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

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.

Efflux Pump Inhibitors against Nontuberculous Mycobacteria

“…The presence of two other putative MmpS5/MmpL5-coding operons in M. abscessus may represent a resistance mechanism through efflux of further antibiotics with anti-M. abscessus activity. Moreover, the presence of multiple efflux pumps of the MmpL family that participate to low-resistance levels to clofazimine and bedaquiline was reported [25]. In addition, an association between overexpression of the MFS pumps MAB_3142 (homologue of M. tuberculosis P55) and MAB_1409 (homologue of M. avium MAV_1406) and clarithromycin resistance was demonstrated in all the three subspecies of M. abscessus [33].…”

“…The presence of several EPs has also been showed in NTM [16]. The best characterized EPs in NTM are those present in M. fortuitum conferring resistance to tetracycline and aminoglycoside [20,21], those present in Mycobacterium smegmatis conferring resistance to fluoroquinolones, rifamycins, and isoniazid [22,23], those present in MAC conferring macrolide resistance [18,24], and those present in M. abscessus conferring resistance to clofazimine and bedaquiline [25]. Overexpression or the increased activity of existing EPs in response to prolonged exposure to the subeffective levels of antimycobacterial drugs, such as may be present during management of the NTM-infected patient, may render an organism increasingly resistant to one or more drugs employed in therapy [26].…”

Differentialin vitroactivity of individual drugs and bedaquiline-rifabutin combinations against actively multiplying and nutrient-starvedMycobacterium abscessus

“…If bedaquiline induces a similar transcriptional response in M. abscessus, it is possible that co-exposure to rifabutin could inhibit this response, thus rendering the bacterial population more vulnerable to ATP depletion and killing by bedaquiline. Likewise, rifabutin may also suppress expression of genes encoding efflux pumps, such as MmpS-MmpL pump systems which are known to be involved in the efflux of and resistance to bedaquiline in M. abscessus (58, 59).…”

Dissecting erm (41)-Mediated Macrolide-Inducible Resistance in Mycobacterium abscessus

“…Unmarked deletion of erm (41) in M. abscessus. The pUX1-katG-erm(41) was generated following the same strategy as previously described (13,14) using the primers listed in Table S2 and introduced into M. abscessus S. Selection of bacteria having undergone the first homologous recombination was done by screening for red fluorescent colonies on 7H10 OADC supplemented with 200 g/ml kanamycin. To promote the second homologous recombination event, the selected clones were subcultured overnight in 7H9 OADC in the absence of kanamycin.…”

“…afford low-level resistance to clofazimine and bedaquiline (13,14). Therapeutic treatments require prolonged courses of multiple antibiotics, usually combining a macrolide, a ␤-lactam (cefoxitin or imipenem), and an aminoglycoside (15).…”