The putative mitochondrial serine protease Pim1 regulates azole tolerance through redox homeostasis in Aspergillus fumigatus.

Aspergillus fumigatus has been included in the World Health Organization's (WHO) inaugural list of priority fungal pathogens for immunosuppressed individuals. Mitochondrial function is closely associated with antifungal susceptibility, and mutations in mitochondrial genes can modulate drug responses in Cyp51A-independent mutants. Using a previously constructed random insertional mutagenesis library, we identified pim1, a gene encoding a putative mitochondrial serine protease, whose mutation via T-DNA insertion regulates azole tolerance in the A. fumigatus Af293 strain. We found that deletion of pim1 in the A1160 background was not essential for fungal viability but increased azole tolerance and attenuated virulence in Galleria mellonella. Pim1 was localized to mitochondria. Further mechanistic analysis revealed that the Δpim1 mutation did not affect intracellular ergosterol content or itraconazole (ITC) retention. Instead, it reduced intracellular reactive oxygen species (ROS) levels, and RNA-seq analysis revealed altered expression of genes involved in anaerobic respiration, ROS/aldehyde scavenging and proline metabolism, which may contribute to azole tolerance. Further experiments supported a role for oxidative homeostasis in this phenotype, as ROS scavenger vitamin C increased ITC tolerance in the WT strain but not in the mutant, whereas H2O2 diminished ITC tolerance in the Δpim1 mutant. Collectively, these findings provide new insight into non-Cyp51A-dependent mechanisms of azole tolerance.