Identification of Amino Acid Substitutions in Cyp51A Contributing to Reduced Voriconazole Susceptibility in...

Aspergillus fumigatus strains cause severe mycosis in humans. Strains belonging to the cryptic species of A. fumigatus, such as Aspergillus lentulus, are also pathogenic and tend to exhibit resistance to azole drugs. Although the contribution of cytochrome P450 14-α sterol demethylase (Cyp51A) to azole resistance in A. lentulus has been highlighted, the molecular mechanisms behind it remain unknown. In this study, we performed functional analysis of the CYP51 gene derived from A. lentulus (Al-cyp51A) to identify substitutions affecting azole susceptibility. We constructed a CRISPR/Cas9-based genome editing method that enabled one-step replacement of the cyp51A locus in A. fumigatus. Using this system showed that the expression of Al-cyp51A in the A. fumigatus strain increased its resistance to voriconazole (VRCZ) in comparison with the cyp51A of A. fumigatus (Af-cyp51A). By screening chimeric Al-cyp51A harboring partial sequences of Af-cyp51A, we successfully identified amino acid residues in Al-Cyp51A that contributed to VRCZ resistance. Molecular dynamics simulations showed that these substitutions may reduce the inhibitory effect of the drug. Our findings provide further insights into the molecular mechanisms of azole resistance in cryptic A. fumigatus and highlight the functional role of cyp51A mutations.