Directed evolution of the pathogenic mold Aspergillus fumigatus reveals novel genes contributing to triazole resistance.

Aspergillus fumigatus is the leading cause of invasive mold infections in immunocompromised patients. Current antifungal treatment primarily depends on the triazole antifungals, which act by inhibiting Erg11/Cyp51, a key enzyme in the ergosterol biosynthetic pathway. However, resistance is emerging at an increasing rate, reducing treatment efficacy and patient survival. Confirmed resistance mechanisms in clinical isolates include mutations in cyp51A, cyp51B, hmg1, hapE, cox10, and the overexpression of drug efflux pumps. To identify additional determinants of triazole resistance, we grew A. fumigatus wild-type and Δcyp51A mutant strains under increasing concentrations of voriconazole. Sequencing of the resultant resistant strains identified known mutations in cyp51A and cyp51B, and novel mutations in hmg1, abcC (cdr1B), ptaB, erg25B, and srbA. Mutations of hmg1 and ptaB occurred early during evolution, while mutations of erg25B and srbA occurred later. Reintroduction of the novel mutations in hmg1, abcC, ptaB, and erg25B into wild-type A. fumigatus and correction of the srbA mutation in the evolved strain validated their contribution toward triazole resistance. Sterol profiling analysis indicated that mutation or deletion of erg25B is associated with a decrease in the accumulation of methylated sterols. Mutation or deletion of ptaB resulted in increased cyp51A, cyp51B, and erg25A expression. Sequence analysis of clinical isolates revealed enrichment of missense mutations in ptaB, hmg1, abcC, and srbA among triazole-resistant strains.