S9.2d Directed evolution of voriconazole resistance in Aspergillus fumigatus identifies novel mutations responsible for triazole resistance

Abstract

S9.2 Azole resistance in Aspergillus fumiagatus: how hot is your hotspot?, September 23, 2022, 4:45 PM - 6:15 PM Aspergillus fumigatus is the leading invasive mold pathogens in humans. The first line of treatment for invasive A. fumigatus infections are the triazole antifungals that inhibit Erg11/Cyp51 lanosterol demethylase activity, blocking ergosterol biosynthesis. In recent years, triazole resistance of A. fumigatus has been increasingly reported, both as a result of widespread agricultural use of fungicidal triazoles and long-term treatment in patients with chronic aspergillosis. To date, the most common triazole resistance mechanisms in A. fumigatus are alterations in the erg11A/cyp51A gene or promoter, followed by overexpression of efflux pumps and mutations in hmg1, encoding HMG-CoA reductase. To identify novel triazole resistance mechanisms, we passaged A. fumigatus wild type and cyp51A-null strains under increasing concentrations of voriconazole (0.25 μg/ml-20 μg/ml) to generate resistant strains. Resistant isolates were whole-genome sequenced and compared with untreated controls. We identified known cyp51A and cyp51B mutations, and novel mutations in HMG1 and in previously uncharacterized genes in the ergosterol biosynthesis pathway as well as several efflux pumps. We identified at which stage of evolution each of the mutations occurred as well as their contribution to the resistance phenotype by re-introduction, alone and in combination, into the susceptible parental strain. Our study identified novel genes conferring triazole resistance and helps outline the complex stepwise evolutionary paths by which A. fumigatus develops resistance.