The alkylphosphocholine miltefosine exerts broad-spectrum fungicidal activity by disrupting membrane integrity and mitochondrial function

Miltefosine (MFS), the only orally available treatment for visceral leishmaniasis, has emerged as a promising repurposed antifungal agent with activity against diverse fungal pathogens, yet its precise mechanisms of action remain incompletely understood. Here, we employed a multi-species chemogenomics approach integrating barcoded deletion libraries from Aspergillus fumigatus, homozygous and haploinsufficient libraries from Saccharomyces cerevisiae, and a conditional GRACE library from Candida albicans, coupled with transcriptional profiling and whole-genome sequencing of MFS-resistant mutants. We demonstrate that MFS exhibits species-dependent fungicidal activity, with partial fungistatic effects against A. fumigatus and S. cerevisiae but potent fungicidal activity against Candida species and Cryptococcus neoformans. Chemogenomic profiling revealed that MFS susceptibility is governed by conserved eukaryotic processes, including membrane lipid homeostasis, vesicular trafficking (ER-to-Golgi, retromer complex), and sphingolipid biosynthesis, with inositolphosphorylceramide synthase (AUR1) emerging as a critical determinant in C. albicans. Whole-genome sequencing of MFS-resistant S. cerevisiae isolates identified loss-of-function mutations in LEM3, which encodes a phospholipid translocase accessory protein, and C. albicans LEM3 mutants similarly exhibited MFS resistance, establishing reduced drug uptake as a conserved resistance mechanism. Transcriptional profiling revealed rapid compensatory responses, including upregulation of ergosterol biosynthesis genes (via UPC2), sphingolipid modifiers, and efflux pumps in C. albicans, while A. fumigatus mounted a massive secretory response, accompanied by downregulation of protein synthesis. We characterized an RTA1-domain protein (RtaB) unique to fungi that confers MFS sensitivity in A. fumigatus and is directly regulated by the transcription factor SmiA. Comparative analysis identified species-specific adaptations: A. fumigatus relies heavily on sulfur assimilation and G-protein signaling, S. cerevisiae is vulnerable to disruptions in amino acid biosynthesis and RNA processing, and C. albicans reveals critical roles for sphingolipid metabolism. Our findings establish that MFS exerts broad-spectrum antifungal activity through coordinated disruption of membrane integrity,