Agricultural SDHIs Induce Azole Resistance in Aspergillus fumigatus via Mitochondrial Sdh1 Suppression

Introduction: Aspergillus fumigatus poses significant clinical challenges due to its increasing azole resistance. This study investigates the sdh1 gene's role in regulating azole susceptibility, mitochondrial function, and virulence. Materials and Methods: Fungal strains were co-cultured with varying concentrations of succinate dehydrogenase inhibitors (SDHIs). Post-treatment azole minimum inhibitory concentrations (MICs) were determined using broth microdilution method, while succinate dehydrogenase subunit (SDH) expression changes were analyzed via RT-qPCR. Using A. fumigatus MFIG001 as the parental strain, sdh1 knockout mutant (Δ sdh1 ) and complemented strain (Δ sdh1::sdh1 ⁺ ) were constructed through homologous recombination. Detect the hyphal growth rate of Δ sdh1 , MICs and the changes in virulence within the Galleria mellonella infection model. Mitochondrial function was evaluated by measuring SDH activity, ATP content, and reactive oxygen species (ROS) levels. Transcriptomic changes were analyzed using RNA-seq and RT-qPCR, with efflux pump activity validated through Rhodamine 6G accumulation assays. Results: Exposure to subinhibitory concentrations of SDHIs induced azole resistance in A. fumigatus , with 4.12% of strains exhibiting reduced susceptibility to voriconazole, itraconazole , and posaconazole. RT-qPCR analysis revealed significant downregulation of sdh1 in resistant strains, implicating its role in resistance development. Deletion of sdh1 resulted in an 8- to 16-fold increase in triazole MICs, confirming its role as a negative regulator of azole susceptibility. Phenotypically, the Δ sdh1 strain exhibited impaired growth, reduced sporulation, and diminished efficacy of azole treatment in the G. mellonella infection model. Furthermore, Δ sdh1 exhibited severe mitochondrial dysfunction, including reduced SDH activity, decreased ATP levels, elevated ROS, and impaired antioxidant defenses. RNA-seq analysis revealed that the deletion of sdh1 upregulated the expression of efflux pump genes (e.g., cdr1B , abcB , mdr4 ), while Rhodamine 6G efflux assays demonstrated significantly enhanced efflux activity. Discussion: These results identify sdh1 as a critical determinant of azole susceptibility through dual mechanisms: mitochondrial function maintenance and efflux pump regulation. The observed SDHI-induced cross-resistance suggests agricultural fungicides may drive environmental selection of azole-resistant strains. While sdh1 deletion increased drug tolerance through efflux activation, the concurrent mitochondrial damage reduced pathogenic fitness, revealing compensatory evolutionary constraints. This work highlights the need to monitor non-target effects of agricultural SDHIs on clinical antifungal resistance.