Berberine-fluconazole synergy against drug-resistant Candida albicans: association with disruption of the Ire1-mediated UPR pathway

Abstract

Candida albicans -associated invasive candidiasis exhibits high mortality. Azole resistance and biofilm-mediated persistent infections greatly weaken clinical antifungal efficacy, necessitating new therapeutic approaches. This study investigated the synergistic antifungal and antibiofilm activities of berberine (BBR) combined with fluconazole (FCZ) against drug-resistant C. albicans , and explored potential pathways correlated with this synergistic phenotype. Synergistic antifungal effects were verified via fractional inhibitory concentration index and time-kill curve assays. Crystal violet staining and Spider plate culture evaluated biofilm formation and fungal morphology. Transcriptome sequencing was performed to screen potential regulatory pathways and drug targets. Endoplasmic reticulum (ER) homeostasis was assessed by examining ultrastructural changes, misfolded protein accumulation, stress sensitivity and secreted protease activity. HAC1 splicing and unfolded protein response (UPR)-related gene expression were analyzed to characterize UPR signaling alterations. Autophagy activation was evaluated via ultrastructural observation, vacuole staining, pharmacological inhibition, and detection of autophagy-related gene transcription and Atg1 protein abundance. BBR combined with FCZ exerted synergistic antifungal effects against drug-resistant C. albicans and inhibited biofilm formation. Combined treatment disrupted ER homeostasis and attenuated the Ire1-mediated UPR signaling response in C. albicans . It induced autophagy-associated alterations, accompanied by vacuolar structural impairment, up-regulated transcription of autophagy-related genes, and increased Atg1 protein abundance. The synergistic effect of BBR combined with FCZ is associated with ER stress pathway dysregulation and autophagy activation in C. albicans . This study provides a potential therapeutic strategy for azole-resistant candidiasis, while further investigations are required to elucidate the precise underlying molecular mechanisms.