Synergistic antifungal activity and mechanisms of dehydronuciferine combined with fluconazole against drug-resistant Candida albicans.

The increasing resistance of Candida albicans to conventional antifungal agents poses a major clinical challenge, underscoring the urgent need for novel therapeutic strategies. Drug combination therapy has emerged as a promising approach to overcome antifungal resistance. In this study, we evaluated the synergistic antifungal activity of dehydronuciferine (DHN), a natural aporphine alkaloid, in combination with fluconazole (FLC) against six clinically drug-resistant C. albicans isolates. Strong synergism was confirmed by checkerboard microdilution assay, with fractional inhibitory concentration index (FICI) values ranging from 0.063 to 0.265. Notably, for strain CA10, the minimum inhibitory concentration of FLC was reduced from >512 to 0.25 μg/mL in the presence of DHN (FICI = 0.125), representing a >2,000-fold reversal of resistance. In a Galleria mellonella infection model, the DHN + FLC combination significantly improved larval survival compared with either monotherapy (P < 0.01). Phenotypic assays demonstrated that the combination markedly suppressed key virulence traits, including hyphal growth, biofilm formation, and epithelial adhesion. Transcriptomic profiling revealed that these phenotypic changes were associated with the downregulation of hypha- and adhesion-related genes and the ergosterol biosynthesis pathway. Together, these findings identify dehydronuciferine as a promising antifungal chemosensitizer that enhances fluconazole efficacy against drug-resistant C. albicans through multitarget perturbation of resistance- and virulence-associated processes. Further mechanistic and preclinical studies are warranted to evaluate its translational potential.IMPORTANCEDrug-resistant Candida albicans infections are increasingly difficult to treat, and the pipeline for new antifungal drugs remains limited. In this study, we show that the natural aporphine alkaloid dehydronuciferine (DHN) acts synergistically with fluconazole to potently inhibit drug-resistant C. albicans by simultaneously suppressing hyphal growth, biofilm formation, and epithelial adhesion. In a Galleria mellonella infection model, the DHN-fluconazole combination significantly improved survival compared with either agent alone. This combination strategy, which pairs an approved antifungal with a natural compound, offers a promising approach to combat antifungal resistance and may accelerate the development of alternative therapeutic options without the need for entirely new drug classes.