Osh2 mediates Candida species resistance to miltefosine by regulating zymosterol accumulation.

Invasive candidiasis poses a growing threat to global public health, compounded by the scarcity of effective antifungal treatments. Miltefosine exhibits broad-spectrum antifungal activity, yet its mechanisms of antifungal action and the development of resistance remain poorly understood. Here, we first generated miltefosine-resistant strains of Candida glabrata through stepwise exposure to increasing drug concentrations. Whole-genome sequencing revealed that nonsense mutations in the OSH2 gene (193C > T and 3177C > A) were key drivers of resistance. Functional validation in Candida albicans confirmed that these OSH2 mutations conferred miltefosine resistance, demonstrating the conserved role of Osh2 across species. Multi-omics profiling of the osh2Δ/Δ mutant revealed significant upregulation of ergosterol biosynthesis genes, including ERG6 and ERG11, and the accumulation of zymosterol, an intermediate in the ergosterol pathway. Chemogenetic dissection further elucidated the role of sterol metabolism in resistance: erg11Δ/Δ mutants, which are unable to synthesize zymosterol, exhibited hypersusceptibility to miltefosine, whereas erg6Δ/Δ strains, which accumulate zymosterol, showed innate resistance. Exogenous supplementation of zymosterol dose dependently increased the minimum inhibitory concentration of miltefosine in C. albicans and C. glabrata, confirming that zymosterol accumulation is a key determinant of resistance. Our findings establish Osh2 as a critical regulator of membrane sterol flux and demonstrate that fungal lipid metabolic plasticity enables evasion of membrane-targeting antifungals. Therapeutic targeting of zymosterol biosynthesis enzymes may overcome such adaptive resistance mechanisms in invasive candidiasis, providing a new strategy to combat drug-resistant fungal infections.