Afu-Emi1 Contributes to Stress Adaptation and Voriconazole Susceptibility in Aspergillus fumigatus

Invasive aspergillosis (IA) is the second most common invasive fungal disease and is associated with high mortality rates. Aspergillus fumigatus is the predominant causal agent of this life-threatening infection. Triazoles are still the cornerstone of antifungal treatment, and voriconazole remains the first-line choice. However, voriconazole resistance has been increasingly reported, which results in significantly higher mortality rates for IA and is particularly problematic. In the present study, we report the identification and functional study of a protein with previously unknown function that is encoded by the gene designated Afu-emi1 (AFUA_1G07360). High-throughput gene replacement technology was applied to construct the knockout ΔAfu-emi1 strain and a revertant strain. The MICs for azoles, including posaconazole, itraconazole, and voriconazole, were evaluated via the broth microdilution method and E-tests, which revealed that disruption of Afu-emi1 resulted in 4-fold increased susceptibility to voriconazole. Colony growth in the presence of oxidants, namely, H(2)O(2) and menadione, and osmotic pressure-altering agents, namely, NaCl and d-sorbitol, was measured. The Afu-emi1 mutant strain exhibited a significant growth defect under oxidative and osmotic stress. The reactive oxygen species (ROS) production levels with or without voriconazole pretreatment were determined, and the Afu-emi1 mutant strain exhibited significantly lower ROS production levels. The effects of Afu-emi1 disruption on voriconazole susceptibility, growth under stress, and ROS production were restored in the revertant strain. In addition, the expression of cyp51A, AfuMDR2, AfuMDR3, AfuMDR4, and cdr1b in the ΔAfu-emi1 strain was significantly reduced. In conclusion, deletion of the gene Afu-emi1 resulted in increased voriconazole susceptibility, attenuated ability for oxidative and osmotic stress adaptation, decreased ROS production, and downregulation of cyp51A, AfuMDR2, AfuMDR3, AfuMDR4, and cdr1b expression, suggesting that Afu-Emi1 is an important regulator of stress adaptation and cyp51A and efflux pump expression in this medically important fungus. IMPORTANCE Voriconazole is the first-line choice for IA, a life-threatening disease. Therefore, voriconazole resistance has become particularly problematic. Disruption of Afu-emi1 resulted in increased susceptibility to voriconazole, a significant growth defect under oxidative and osmotic stress, and downregulation of target enzyme Cyp51A and efflux pump expression, suggesting that Afu-Emi1 is an important regulator of stress adaptation and cyp51A and efflux pump expression in this medically important fungus. Targeting Afu-Emi1 might help to enhance azole therapeutic efficacy and impede azole resistance.