转录因子RV0678通过调控转运蛋白MmpS3/MmpL3表达介导脓肿分枝杆菌对克拉霉素耐药的机制

脓肿分枝杆菌感染治疗困难，克拉霉素是核心治疗药物，但耐药性的产生是临床难题。目前已知的克拉霉素耐药机制不能完全解释临床菌株的耐药现象。课题组前期研究发现，RV0678突变伴MmpS3/MmpL3表达异常升高特异地存在于部分已知机制无法解释的克拉霉素耐药株。MmpS3/MmpL3可以通过促进脂质合成加固细胞壁抵抗药物进入细胞，RV0678是MmpS/MmpL家族的抑制子。我们推测：野生型RV0678能够抑制MmpS3/MmpL3的表达；突变型RV0678的抑制子功能失活，克拉霉素作用下菌株MmpS3/MmpL3表达异常升高，导致细胞壁通透性降低，从而菌株产生耐药。本项目拟通过基因敲除及回补、组学测序、电镜、质谱分析、细胞壁通透性检测、凝胶迁移等实验，阐释MmpS3/MmpL3在脓肿分枝杆菌对克拉霉素耐药中的作用，明确RV0678对MmpS3/MmpL3的调控作用和调控机制，具理论及实用价值。

The treatment of Mycobacterium abscess (Mab) infection is challenging. Typically clarithromycin (CLA) plays a central role in the therapy, but the resistance is a serious clinical problem and cannot be fully explained by current known mechanisms. We previously screened the susceptibility and resistance mechanism of 162 Mab isolates to CLA, and discovered that RV0678 mutation with abnormal high expression of MmpS3/MmpL3 specifically existed in some CLA resistant strains that could not be explained by current known mechanisms. MmpS3/MmpL3 can reinforce cell wall by promoting lipid synthesis so as to resist agents into the cell. RV0678 is an inhibitor of partial MmpS/MmpL family transporters. Therefore, we hypothesized that wild-type RV0678 could inhibit the expression of MmpS3/MmpL3. When RV0678 was mutated, its inhibitory function was inactivated, the expression of MmpS3/MmpL3 was abnormally elevated when CLA was applied, resulting in decreased cell wall permeability and finally caused resistance to CLA. We will use gene knock out/in, transcriptome and proteome approaches, cell wall permeability assay, electron microscopy and mass spectrometry, and EMSA, to explore the function of RV0678 and MmpS3/MmpL3 in CLA resistance, elucidate the molecular mechanism of RV0678 and MmpS3/MmpL3 signaling. Outcome will not only improve our understanding in antibiotic resistance, but also reveal new targets for drug developing.