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  

Construction of enterhemorrhagic Escherichia coli strain deleted for espO gene and analysis of its biological functions

作　　者：雷巧玲 薛娟 潘兴 吕均 杨瑾 朱平 孟昆 李姗 Lei Qiaoling;Xue Juan;Pan Xing;Lyu Jun;Yang Jin;Zhu Ping;Meng Kun;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 Scierue and Technology,Huazhong Agricultural University,Wuhan 430070,China)

机构地区：[1]湖北医药学院附属太和医院,感染与免疫性疾病研究所,十堰442000 [2]华中农业大学生物医学中心,武汉430070 [3]华中农业大学生命科学技术学院,武汉430070

出　　处：《中华微生物学和免疫学杂志》2021年第2期88-96,共9页Chinese Journal of Microbiology and Immunology

基　　金：湖北省教育厅创新团队项目(T201713);湖北省教育厅科学研究计划指导性项目(B2020102)。

摘　　要：目的检测espO基因敲除对肠出血性大肠埃希菌(EHEC)生物学特性的影响。方法自杀质粒pCVD442-ΔespO介导的两步法构建espO基因敲除菌株(ΔespO),pTrc99a质粒构建espO基因回补突变株(CΔespO),不同EHEC菌株感染HeLa细胞以了解espO基因在感染过程中的生物学功能与致死作用。结果PCR和电泳以及基因测序结果表明,ΔespO和CΔespO突变株构建成功。与野生株相比,ΔespO和CΔespO突变株的生长速度无明显差异,表明espO基因不影响细菌的生长与繁殖。此外,EspO可激活肿瘤坏死因子(TNF)诱导的NF-κB信号通路,而效应蛋白NleB能够抑制该激活过程。在细菌感染细胞试验中,EspO不能抑制TNF和TRAIL(TNF-related apoptosis-inducing ligand)诱导的细胞死亡。结论本研究成功构建了EHEC espO基因敲除和回补突变株菌株,初步探究了espO与宿主细胞的相互作用及该基因在感染过程中对细胞凋亡的影响,为后续深入研究细菌EspO蛋白的体外生物学活性以及体内感染过程中的致病作用提供实验依据。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-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.Results PCR,electrophoresis and gene sequencing showed that theΔespO and CΔespO mutants were successfully constructed.Compared with the wild-type strain,neither theΔespO 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.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.

关 键 词：肠出血性大肠埃希菌 EspO 基因敲除 细胞死亡 

分 类 号：R378.21[医药卫生—病原生物学]