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

Abstract

<i>Mycobacterium abscessus</i> 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 <i>M. abscessus</i>-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 <i>M. abscessus</i> pulmonary disease. To get insights into their mechanisms of resistance in <i>M. abscessus</i>, we previously characterized the TetR transcriptional regulator MAB_2299c, which controls expression of the <i>MAB_2300-MAB_2301</i> genes, encoding an MmpS-MmpL efflux pump. Here, <i>in silico</i> studies identified a second <i>mmpS-mmpL</i> (<i>MAB_1135c-MAB_1134c</i>) target of MAB_2299c. A palindromic DNA sequence upstream of <i>MAB_1135c</i>, sharing strong homology with the one located upstream of <i>MAB_2300</i>, was found to form a complex with the MAB_2299c regulator in electrophoretic mobility shift assays. Deletion of <i>MAB_1135c-1134c</i> 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 <i>MAB_2299c</i> as well as <i>MAB_2300-MAB_2301</i> further exacerbated the sensitivity of this strain to both drugs <i>in vitro</i> and inside macrophages. Overall, these results indicate that <i>MAB_1135c-1134c</i> 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 <i>MAB_2299c</i> as a marker of resistance to be considered when assessing drug susceptibility in clinical isolates.