Fluorescent Probes Derived from the Polyene Class of Antifungal Drugs Reveal Distinct Localization Patterns and Resistance‐Associated Vacuolar Sequestration in Candida Species

Amphotericin B, nystatin, and natamycin are the only polyene antifungals in clinical use and remain last‐resort therapies for severe fungal infections. These agents disrupt fungal membranes through ergosterol binding, but how structural differences between the three polyenes influence subcellular distribution, and whether such distribution is linked to resistance, remains uncharacterized. To investigate these relationships, we developed fluorescent probes from each clinically used polyene by conjugating a common fluorophore to a conserved functional group, preserving the amphoteric nature and ergosterol‐dependent antifungal activity of the parent drug while enabling live‐cell imaging. This unified design allowed direct comparison of localization and trafficking across Candida species, including sterol biosynthesis mutants with a high level of polyene resistance. The probes revealed structure‐specific distribution patterns, with resistant strains showing enhanced accumulation within the vacuole lumen for all three polyenes. These findings suggest that vacuolar sequestration contributes to polyene resistance and may represent an unrecognized aspect of the fungal stress response. More broadly, this work provides the first direct visual evidence that polyene structure dictates subcellular distribution patterns in fungal cells.