Expression Profiling and Molecular Modeling Analysis of Cyp51C 14α-Demethylase Associated with Azole Resistance in Clinical Aspergillus flavus Isolates

Invasive infections caused by Aspergillus flavus are more common in tropical and subtropical countries. The emergence of azole resistance in A. flavus complicates the management of aspergillosis, as azoles are the first-line and empirical therapy. The aim of this study was to investigate the molecular mechanisms underlying azole resistance in A. flavus, focusing on the cyp51C gene. We screened 34 molecularly confirmed A. flavus isolates obtained from patients with invasive aspergillosis for cyp51C gene expression by real-time RT-qPCR and for mutations by PCR sequencing. Molecular modeling and docking studies were performed using SWISS-MODEL, SwissDock, and I-TASSER software. Susceptibility testing revealed that 14.71% and 8.82% of isolates were resistant to itraconazole and posaconazole, respectively, with 5.88% exhibiting cross-resistance. The mRNA expression of cyp51C was upregulated (>2.5-fold) in five of the six resistant strains (83.33%). Hyperexpression of cyp51C was significantly more frequent among resistant isolates than among susceptible isolates (Fisher’s exact test, p = 0.014). Sequencing identified ten point mutations, including six synonymous and four non-synonymous substitutions. The non-synonymous mutations M54T and S240A were detected in the protein sequences of both resistant and susceptible isolates. Notably, D254N and I285V were observed exclusively in resistant isolates and in susceptible isolates with itraconazole MICs near the epidemiological threshold. Homology modeling and 3D structure prediction of the mutated Cyp51C protein demonstrated interactions with itraconazole, posaconazole, and voriconazole. Importantly, I-TASSER analysis indicated that the I285V substitution is located near the itraconazole binding site. Simultaneous overexpression of the cyp51A, cyp51B and cyp51C genes was observed in 33.33% of resistant isolates. These findings suggest that multiple target genes and mechanisms may act concurrently to confer azole resistance in A. flavus. Overall, this study supports the hypothesis that azole resistance in A. flavus is multifactorial and highlights the potential value of combining mutation analysis, gene expression profiling, and structural modeling for improved molecular surveillance and antifungal resistance monitoring.