Directed evolution of the pathogenic mold <i>Aspergillus fumigatus</i> reveals novel genes contributing to triazole resistance

<i>Aspergillus fumigatus</i> 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 <i>cyp51A</i>, <i>cyp51B</i>, <i>hmg1</i>, <i>hapE</i>, <i>cox10</i>, and the overexpression of drug efflux pumps. To identify additional determinants of triazole resistance, we grew <i>A. fumigatus</i> wild-type and <i>Δcyp51A</i> mutant strains under increasing concentrations of voriconazole. Sequencing of the resultant resistant strains identified known mutations in <i>cyp51A</i> and <i>cyp51B</i>, and novel mutations in <i>hmg1</i>, <i>abcC (cdr1B)</i>, <i>ptaB, erg25B,</i> and <i>srbA</i>. Mutations of <i>hmg1</i> and <i>ptaB</i> occurred early during evolution, while mutations of <i>erg25B</i> and <i>srbA</i> occurred later. Reintroduction of the novel mutations in <i>hmg1</i>, <i>abcC</i>, <i>ptaB,</i> and <i>erg25B</i> into wild-type <i>A. fumigatus</i> and correction of the <i>srbA</i> mutation in the evolved strain validated their contribution toward triazole resistance. Sterol profiling analysis indicated that mutation or deletion of <i>erg25B</i> is associated with a decrease in the accumulation of methylated sterols. Mutation or deletion of <i>ptaB</i> resulted in increased <i>cyp51A</i>, <i>cyp51B</i>, and <i>erg25A</i> expression. Sequence analysis of clinical isolates revealed enrichment of missense mutations in <i>ptaB</i>, <i>hmg1</i>, <i>abcC</i>, and <i>srbA</i> among triazole-resistant strains.