Mechanistic and Structural Insights Into the Unique TetR-Dependent Regulation of a Drug Efflux Pump in .

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 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 IR located upstream of . Introduction of point mutations within IR identified the sequence requirements for optimal binding of the regulator. Moreover, formation of the protein/IR complex was severely impaired for MAB_4384 harboring D14N or F57L substitutions. IR reporter fusions in 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 IR 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.