Integrated transcriptome and mitochondrial respiratory activity analysis reveals oxidative stress adaptation in azole-resistant Aspergillus fumigatus

ABSTRACT Azole resistance in Aspergillus fumigatus is a major challenge in the treatment of invasive aspergillosis. Although cyp51A mutations are well characterized, the global transcriptional responses and physiological adaptations associated with resistance remain incompletely understood. RNA sequencing (RNA-seq) was performed to compare the transcriptomes of the azole-susceptible strain AF293 and four azole-resistant clinical isolates with distinct resistance mechanisms. Voriconazole was used for all antifungal exposure experiments. Differentially expressed genes (DEGs) were analyzed by GO and KEGG enrichment analyses. Mitochondrial respiratory chain Complex I and Complex III activities were further evaluated in SHJT42B, a representative cyp51A-mediated resistant isolate. Azole-resistant isolates exhibited extensive transcriptional reprogramming following voriconazole exposure, with enrichment of oxidative stress-related pathways. Oxidative stress-associated genes showed differential regulation in both cyp51A -dependent and cyp51A -independent resistant isolates, suggesting a conserved stress-response adaptation during azole resistance. Functional analyses revealed that SHJT42B exhibited altered mitochondrial respiratory activity, characterized by reduced Complex I and Complex III activities and increased intracellular reactive oxygen species (ROS) levels compared with AF293. These findings suggest that mitochondrial respiratory alterations may be associated with oxidative stress adaptation in azole-resistant A. fumigatus . Our study integrates transcriptomic and functional analyses to identify oxidative stress adaptation as an important physiological response associated with azole resistance in A. fumigatus . The findings further suggest that mitochondrial homeostasis may represent a potential vulnerability for future combination antifungal strategies, although additional studies across diverse resistant isolates are required. IMPORTANCE Azole-resistant Aspergillus fumigatus is an increasing threat to patients with weakened immune systems, as it limits the effectiveness of first-line antifungal treatments and leads to high mortality. Most previous studies have focused on single resistance genes, leaving the broader biological changes in resistant fungi poorly understood. In this study, we show that azole-resistant strains undergo extensive transcriptional changes involving cellular stress responses and energy metabolism. By integrating transcriptomic analysis with functional validation, we reveal oxidative stress-associated adaptations across resistant isolates and identify altered mitochondrial respiratory activity in the representative resistant isolate SHJT42B. These findings suggest that antifungal resistance involves not only genetic determinants but also broader cellular adaptations that promote survival under drug pressure. Targeting mitochondrial homeostasis or oxidative stress response pathways may provide potential strategies for enhancing antifungal efficacy and warrants further investigation.