Bacteriology
Construction of enterhemorrhagic Escherichia coli strain deleted for espO gene and analysis of its biological functions
Lei Qiaoling, Xue Juan, Pan Xing, Lyu Jun, Yang Jin, Zhu Ping, Meng Kun, Li Shan
Published 2021-02-28
Cite as Chin J Microbiol Immunol, 2021, 41(2): 88-96. DOI: 10.3760/cma.j.cn112309-20201006-00461
Abstract
ObjectiveTo analyze the effects of espO gene knockout on the biological characteristics of enterhemorrhagic Escherichia coli (EHEC).
MethodsTwo-step methods mediated by the suicide plasmid pCVD442-ΔespO and plasmid pTrc99a were used to construct the espO gene-deleted strain (ΔespO) and the complemented mutant (CΔespO), respectively. HeLa cells were infected with different EHEC strains to analyze the biological functions and lethal effects of espO gene during infection.
ResultsPCR, electrophoresis and gene sequencing showed that the Δ espO and CΔespO mutants were successfully constructed. Compared with the wild-type strain, neither the Δ espO nor CΔespO 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.
ConclusionsIn 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.
Key words:
Enterhemorrhagic Escherichia coli; EspO; Gene knockout; Cell death
Contributor Information
Lei Qiaoling
Institute of Infection and Immunity, Taihe Hospital Affiliated to Hubei University of Medicine, Shiyan 442000, China
Xue Juan
Institute of Infection and Immunity, Taihe Hospital Affiliated to Hubei University of Medicine, Shiyan 442000, China
Pan Xing
Institute of Infection and Immunity, Taihe Hospital Affiliated to Hubei University of Medicine, Shiyan 442000, China
College of Biomedicine and Health, Huazhong Agricultural University, Wuhan 430070, China
College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China
Lyu Jun
Institute of Infection and Immunity, Taihe Hospital Affiliated to Hubei University of Medicine, Shiyan 442000, China
Yang Jin
Institute of Infection and Immunity, Taihe Hospital Affiliated to Hubei University of Medicine, Shiyan 442000, China
Zhu Ping
Institute of Infection and Immunity, Taihe Hospital Affiliated to Hubei University of Medicine, Shiyan 442000, China
Meng Kun
Institute of Infection and Immunity, Taihe Hospital Affiliated to Hubei University of Medicine, Shiyan 442000, China
Li Shan
Institute of Infection and Immunity, Taihe Hospital Affiliated to Hubei University of Medicine, Shiyan 442000, China
College of Biomedicine and Health, Huazhong Agricultural University, Wuhan 430070, China
College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China
