结核分枝杆菌膜蛋白MmpS5-MmpL5的表达及功能研究

Study of expression and function of Mycobacterium tuberculosis membrane protein MmpL5-MmpS5

目的:表达结核分枝杆菌膜蛋白MmpL5及MmpS5-MmpL5外排泵系统,探讨其在贝达喹啉耐药中的作用。方法:构建结核分枝杆菌外排泵蛋白MmpL5及MmpS5-MmpL5的乙酰胺诱导型表达载体,在耻垢分枝杆菌中诱导表达MmpL5蛋白及MmpS5-MmpL5蛋白,测定表达菌株的生长特性及其外排能力,同时探讨贝达喹啉对表达菌株最小抑菌浓度的变化与对菌体三磷酸腺苷(adenosine triphosphate,ATP)含量的影响。结果:成功在耻垢分枝杆菌中表达结核分枝杆菌膜蛋白MmpL5及MmpS5-MmpL5,表达菌株的生长不受影响,共表达MmpS5-MmpL5蛋白的菌株外排能力增加,导致其对溴化乙锭的积累量(△荧光强度为395)较表达MmpL5蛋白组(△荧光强度为655)和携带pMV261s空质粒组(△荧光强度为642)减少约40%。贝达喹啉对MmpS5-MmpL5重组耻垢分枝杆菌的最低抑菌浓度由0.25μg/ml提高到2μg/ml,增加了8倍。贝达喹啉将MmpS5-MmpL5重组耻垢分枝杆菌菌体内ATP含量[(0.197±0.018)μmol/L]降低36.49%[(0.125±0.010)μmol/L]。MmpL5单表达不能形成外排泵,也对贝达喹啉无影响。结论:结核分枝杆菌的膜蛋白MmpL5和MmpS5需要共表达才能在耻垢分枝杆菌中发挥作用,降低贝达喹啉的药物敏感性。MmpS5-MmpL5蛋白表达体系的建立为进行贝达喹啉的具体转运机制研究奠定了基础。

Objective:To express the Mycobacterium tuberculosis membrane protein MmpL5 and MmpS5-MmpL5 efflux pump system and explore its role in the drug resistance of bedaquiline.Methods:Induced expression of proteins MmpL5 and MmpS5-MmpL5 in Mycobacterium smegmatis were achieved based on the construction of acetamide-induced expression vector.The growth characteristics and efflux capacity of the expressed strains were measured,and the change of minimum inhibitory concentration(MIC)of bedaquiline on the expressed strain and its effect on the and content of adenosine triphosphate(ATP)were investigated.Results:MmpL5 and MmpS5-MmpL5 proteins of Mycobacterium tuberculosis were successfully expressed in Mycobacterium smegmatis.The growth of expressed strains was not affected by MmpL5 and MmpS5-MmpL5,but the efflux capacity of co-expressing MmpS5-MmpL5 strains increased.Compared with the MmpL5-expressing group(△Fluoresce intensity=655)and pMV261s carrying empty plasmid group(△Fluoresce intensity=642),the accumulation of ethidium bromide in co-expressing MmpS5-MmpL5(△Fluoresce intensity=395)was reduced by 40%.The MIC of bedaquiline to MmpS5-MmpL5 recombinant strain was increased 8-fold than the control(2μg/ml vs.0.25μg/ml).The content of ATP in MmpS5-MmpL5 recombinant Mycobacterium smegmatis((0.197±0.018)μmol/L)decreased by 36.49%((0.125±0.010)μmol/L)after bedaquiline treatment.Single expression of MmpL5 could not form efflux pump and had no effect on bedaquiline.Conclusion:Membrane protein MmpL5 and MmpS5 of Mycobacterium tuberculosis needed to be co-expressed to reduce the drug sensitivity of bedaquiline in Mycobacterium smegmatis.The establishment of the MmpS5-MmpL5 protein expression system laid the foundation for the study of the transport mechanism of bedaquiline.