结核分枝杆菌膜蛋白MmpS5/MmpL5与贝达喹啉的相互作用研究

doi:10.19982/j.issn.1000-6621.20240584

摘要/Abstract

摘要：

目的:表达和纯化结核分枝杆菌膜蛋白MmpS5和MmpL5,并检测二者与贝达喹啉(Bdq)之间的相互作用,探究MmpS5/MmpL5外排泵系统导致Bdq耐药的具体转运机制。方法:构建MmpS5和MmpL5蛋白的表达质粒,在HEK293F真核细胞和耻垢分枝杆菌中分别诱导表达纯化的MmpS5和MmpL5蛋白。采用差示扫描荧光法(differential scanning fluorimetry, DSF)和竞争性酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)检测Bdq与MmpS5和MmpL5蛋白之间的相互作用。结果:成功构建了MmpS5和MmpL5蛋白的表达质粒,并在HEK293F细胞中表达纯化MmpS5蛋白,在耻垢分枝杆菌中表达纯化MmpL5蛋白。DSF实验显示,随着Bdq浓度的增加,MmpS5的蛋白熔解温度(melting temperature,Tm)略有降低,而MmpL5的Tm明显上升,最大增幅达5.98℃,提示MmpL5蛋白与Bdq存在结合;进一步行竞争性ELISA检测,显示随着未标记Bdq浓度的增加,MmpL5的吸光度值显著降低,表明MmpL5与生物素标记的Bdq存在相互作用,且该相互作用可被未标记Bdq竞争性抑制。结论:在MmpS5/MmpL5外排泵系统中,MmpL5蛋白与Bdq存在直接相互作用,而MmpS5蛋白与Bdq无明显相互作用,提示MmpL5可能直接外排Bdq,而MmpS5蛋白主要发挥辅助作用,不直接参与外排Bdq。这为深入理解MmpS5/MmpL5外排泵系统在Bdq耐药性中的作用机制提供了重要的研究基础。

关键词: 分枝杆菌,结核, 膜蛋白质类, 药理作用分子作用机制, 贝达喹啉

Abstract:

Objective: To express and purify the Mycobacterium tuberculosis membrane proteins MmpS5 and MmpL5, investigate their interaction with bedaquiline (Bdq), and elucidate the specific transport mechanism of the MmpS5/MmpL5 efflux pump system in conferring Bdq resistance. Methods: Expression plasmids for MmpS5 and MmpL5 were constructed. MmpS5 and MmpL5 proteins were expressed and purified in HEK293F mammalian cells and Mycobacterium smegmatis, respectively. The interactions between Bdq and MmpS5/MmpL5 were assessed using differential scanning fluorimetry (DSF) and competitive enzyme-linked immunosorbent assay (ELISA). Results: The expression plasmids for MmpS5 and MmpL5 were successfully constructed. MmpS5 was purified in HEK293F cells, and MmpL5 was purified in Mycobacterium smegmatis. DSF results showed that as the concentration of Bdq increased, the melting temperature (Tm) of MmpS5 slightly decreased. Whereas the Tm of MmpL5 significantly increased, by up to 5.98 ℃, suggesting a binding interaction between MmpL5 and Bdq. Competitive ELISA further demonstrated that the absorbance of MmpL5 decreased significantly with increasing concentrations of unlabeled Bdq, indicating that MmpL5 interacts with biotin-labeled Bdq, and this interaction can be competitively inhibited by unlabeled Bdq. Conclusion: In the MmpS5/MmpL5 efflux pump system, MmpL5 directly interacts with Bdq, while MmpS5 shows no significant interaction. These findings suggest that MmpL5 may directly mediate the efflux of Bdq, whereas MmpS5 likely plays an auxiliary role without direct involvement in Bdq transport. This study provides a critical foundation for understanding the mechanistic role of the MmpS5/MmpL5 efflux pump in Bdq resistance.

Key words: Mycobacterium tuberculosis, Membrane proteins, Molecular mechanisms of pharmacological action, Bedaquiline

中图分类号:

郑壮彬, 毕利军, 张立群. 结核分枝杆菌膜蛋白MmpS5/MmpL5与贝达喹啉的相互作用研究[J]. 中国防痨杂志, 2025, 47(7): 884-892. doi: 10.19982/j.issn.1000-6621.20240584

Zheng Zhuangbin, Bi Lijun, Zhang Liqun. Study on the interaction between Mycobacterium tuberculosis membrane protein MmpS5/MmpL5 and bedaquiline[J]. Chinese Journal of Antituberculosis, 2025, 47(7): 884-892. doi: 10.19982/j.issn.1000-6621.20240584

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