Atorvastatin增加结核分枝杆菌对乙胺丁醇敏感性作用和机制研究

Tuberculosis treatment has been facing the challenge from drug resistance, especially multidrug resistance. It is thus in urgent need to develop new drugs and therapeutic regimens. In the treatment of infection caused by bacteria, the combined usage of antibiotics and sensitizing agent is one important approach and direction of research. Because 60% dry weight of the cell wall of Mycobacterium tuberculosis（M. tuberculosis） is lipid, the hypolipidemic drug atorvastatin that affects the lipid metabolism has the potential to become the drug that targets the flotillin of M. tuberculosis. We found that although atorvastatin alone could not kill or inhibit the growth of M. tuberculosis, the combined usage of atorvastatin could significantly improve the efficient of ethambutol. What is more the combination could obviously reduce drug resistant level of the muti-drug resistant M. tuberculosis to ethambutol but not rifampin. Based on these results, we hypothesized that atorvastatin enhances the sensitivity of M.tuberculosis to ethambutol via affecting the flotillin structure in the cell wall of M. tuberculosis. We aim to characterize the optimal portion between atorvastatin and ethambutol. Then the underlying mechanism of synergistic effects of atorvastatin will be clarified by testing the integrity of the cell wall, the efficacy of efflux pump and the level of ROS under the pressure of atorvastatin. Finally we will use the M. tuberculosis transposon library and RNAseq to find out the potential targets of atorvastatin in M. tuberculosis, providing scientific evidence for the development of sensitizer.

耐药尤其是耐多药是当前结核病治疗最严重的挑战，亟需研发新药物及治疗方案；而联用抗菌增敏剂是治疗细菌感染重要途径及研发方向。结核菌细胞壁干重的60%为脂质，因此影响脂类代谢的药物阿伐他汀有成为针对结核菌细胞壁脂筏药物的潜力。我们前期研究发现，单独使用阿伐他汀不能抑制结核菌的生长，但是联合使用阿伐他汀可以显著增加结核菌对靶向细胞壁抗结核药物乙胺丁醇的敏感性；同时，显著降低耐多药结核菌对乙胺丁醇的耐药程度，但不影响非靶向细胞壁药物利福平的抗菌活性。基于此，我们提出阿伐他汀通过影响结核菌细胞壁表面脂筏，增加结核菌对乙胺丁醇敏感性的假说。本项目拟在前期基础上，筛选出最佳联合用药比例，通过检测阿伐他汀压力下结核菌细胞壁完整性，外排泵活性，ROS反应程度，明确阿伐他汀增强乙胺丁醇抗结核菌能力的作用机制；并通过在阿伐他汀压力下的转座子测序及RNAseq找出其在结核菌中的作用靶点，为增敏剂开发奠定基础。

全球传染病头号杀手的位置虽然曾被新型冠状病毒COVID-19取代，但结合分枝杆菌仍然是非常重要人类致病菌，特别是耐药结核分枝杆菌导致的结核病仍然是人类健康的主要杀手。本研究发现阿伐他汀处理能增强乙胺丁醇对结核分枝杆菌的治疗效果，而且能增加乙胺丁醇对滞留菌处理效果，特别是阿伐他汀能逆转对乙胺丁醇耐药的结核分枝杆菌对乙胺丁醇的敏感性，使其恢复对乙胺丁醇的敏感性，为本研究的主要发现之一。同时本研究探索了不同浓度比例阿伐他汀与乙胺丁醇处理分枝杆菌，筛选出最适的组合比例。为了解析阿伐他汀的作用机制，本研究发现阿伐他汀影响了结核分枝杆菌细胞壁，特别是细胞壁中脂质成分的组成，但未显著改变药物进入菌体及在菌体内蓄积的能力。为了明确阿伐他汀增强乙胺丁醇抗结核菌能力的作用机制，本研究通过同位素代谢流进一步解析细胞壁的具体变化，发现阿伐他汀通过改变细胞壁成分PI（乙胺丁醇作用靶点LAM的上游）从而增强了乙胺丁醇对抑制结核分枝杆菌的效果。该发现较好的解释了阿伐他汀与乙胺丁醇的协同作用。本研究继续通过了转录组分析和biotin探针标记的阿伐他汀探针pull down结核分枝杆菌蛋白明确了阿伐他汀的作用靶点。