Non-ergosterol lipid remodeling governs amphotericin B susceptibility via TOR–calcineurin signaling in Candida glabrata

Understanding the molecular determinants of antifungal drug susceptibility is essential for addressing rising resistance in fungal pathogens. Here, we uncover a central role for sphingolipid (SL) metabolism in modulating susceptibility to amphotericin B (AmB) in Candida glabrata . Through targeted lipidomics, we show that C. glabrata lacks the glucosylceramide biosynthetic branch and is enriched in long-chain phytoceramides. Deleting the inositol phosphosphingolipid phospholipase C gene ( CgISC1 ) resulted in elevated AmB sensitivity, independent of ergosterol biosynthesis, and was associated with altered SL profiles and disrupted ceramide chain-length ratios. We further demonstrate that this phenotype is regulated by the TOR and calcineurin signaling pathways. Pharmacological inhibition and genetic interaction studies reveal that TOR-dependent regulators ( CgORM2 ) and calcineurin effectors, including the CK2 subunit CgCKB s, modulate SL biosynthesis. Deletion of CgCKB2 in the Cgisc1 Δ background reverses AmB susceptibility, whereas deletion of CgCKB1 in Cgisc1 Δ cells further increases susceptibility to AmB. These findings define a signaling lipid regulator that governs membrane composition and AmB sensitivity, providing a framework for targeting sphingolipid pathways to potentiate antifungal therapies.