Thirty years of fluconazole therapy selects an azole-resistant Candida albicans isolate with a pre-adapted physiological, metabolic and structural state

Abstract

Azole resistance in Candida albicans is traditionally attributed to alterations in drug targets and efflux mechanisms; however, how long-term antifungal exposure reshapes fungal physiology remains incompletely understood. Here, we characterize a fluconazole-resistant C. albicans clinical isolate (PUJ256) recovered from a patient with chronic mucocutaneous candidiasis after more than 30 years of continuous fluconazole therapy. Compared with the reference strain SC5314, the resistant isolate exhibited a clear fitness trade-off, with reduced filamentation and increased membrane vulnerability under basal conditions, yet enhanced fitness in the presence of fluconazole. Integration of phenotypic analyses with label-free quantitative proteomics revealed extensive metabolic remodelling, including coordinated regulation of central carbon metabolism, ergosterol biosynthesis and redox homeostasis. Notably, mitochondrial membrane potential was preserved in the resistant isolate under fluconazole stress, whereas the susceptible strain exhibited mitochondrial depolarization together with activation of MAPK signalling pathways. In addition, the resistant isolate displayed reduced susceptibility to phagocytosis under antifungal exposure, consistent with an increased capacity for persistence in the context of ongoing treatment. Collectively, our findings indicate that long-term azole resistance in C. albicans PUJ256 is associated with a stable, pre-adapted physiological state characterized by metabolic reprogramming and mitochondrial resilience. The prolonged antifungal exposure of this isolate provides a valuable opportunity to explore adaptative strategies that extend beyond canonical mechanisms, pointing to mitochondrial function and metabolic plasticity as potential targets for therapeutic intervention.