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

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MsmEFG1(MSMEG_1400)和MsmEFG2(MSMEG_6535)，fusA1(Rv0684)和fusA2(Rv0120c)。基因突变库和生物信息学推测MsmEFG1(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。

Abstract:

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,MsmEFG1(MSMEG_1400) and MsmEFG2(MSMEG_6535),fusA1(Rv0684) and fusA2(Rv0120c),respectively.MsmEFG1(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.