CYP51C Y319H and H349R substitutions cannot confer azole resistance in Aspergillus flavus NRRL 3357-5

Azole antifungals inhibit the cytochrome P450-dependent lanosterol 14-α-demethylase (CYP51), an essential enzyme in the ergosterol biosynthetic pathway. While azole resistance mechanisms are well established in Aspergillus fumigatus, the molecular basis of resistance in Aspergillus flavus remains less defined. Previous studies identified nonsynonymous substitutions Y319H and H349R in CYP51C of azole-resistant A. flavus isolates, but their functional significance had not been experimentally confirmed. In this study, we used targeted gene replacement to introduce these mutations individually into a wild-type A. flavus background and assessed their impact on azole susceptibility and fitness. Sequence alignment revealed that residues Y319 and H349 are not conserved among fungal CYP51 (synonym "ERG11") homologs. Both mutant strains exhibited the same susceptibility profiles to itraconazole, voriconazole, posaconazole, isavuconazole, echinocandins, and amphotericin B as the wild-type control. Moreover, colony growth and sporulation were comparable across all strains, indicating no measurable fitness cost associated with either substitution. These results indicate that CYP51C Y319H and H349R may not confer azole resistance or affect fungal fitness, underscoring the necessity of functional validation to distinguish neutral polymorphisms from true resistance determinants in A. flavus.IMPORTANCEAzole resistance in Aspergillus flavus is an emerging clinical concern, yet its molecular basis remains poorly defined. This study demonstrates that two previously reported CYP51C substitutions (Y319H and H349R) do not confer azole resistance or affect fungal fitness when tested experimentally. Our findings highlight that the presence of mutations in resistant isolates does not necessarily imply causality. By emphasizing the need for functional validation, this work helps prevent misinterpretation of neutral polymorphisms as resistance markers and improves the accuracy of molecular diagnostics and resistance surveillance in A. flavus.