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Mycobacterium abscessus is an emerging human pathogen causing severe pulmonary infections and is refractory to standard antibiotherapy, yet few drug resistance mechanisms have been reported in this organism. Recently, mutations in MAB_4384 leading to up-regulation of the MmpS5/MmpL5 efflux pump were linked to increased resistance to thiacetazone derivatives. Herein, the DNA-binding activity of MAB_4384 was investigated by electrophoretic mobility shift assays using the palindromic sequence IRS5/L5 located upstream of mmpS5/mmpL5. Introduction of point mutations within IRS5/L5 identified the sequence requirements for optimal binding of the regulator. Moreover, formation of the protein/IRS5/L5 complex was severely impaired for MAB_4384 harboring D14N or F57L substitutions. IRS5/L5/lacZ reporter fusions in M. abscessus demonstrated increased β-galactosidase activity either in strains lacking a functional MAB_4384 or in cultures treated with the TAC analogs. In addition, X-ray crystallography confirmed a typical TetR homodimeric structure of MAB_4384 and unraveled a putative ligand binding site in which the analogs could be docked. Overall, these results support drug recognition of the MAB_4384 TetR regulator, alleviating its binding to IRS5/L5 and steering up-regulation of MmpS5/MmpL5. This study provides new mechanistic and structural details of TetR-dependent regulatory mechanisms of efflux pumps and drug resistance in mycobacteria.

结核分枝杆菌（Mycobacterium abscessus）作为一种新兴的人体致病菌，能够引发严重的肺部感染，且对常规抗菌疗法具有抗药性，然而关于该菌的耐药机制报道甚少。近期研究表明，MAB_4384基因突变导致MmpS5/MmpL5外排泵的上调，进而增强了对抗硫乙酰胺衍生物的耐药性。本研究通过电泳迁移率变动分析，利用位于mmpS5/mmpL5上游的回文序列IRS5/L5，对MAB_4384的DNA结合活性进行了探究。在IRS5/L5中引入点突变，确定了调节因子最佳结合的序列要求。此外，携带D14N或F57L替换突变的MAB_4384，其蛋白/IRS5/L5复合物的形成受到严重抑制。在结核分枝杆菌中，IRS5/L5/lacZ报告基因融合体在缺乏功能MAB_4384的菌株或经TAC类似物处理的培养物中显示出β-半乳糖苷酶活性的增加。此外，X射线晶体学证实了MAB_4384典型的TetR同源二聚体结构，并揭示了潜在的配体结合位点，其中类似物可以对接。总体而言，这些结果支持了对MAB_4384 TetR调节因子的药物识别，减轻了其与IRS5/L5的结合，并引导MmpS5/MmpL5的上调。本研究为结核分枝杆菌中TetR依赖性外排泵调节机制和耐药性的分子机制提供了新的结构和功能细节。