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

Polyene antifungals, including amphotericin B, nystatin, and natamycin, remain essential for treating life-threatening fungal infections, yet the relationship between their chemical structures, subcellular behavior, and mechanisms of resistance remains poorly understood. Although structurally distinct, all three drugs interact with ergosterol in the fungal plasma membrane to form assemblies that disrupt membrane integrity and function. To investigate how polyene structure influences intracellular distribution and its potential association with resistance, we developed a comprehensive set of fluorescent probes derived from each clinically used polyene. Each probe features the same fluorophore conjugated to a conserved functional group, preserving the amphoteric nature of the parent drugs and enabling live-cell imaging. This unified design allowed direct comparison of subcellular localization and trafficking across Candida species, including sterol biosynthesis mutants with high-level polyene resistance. The probes maintained ergosterol-dependent antifungal activity in vitro and revealed structure-specific subcellular distribution patterns. In highly resistant strains, all three probes showed enhanced accumulation within the vacuole lumen. These findings suggest that vacuolar sequestration contributes to polyene resistance and may reflect a previously unrecognized aspect of the fungal stress response to polyenes. More broadly, this work provides the first direct visual evidence that polyene structure influences intracellular fate and membrane trafficking in fungal cells. The fluorescent probes developed here offer a broadly applicable platform for analyzing polyene distribution, tracking sterol-associated membrane remodeling, and detecting resistance-associated changes in sterol composition in pathogenic fungi.