The TetR-family transcription factor MAB_2299c regulates the expression of two distinct MmpS-MmpL efflux pumps involved in cross-resistance to clofazimine and bedaquiline in Mycobacterium abscessus

: Mycobacterium abscessus is a human pathogen responsible for severe respiratory infections, particularly in patients with underlying lung disorders. Notorious for being highly resistant to most antimicrobials, new therapeutic approaches are needed to successfully treat M. abscessus-infected patients. Clofazimine (CFZ) and bedaquiline (BDQ) are two antibiotics used for the treatment of multidrug-resistant tuberculosis and are considered alternatives for the treatment of M. abscessus pulmonary disease. To get insights into their mechanisms of resistance in M. abscessus, we previously characterized the TetR transcriptional regulator MAB_2299c, which controls expression of the MAB_2300-MAB_2301 genes, encoding an MmpS-MmpL efflux pump. Here, in silico studies identified a second mmpS-mmpL (MAB_1135c-MAB_1134c) target of MAB_2299c. A palindromic DNA sequence upstream of MAB_1135c, sharing strong homology with the one located upstream of MAB_2300, was found to form a complex with the MAB_2299c regulator in electrophoretic mobility shift assays. Deletion of MAB_1135c-1134c in a wild-type strain led to increased susceptibility to both CFZ and BDQ. In addition, deletion of these genes in a CFZ/BDQ-susceptible mutant lacking MAB_2299c as well as MAB_2300-MAB_2301 further exacerbated the sensitivity of this strain to both drugs in vitro and inside macrophages. Overall, these results indicate that MAB_1135c-1134c encodes a new MmpS-MmpL efflux pump system involved in the intrinsic resistance to CFZ and BDQ. They also support the view that MAB_2299c controls the expression of two separate MmpS-MmpL efflux pumps, substantiating the importance of MAB_2299c as a marker of resistance to be considered when assessing drug susceptibility in clinical isolates.