Mitochondrial iron homeostasis is required for azole tolerance in Candida albicans

Antimicrobial resistance is an escalating global concern with the human fungal pathogen, Candida albicans, increasingly exhibiting resistance to fluconazole (FLC). Together with the high mortality associated with bloodstream infections, this growing resistance contributed to the classification of C. albicans as a critical priority pathogen by the World Health Organisation (WHO). Notably, C. albicans can survive high concentrations of fluconazole (FLC) through a phenomenon termed tolerance, which is different from resistance as tolerant cells grow slowly. Tolerance is considered a precursor to resistance development. Previous work from our laboratory demonstrated that intracellular iron homeostasis, particularly the mobilisation of vacuolar iron stores, modulates the synergistic interaction between doxycycline, an iron chelator, and FLC. Building on this, we hypothesised that mitochondrial iron plays a central role in determining FLC susceptibility, supported by emerging links between mitochondrial function, iron metabolism, and azole response. Mitochondrial iron import is mediated by MRS4, after which iron is primarily incorporated into iron-sulfur clusters and haem. Here, we investigated the role of mitochondrial iron import via MRS4 in azole susceptibility phenotypes, including tolerance, synergy and cidality. We demonstrate that MRS4 is essential for FLC tolerance under iron-chelating conditions, despite minimal effects on minimal inhibitory concentration or drug synergy. Absence of MRS4 results in mitochondrial dysfunction and accumulation of toxic sterol intermediates during FLC treatment. Collectively, our findings reveal that mitochondrial iron homeostasis critically governs azole tolerance and highlights toxic sterol accumulation as a driver of fungicidal outcomes.