AX-35 Targets the Cytochrome bc1 Complex in Mycobacterium abscessus and Reveals a Novel Resistance Mechanism

Abstract In patients with cystic fibrosis, nontuberculous mycobacteria are opportunistic, intracellular and extracellular pulmonary pathogens that contribute to deterioration of lung function and, ultimately, morbidity from respiratory failure. Among nontuberculous mycobacterial respiratory pathogens, members of the Mycobacterium abscessus complex pose additional treatment complications as they are notoriously drug-resistant. As part of our routine screening against mycobacterial species, one of the lead antituberculosis compounds, the cytochrome bc1 inhibitor AX-35, was found to be active against isolates from the Mycobacterium abscessus complex in vitro (MIC ≤ 15 μg/mL) and ex vivo in infected macrophages. Isolation and characterization of AX-35-resistant mutants in M. abscessus ATCC 19977 (MIC AX-35 > 100 μg/mL) revealed the presence of a chimeric gene resulting from the recombination of 737 bps of MAB_2467 into MAB_1966c. In M. abscessus, both genes encode for the b subunit of the cytochrome bc1 oxidase, also known as QcrB. Further analysis, comprising gene inactivation, ATP-depletion assays, AlphaFold3-based structural modeling, and molecular docking, suggested that the product of MAB_1966c is the target of AX-35. Interestingly, transcriptomic analysis of M. abscessus exposed to AX-35 revealed that the compound induced expression of MAB_2467 and the alternate terminal oxidase, cytochrome bd. In M. tuberculosis, in the absence of the cytochrome bd oxidase, QcrB inhibitors, including AX-35, are bactericidal. However, in M. abscessus, AX-35 had bacteriostatic activity. These results suggested that, in the presence of AX-35, MAB_2467 may compensate for the absence of the cytochrome bd oxidase and that M. abscessus ATCC 19977 genome encodes two QcrB paralogs.