New gene linked to clofazimine resistance in Mycobacterium intracellulare

Clofazimine (CFZ) is being repurposed as a promising treatment for Mycobacterium avium - intracellulare complex pulmonary disease (MAC-PD), but we still know little about how M. intracellulare becomes resistant to this drug. To tackle that gap, a team led by Ying Zhang created CFZ-resistant strains of M. intracellulare in the laboratory. The researchers deliberately generated 36 CFZ-resistant M. intracellulare mutants in vitro and then searched the genomes of those mutants for changes that could explain resistance. By comparing the genomes of resistant and non-resistant bacteria, the team aimed to find genetic changes tied to CFZ resistance and then test whether those changes actually cause resistance. This approach—making resistant mutants, sequencing their genomes, and testing suspect genes—lets researchers move from observing resistance to identifying specific genetic culprits. Ying Zhang and colleagues focused on mutations that appeared repeatedly among independent resistant mutants, reasoning that repeated changes are likely to matter most for how CFZ stops working against M. intracellulare.

The central tool the team used was whole-genome sequencing to catalog mutations in their 36 CFZ-resistant mutants. They found that mutations in the marR gene (WP_009952290.1) appeared in 61% of resistant mutants, making marR the most common site of change. Additional mutations were identified in genes encoding flavin-dependent oxidoreductase (ssuD, C67A), membrane lipoprotein (lppI, C207 deletion), glucose-methanol-choline oxidoreductase (G157 deletion), MASE1 domain-containing protein (C62G), and a PPE family protein (222C deletion). To test whether marR changes actually cause resistance, the researchers performed gene complementation experiments: introducing a wild-type marR into CFZ-resistant strains L72 and L74. This intervention reduced CFZ minimum inhibitory concentrations (MICs) from 1 μ g/mL to the susceptible baseline (0.25 μ g/mL), confirming that marR mutations can drive CFZ resistance. Importantly, the M. intracellulare MarR identified here lacks homology to the M. tuberculosis MarR family protein Rv0678 (MmpR), is flanked by non-efflux pump genes dhmA and doxX, and its mutation did not cause bedaquiline cross-resistance.

These findings point to marR as a key determinant of CFZ resistance in M. intracellulare and suggest that the mechanism is distinct from what is seen in M. tuberculosis. Because the M. intracellulare MarR lacks similarity to Rv0678 (MmpR) and sits next to dhmA and doxX rather than efflux pump genes, the regulatory changes that lead to CFZ resistance here appear to follow a different path. The absence of bedaquiline cross-resistance when marR is mutated is clinically relevant because it suggests that resistance to CFZ does not automatically mean loss of bedaquiline activity in M. intracellulare, unlike some patterns reported in other species. The study underscores the need for further mechanistic research to map exactly how marR and the other mutated genes alter bacterial response to CFZ. Those future studies could inform diagnostic tests that detect resistance and guide therapeutic strategies for MAC-PD by identifying when CFZ is likely to succeed or fail.