耻垢分枝杆菌EF-G敲低菌株的构建和耐药分析  

Construction of EF-G knockdown strain of Mycobacterium smegmatis and drug resistance analysis

作　　者：狄玉昌 白嘉诚 迟明哲 范伟兴[2] 张雪莲[1] DI Yuchang;BAI Jiacheng;CHI Mingzhe;FAN Weixing;ZHANG Xuelian(State Key Laboratory of Genetic Engineering,School of Life Sciences,Fudan University,Shanghai 200433,China;Laboratory of Zoonosis,China Animal Health and Epidemiology Center,Qingdao 266032,Shangdong,China)

机构地区：[1]复旦大学生命科学学院遗传工程国家重点实验室,上海200433 [2]中国动物卫生与流行病学中心,山东青岛266032

出　　处：《生物工程学报》2022年第3期1050-1060,共11页Chinese Journal of Biotechnology

基　　金：国家自然科学基金(81971898,81673482);国家重点研发计划(2016YFA0500601);新疆建设兵团重点科技攻关项目(2020AB015)。

摘　　要：GTPase延伸因子G(elongation factor G,EF-G)是蛋白质翻译过程中重要的翻译因子。作为唯一在翻译延伸和核糖体再生2个翻译环节发挥重要功能的翻译因子,EF-G成为潜在的抗菌药物作用靶点。耻垢分枝杆菌(Mycobacterium smegmetics,Msm)和结核分枝杆菌(Mycobacterium tuberculosis,Mtb)基因组中均存在2个EF-G同源编码基因,分别为Msm EFG1(MSMEG_1400)和Msm EFG2(MSMEG_6535),fusA1(Rv0684)和fusA2(Rv0120c)。基因突变库和生物信息学推测Msm EFG1(MSMEG_1400)和fusA1(Rv0684)是生长必需基因。为探究分枝杆菌中EF-G的生物学功能及特点,利用成簇的规律间隔的短回文重复序列干扰(clustered regularly interspaced short palindromic repeats interference,CRISPRi)技术构建了耻垢分枝杆菌中2个EF-G诱导型敲低菌株(Msm-ΔEFG1(KD)和Msm-ΔEFG2(KD)),研究发现EF-G2的敲低对细菌生长无影响,而EF-G1的敲低显著影响分枝杆菌的生长,成膜能力显著减弱、菌落形态显著变化、菌体长度显著增长,推测EF-G可能与细菌的分裂相关。最低抑菌浓度(minimal inhibitory concentration,MIC)实验结果表明,抑制EF-G1的表达可增强分枝杆菌对利福平、异烟肼、红霉素、夫西地酸、卷曲霉素等抗菌药物的敏感性,提示EF-G1可能成为未来抗结核药物筛选的潜在靶标,为探究EF-G在分枝杆菌中的生理功能及作为潜在药物靶标提供基础。As the only translational factor that plays a critical role in two translational processes(elongation and ribosome regeneration),GTPase elongation factor G(EF-G)is a potential target for antimicrobial agents.Both Mycobacterium smegmatis and Mycobacterium tuberculosis have two EF-G homologous coding genes,Msm EFG1(MSMEG_1400)and Msm EFG2(MSMEG_6535),fusA1(Rv0684)and fusA2(Rv0120 c),respectively.Msm EFG1(MSMEG_1400)and fusA1(Rv0684)were identified as essential genes for bacterial growth by gene mutation library and bioinformatic analysis.To investigate the biological function and characteristics of EF-G in mycobacterium,two induced EF-G knockdown strains(Msm-ΔEFG1(KD)and Msm-ΔEFG2(KD))from Mycobacterium smegmatis were constructed by clustered regularly interspaced short palindromic repeats interference(CRISPRi)technique.EF-G2 knockdown had no effect on bacterial growth,while EF-G1 knockdown significantly retarded the growth of mycobacterium,weakened the film-forming ability,changed the colony morphology,and increased the length of mycobacterium.It was speculated that EF-G might be involved in the division of bacteria.Minimal inhibitory concentration assay showed that inhibition of EF-G1 expression enhanced the sensitivity of mycobacterium to rifampicin,isoniazid,erythromycin,fucidic acid,capreomycin and other antibacterial agents,suggesting that EF-G1 might be a potential target for screening anti-tuberculosis drugs in the future.

关 键 词：耻垢分枝杆菌 EF-G 成簇规律间隔短回文重复序列干扰(CRISPRi) 最小抑菌浓度 耐药 

分 类 号：R378.91[医药卫生—病原生物学]