showed significant difference in growth rate, indicating that the espO gene had no influence on the growth and replication of EHEC. Furthermore, EspO could activate the tumor necrosis factor receptor (TNF)-induced NF-κB signaling pathway, while the effector protein NleB could inhibit the process. EspO could not inhibit the death of HeLa cells induced by TNF or TNF-related apoptosis-inducing ligand (TRAIL) after EHEC infection. Conclusions:In this study, we successfully constructed the espO gene-deleted and complemented mutants of EHEC and preliminarily analyzed the interaction between espO gene and host cells and the effects of espO gene on cell apoptosis during infection, which provided reference for further research on the in vitro biochemical activity and in vivo pathogenic roles of EspO.
