Miltefosine regulates gene expression related to morphogenesis and zinc metabolism in Candida albicans and Candida tropicalis

ABSTRACT Candida albicans and Candida tropicalis are major hyphae-forming pathogens causing candidiasis. The rise of antifungal resistance underscores the need for new therapeutic strategies. Miltefosine (MFS), an alkylphosphocholine designated as an orphan drug for invasive candidiasis, has shown inhibitory, fungicidal, and antibiofilm effects against Candida spp. In this study, we used microbiological and transcriptomic approaches to investigate the mechanisms underlying inhibition of growth and filamentation by MFS in C. albicans and C. tropicalis . The minimum inhibitory concentrations of MFS for all isolates ranged from 1 to 4 µg/mL, and antibiofilm effects were observed at 2–8 µg/mL (biofilm formation), and at 16–128 µg/mL (mature biofilm). MFS impaired yeast adhesion on polystyrene, inhibited the yeast-to-hyphae transition on different filament-inducing media, and induced alterations in the chitin and chitosan exposure in the cell wall. The inhibitory effect on filamentation promoted by MFS can be explained, in part, by the negative regulation of gene expression associated with virulence and transcriptional factors, such as ECE1 , HWP1 , ALS3 , EFG1 , RAS1 , CYR1 , TEC1 , NGT1 , and CEK1 for C. albicans and ALS1 , SAP3 , EFG1 , BCR1 , and NGT1 for C. tropicalis . In addition, the RNA-seq and qPCR analyses highlighted the most enriched upregulated genes in response to MFS involved in zinc metabolism, especially zincophore PRA1 and zinc transporters ZRT1 and Z RT2 , and improved fungal growth was observed under MFS treatment in zinc-supplemented media. Our data provided further evidence of the mode of action of MFS in inhibiting growth and filamentation, particularly concerning the involvement of zinc metabolism in MFS-induced stress. IMPORTANCE Miltefosine is a repurposed antifungal agent that effectively targets molds and yeasts, including Candida albicans and Candida tropicalis . By integrating phenotypic and transcriptomic approaches, we demonstrate that miltefosine exerts fungicidal activity while broadly impairing key virulence attributes, including adhesion, biofilm development, and yeast-to-hyphae transition. The transcriptional profiling revealed upregulation of filamentation-blockers and zinc machinery genes, and downregulation of virulence factor genes alongside modulation of cell wall component exposure. RNA-seq further highlighted a massive response involving zinc homeostasis, particularly the zincophore and zinc transporters, suggesting metal metabolism as part of the drug’s mechanism of action. Given the limited antifungal arsenal and the clinical burden of invasive candidiasis, these findings expand the mechanistic framework underlying miltefosine activity and reinforce its repositioning as a candidate for antifungal therapy in two highly relevant fungal pathogens. Collectively, this work advances our understanding of antivirulence strategies and supports drug repurposing to address antifungal resistance.