The PHO signaling pathway directs lipid remodeling in Cryptococcus neoformans via DGTS synthase to recycle phosphate during phosphate deficiency

The phosphate sensing and acquisition (PHO) pathway of Cryptococcus neoformans is essential for growth in phosphate-limiting conditions and for dissemination of infection in a mouse model. Its key transcription factor, Pho4, regulates expression of genes controlling the acquisition of phosphate from both external and cellular sources. One such gene, BTA1, is highly up-regulated during phosphate starvation. Given that a significant proportion of cellular phosphate is incorporated into phospholipids, and that the Pho4-dependent BTA1 gene encodes an enzyme predicted to catalyse production of a phosphorus-free betaine lipid, we investigated whether phospholipids provide an accessible reservoir of phosphate during phosphate deficiency. By comparing lipid profiles of phosphate-starved WT C. neoformans, PHO4 (pho4Δ) and BTA1 (bta1Δ) deletion mutants using thin layer chromatography and liquid chromatography mass spectrometry, we showed that phosphatidylcholine (PC) is substituted by the phosphorus-free betaine lipids diacylglyceryl-N,N,N-trimethylhomoserine (DGTS) and diacylgyceryl hydroxymethyl-N,N,N-trimethyl-beta-alanine (DGTA) in a Pho4- and Bta1-dependent manner, and that BTA1 encodes a functional DGTS synthase. Synthesis of DGTA tightly correlated with that of DGTS, consistent with DGTS being the precursor of DGTA. Similar to pho4Δ, bta1Δ grew more slowly than WT in cell culture medium (RPMI) and was hypovirulent in a murine model of cryptococcosis. In contrast to pho4Δ, bta1Δ tolerated alkaline pH and disseminated to the brain. Our results demonstrate that Bta1-dependent substitution of PC by betaine lipids is tightly regulated in C. neoformans by the PHO pathway, to conserve phosphate and preserve membrane integrity and function. This phospholipid remodeling strategy may also contribute to cryptococcal virulence during host infection.