Fluorescent Farnesyl-Derived Probes Reveal Distinct Intracellular Localization Associated with Morphogenesis Suppression in Candida albicans

Abstract

Farnesol is a quorum-sensing sesquiterpene produced by Candida albicans that inhibits filamentation, a morphogenetic transition underlying fungal invasion and virulence. Notably, several farnesyl-derived sesquiterpenes bearing functional groups other than the original alcohol of farnesol have also been reported to suppress filamentation, indicating that this activity is not unique to farnesol itself but may instead reflect shared properties of the farnesyl scaffold. This observation prompted us to probe whether differences in the intracellular distribution of farnesyl-derived molecules might accompany differences in their morphogenesis-suppressing activity. To address this question, we synthesized two fluorescent farnesyl-derived probes, F-1 and F-2, together with dodecyl-chain analogues as controls. Both probes preserve the native farnesyl isoprenoid scaffold but differ in fluorophore identity, with F-1 labeled with a tetramethylrhodamine (TAMRA) dye and F-2 labeled with a 7-diethylaminocoumarin dye. Counterintuitively, in live C. albicans cells, F-1, despite bearing the more sterically demanding TAMRA dye, inhibited filamentation and displayed broad cytosolic distribution with enrichment along the tubular mitochondrial network. Inhibition by F-1 was reversed by exogenous dbcAMP, consistent with a mechanism like that of farnesol involving the Ras1-cAMP signaling pathway. In contrast, F-2 showed no antifilamentation activity and localized predominantly to cytosolic membranous compartments with irregular morphology. Notably, F-1 retained mitochondrial enrichment and filamentation-inhibitory activity in drug-resistant strains, and its intracellular accumulation at biologically active concentrations was not substantially diminished by enhanced efflux. Together, these findings demonstrate that chemical modification of farnesyl-derived sesquiterpenes, including incorporation of substituents with substantial molecular weight and steric bulk, can preserve morphogenesis-inhibitory activity. These results support a model in which the pleiotropic morphogenesis-inhibitory effects of farnesyl-derived compounds involve not only plasma membrane-associated signaling but also access to intracellular compartments, including mitochondria.