大肠杆菌机械敏感性离子通道MscS失活特性分析  

Analysis of inactivation characteristics of Escherichia coli mechanosensitive ion channel MscS

作　　者：王晓敏 张怡 吕梦丹 王志坚[1] 应瑾瑶 殷乐依 吴宇涛 刘胜兵[1] 敖雷[1] 郑永霞[1] 徐营[1] 潘巍巍[1] 李月舟 WANG Xiaomin;ZHANG Yi;LV Mengdan;WANG Zhijian;YING Jinyao;YIN Leyi;WU Yutao;LIU Shengbing;AO Lei;ZHENG Yongxia;XU Ying;PAN Weiwei;LI Yuezhou(College of Medicine,Jiaxing University,Jiaxing 314000,Zhejiang,China;The Children's Hospital,Zhejiang University School of Medicine,Hangzhou 310000,Zhejiang,China)

机构地区：[1]嘉兴学院医学院,浙江嘉兴314000 [2]浙江大学医学院附属儿童医院,浙江杭州310000

出　　处：《微生物学报》2022年第9期3529-3541,共13页Acta Microbiologica Sinica

基　　金：科技部重点研发计划(2018YFE0112900);嘉兴市科技计划(2020AD10018);嘉兴学院科研启动项目(CD70519018);嘉兴学院大学生SRT项目(CD8517211489);国家自然科学基金(31871402);浙江省基础公益研究计划(LY21H160047,LGD22H160004);国家大学生创新训练计划(202113291002);浙江省新苗项目(2021R417028)。

摘　　要：【目的】细菌机械敏感性离子通道MscS能够在细菌周围环境渗透压急剧降低时,打开并释放胞内内容物,平衡内外渗透压差,使细菌存活。鉴于其广泛分布在各种细菌中,而在哺乳动物中未发现其同源体,MscS被认为是一种新型抗生素靶点。MscS一个独特的开放特征是具有失活特性,即在持续的机械刺激条件下,MscS从开放状态进入一种非离子通透的失活状态,从而避免因通道持续开放引起大量内容物流失导致细菌死亡。该研究的目的是鉴定影响MscS失活的关键氨基酸,为靶向Msc S的药物设计提供思路。【方法】采用分子克隆方法制备Msc S Cyto-helix(P166−I170)半胱氨酸突变体,利用巯基化合物MTSET^(+)结合半胱氨酸从而对其侧链基团进行修饰,并通过低渗刺激实验,检测表达MscS半胱氨酸突变体的大肠杆菌分别在无或有MTSET^(+)处理下,低渗刺激诱发通道开放后的存活率筛选显著影响通道功能的突变体。利用电生理膜片钳方法检测突变体在MTSET^(+)处理前后通道失活特性的变化,结合定点突变手段进一步探讨失活机制。【结果】MTSET^(+)处理导致表达半胱氨酸突变体G168C-MscS的大肠杆菌在低渗刺激后存活率极大降低;G168C-MscS在结合MTSET^(+)后失去失活特性,保持持续开放,是导致细菌胞内内容物大量流失并死亡的重要原因;酪氨酸突变G168Y-MscS、亮氨酸突变G168L-MscS和赖氨酸突变G168K-MscS的失活特性与野生型WT-MscS一致,而天冬氨酸突变G168D、缬氨酸突变G168V和异亮氨酸突变G168I的失活速率显著降低,尤其是G168I-MscS失去失活特性,表明MscS 168位点是影响通道失活的关键位点,并且通道失活特性与该位点氨基酸侧链基团的大小及电荷性质相关。【结论】G168位点甘氨酸是影响MscS通道失活的关键氨基酸。[Objective]The mechanosensitive channel of small conductance(MscS)in bacteria releases solutes and water when a hypo-osmotic shock raises the pressure in the cells,thereby enabling the survival of bacteria.Given its wide distribution in various bacteria and no homologues found in mammals,MscS is considered a novel antibiotic target.A hallmark of MscS is that it enters a tension-insensitive inactivated state upon prolonged mechanical stimulation,thereby avoiding the loss of a large amount of cell content and preventing cell death.This study aims to identify the key residues related to the inactivation of MscS,which is expected to serve as a reference for the development of MscS-targeting drugs.[Methods]The cysteine mutants of MscS Cyto-helix(P166−I170)were prepared with molecular cloning method.The thiol compound MTSET^(+)binds to cysteine and thus modify cysteine’s side chain group.In this study,osmotic downshock assay was used to examine the viability of Escherichia coli expressing cysteine mutants of MscS Cyto-helix(P166−I170)upon hypotonic stimulation without or with MTSET^(+)treatment and screened for cysteine mutant that significantly affected the channel function.The inactivation of MscS mutants before and after MTSET^(+)treatment was examined by electrophysiological experiments.In addition,the inactivation mechanism of MscS was further explored by eletrophysiology combined with site-directed mutagenesis.[Results]MTSET^(+)led to a great decrease in the survival rate of E.coli expressing G168C-MscS upon hypotonic stimulation.G168C-MscS lost its inactivation property after binding to MTSET^(+)and remained open,resulting in great loss of intracellular contents and bacterial death.The inactivation properties of G168Y-MscS,G168L-MscS,and G168K-MscS mutants were consistent with WT-MscS,while the inactivation rates of the three mutants G168D,G168V,and G168I were significantly reduced,especially G168I-MscS which lost the inactivation properties.Therefore,MscS G168 affected channel inactivation,and the channel

关 键 词：机械敏感性离子通道 MSCS 大肠杆菌 低渗刺激 膜片钳 

分 类 号：Q935[生物学—微生物学]