乙胺丁醇与异烟肼协同对耻垢分枝杆菌生长的影响研究

Ethambutol and isoniazid influence the growth of Mycobacterium smegmatis

摘要:

【目的】异烟肼(isoniazid, INH)和乙胺丁醇(ethambutol, EMB)是治疗结核病(tuberculosis, TB)的2种主要一线药物，虽然这2种药在临床上的联合使用有效地遏制了结核病的蔓延，但是它们之间潜在的协同作用机制却十分不清楚。【方法】本项目以耻垢分枝杆菌为模式菌株，首先，克隆Ms0606基因并以异丙基β-d-1-硫代吡喃半乳糖苷(isopropyl β-d-1-thiogalactopyranoside, IPTG)诱导蛋白质的表达，再采用等温滴定量热法(isothermal titration calorimetry, ITC)证实Ms0606蛋白与EMB存在相互作用，进一步通过凝胶迁移实验(electrophoretic mobility shift assay, EMSA)证明EMB对Ms0606的DNA结合活性的影响，然后检测基因Ms0606超表达菌株在INH及INH+EMB处理下细菌的生长曲线。【结果】最终纯化得到纯度较高的Ms0606蛋白，EMB与Ms0606以1:1的比例结合，并且EMB能特异性地增强Ms0606的DNA结合活性，在非致死剂量EMB存在下，Ms0606过量表达增强了INH对M. smegmatis MC²155的杀菌能力。【结论】发现由Ms0606编码的TetR家族转录调控因子，在2种药物协同作用中发挥作用，进一步研究将有助于我们对细菌抗药性调控机制的了解。

Abstract:

[Objective] Isoniazid (INH) and ethambutol (EMB) are the two main first-line drugs for the treatment of tuberculosis (TB), while the underlying synergistic mechanism remains unclear. [Methods] The Ms0606 gene was cloned from Mycobacterium smegmatis and its expression was induced by the addition of 0.5 mmol/L isopropyl β-d-1-thiogalactopyranoside (IPTG) at 37 °C for 4 h. The interaction between Ms0606 protein and EMB was studied by isothermal titration calorimetry (ITC). The effect of EMB on the DNA binding activity of Ms0606 was examined by electrophoretic mobility shift assay (EMSA). The growth curves of the Ms0606-overexpressing strain treated with INH and INH+EMB were established. [Results] Ms0606 protein was purified with high purity. EMB can bind with Ms0606 at a ratio of 1:1 and specifically enhance the DNA binding activity of Ms0606. In the presence of a non-lethal dose of EMB, the overexpression of Ms0606 enhanced the effect of INH on M. smegmatis MC²155. [Conclusion] The TetR family transcriptional regulator encoded by Ms0606 plays a role in the synergistic effect of EMB and INH, the further research on which will help us to understand the regulatory mechanism of bacterial resistance.