Identification of Novel Mutations Causing Clofazimine Resistance in Mycobacterium intracellulare

Clofazimine (CFZ) is a promising repurposed drug for treating Mycobacterium avium-intracellulare complex pulmonary disease (MAC-PD), but its resistance mechanisms in M. intracellulare remain poorly understood. In this study, we generated 36 CFZ-resistant M. intracellulare mutants in vitro and identified various mutations in the marR gene (WP_009952290.1) in 61% of resistant mutants through whole-genome sequencing. Mutations were identified in additional genes encoding flavin-dependent oxidoreductase (ssuD, C67A), membrane lipoprotein (lppI, C207 deletion), glucose-methanol-choline oxidoreductase (G157 deletion), MASE1 domain-containing protein (C62G), and PPE family protein (222C deletion). Gene complementation experiments demonstrated that introducing the wild-type marR in CFZ-resistant strains (L72 and L74) with marR mutations reduced CFZ minimum inhibitory concentrations (MICs) from 1 g/mL to the susceptible baseline (0.25 g/mL), confirming its critical role in CFZ resistance. Notably, the M. intracellulare MarR lacks homology to M. tuberculosis MarR family protein Rv0678 (MmpR) whose mutations are involved in CFZ and bedaquiline (BDQ) resistance and is flanked by non-efflux pump genes (dhmA and doxX), and unlike M. tuberculosis, its mutation does not cause bedaquiline cross-resistance, indicating a different MarR and distinct regulatory mechanism for CFZ resistance in M. intracellulare. This work highlights marR as a key determinant of CFZ resistance in M. intracellulare and underscores the need for further mechanistic studies with implications for clinical diagnostics and therapeutic strategies.