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 CgCKBs, modulate SL biosynthesis. Deletion of CgCKB2 in the Cgisc1Δ background reverses AmB susceptibility, whereas deletion of CgCKB1 in Cgisc1Δ 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.