Iron starvation confers azole tolerance in Aspergillus fumigatus hyphae via mitochondrial function modulation

Antifungal tolerance, a transient survival state distinct from genetic resistance, poses a significant challenge to antifungal therapy. However, the factors that induce antifungal tolerance and their underlying mechanisms remain poorly understood. Here, we demonstrate that iron starvation exerts a paradoxical effect on azole susceptibility in the human pathogenic filamentous fungus Aspergillus fumigatus by specifically increasing azole tolerance in hyphae while reducing the minimal inhibitory concentration against both conidia and hyphae. This hyphal tolerance could be quantified using a newly developed protoplast-based time-kill assay. The minimum duration required to kill 99% of hyphal cells (MDK 99 ) following azole treatment was significantly prolonged under iron-starved conditions. Further investigations reveal that iron starvation impairs mitochondrial function by reducing the activity of mitochondrial electron transport chain (ETC) complexes I and III. This attenuation leads to a marked reduction in two key hallmarks of azole-induced hyphal death, including reactive oxygen species accumulation and carbohydrate patch formation. Supporting this mechanism, genetic deletion of mba1 , a critical mitochondrial assembly factor essential for the proper biogenesis of ETC complexes I and III, confers azole tolerance in A. fumigatus hyphae even under iron-replete conditions. Importantly, this iron starvation-mediated azole tolerance could be restored through supplementation with the mitochondrial cofactor coenzyme Q10. Overall, these findings identify iron availability as a key environmental modulator of azole fungicidal effect and reveal potential therapeutic targets to counteract this antifungal tolerance in invasive fungal infections. IMPORTANCE Aspergillus fumigatus undergoes an obligate life cycle with distinct morphotypes, and hyphae represent the predominant morphological form of the fungus during invasive pulmonary aspergillosis. However, owing to the multinucleate nature and pronounced physiological heterogeneity of hyphae, it is challenging to achieve quantitative and effective assessment of their drug susceptibility. In this study, by developing a protoplast release-based time-kill assay, we uncovered that iron starvation confers azole tolerance in A. fumigatus hyphae. Importantly, mechanistic investigations further revealed that supplementation with the mitochondrial cofactor coenzyme Q10 restores fungicidal activity of azoles against A. fumigatus hyphae under iron-starved conditions. Overall, this study underpins the elucidation of environmental factor-antifungal drug tolerance associations and offers targeted strategies against this tolerance.