Multi-drug resistant tuberculosis (MDR-TB) represents a major health problem worldwide. Drug efflux and the activity of efflux transporters likely play important roles in the development of drug-tolerant and drug-resistant mycobacterial phenotypes. This study is focused on the action of a mycobacterial efflux pump as a mechanism of drug resistance. Previous studies demonstrated up-regulation of the TetR-like transcriptional regulator MSMEG_3765 in Mycobacterium smegmatis and its ortholog Rv1685c in Mycobacterium tuberculosis (Mtb) in acid-nitrosative stress conditions. MSMEG-3765 regulates the expression of the MSMEG_3762/63/65 operon, and of the orthologous region in Mtb (Rv1687c/86c/85c). MSMEG-3762 and Rv1687c are annotated as ATP-binding proteins, while MSMEG-3763 and Rv1686c are annotated as trans-membrane polypeptides, defining an ABC efflux pump in both M. smegmatis and Mtb. The two putative efflux systems share a high percentage of identity. To examine the role of the putative efflux system MSMEG-3762/63, we constructed and characterized a MSMEG-3763 deletion mutant in M. smegmatis (∆MSMEG_3763). By comparative analysis of wild type, knockout, and complemented strains, together with structural modeling and molecular docking bioinformatics analyses of the MSMEG-3763 trans-membrane protein, we define the protein complex MSMEG-3762/63 as an efflux pump. Moreover, we demonstrate involvement of this pump in biofilm development and in the extrusion of rifampicin and ciprofloxacin (CIP), antimicrobial drugs used in first- and second-line anti-TB therapies.

耐多药结核病（MDR-TB）代表了全球主要的健康问题。药物外排和外向转运蛋白的活性可能在耐药和耐药分枝杆菌表型的发展中起重要作用。这项研究的重点是作为耐药机制的分枝杆菌外排泵的作用。先前的研究表明TetR样转录调节因子的上调MSMEG_3765 在 耻垢分枝杆菌 及其直系同源物 Rv1685c 在 结核分枝杆菌 （山地车）在酸性亚硝化胁迫条件下。MSMEG-3765调节MSMEG_3762 / 63/65 操纵子，以及位于 山地车 （RV1687C / 86C / 85C）。MSMEG-3762和Rv1687c标注为ATP结合蛋白，而MSMEG-3763和Rv1686c标注为跨膜多肽，在两者中均定义了ABC外排泵耻垢分枝杆菌 和 山地车。这两个假定的外排系统具有很高的同一性百分比。为了检查推定外排系统MSMEG-3762 / 63的作用，我们构建并表征了MSMEG-3763 缺失突变体 耻垢分枝杆菌 （ΔMSMEG_3763）。通过对野生型，敲除和互补菌株的比较分析，以及对MSMEG-3763跨膜蛋白的结构建模和分子对接生物信息学分析，我们将蛋白复合物MSMEG-3762 / 63定义为外排泵。此外，我们证明了该泵参与了生物膜的开发以及利福平和环丙沙星（CIP）的挤出，利福平和环丙沙星（一线和二线抗结核疗法中使用的抗菌药物）。