Characteristics of individual <i>cyp51A</i> SNPs and combinations thereof impacting the azole phenotype in TR ₃₄ -mediated resistance genotypes of <i>Aspergillus fumigatus</i>

The World Health Organization has flagged the rise of drug resistance in <i>Aspergillus fumigatus</i> as a critical concern. Elevated mutation rates in this pathogen contribute to the rapid development of resistance, complicating treatment efforts. We conducted a study on the prevalence of azole resistance among clinical <i>A. fumigatus</i> isolates in the Netherlands from 1994 to 2022 and identified 34 <i>cyp51A</i> variants. To investigate the impact of individual single-nucleotide polymorphisms (SNPs) and combinations thereof on the azole phenotype in TR₃₄-mediated resistance genotypes, we focused on novel, recent mutations and explored the effects of SNPs L98H, T289A, I364V, and G448S and the combination of the mutations TR₃₄/L98H, TR₃₄/L98H/T289A/G448S, and TR₃₄/L98H/T289A/I364V/G448S on azole affinity and susceptibility. We created the three-dimensional protein model of the Cyp51A protein with azoles, and the mutation was introduced to the wild-type <i>cyp51A A. fumigatus</i> strain by the CRISPR-Cas9 gene editing technique. Finally, <i>in vitro</i> susceptibility testing of <i>A. fumigatus</i> strains carrying the mutations was conducted to confirm the azole phenotypes observed in clinical isolates. The MICs of all four azoles against the mutated <i>cyp51A</i> strains, which harbored combination mutations, were higher than those of the wild type, with highly elevated MICs of itraconazole, voriconazole, and isavuconazole. Genotypes TR₃₄/L98H/T289A/I364V/G448S mutant showed a consistent phenotype to the clinical strains, which are highly resistant to voriconazole but susceptible to itraconazole. In this study, we show that molecular dynamics simulations of amino acid substitutions in the <i>cyp51A</i> gene correlate to the structure-function relationship of <i>in vitro</i> phenotype.