Abstract
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.
Highlights
Phosphorus in the form of phosphate (PO43, Pi) is essential for cellular growth and function
To determine whether the lipid composition in C. neoformans changes in response to phosphate deprivation, WT was incubated in minimal medium in the presence and absence of phosphate and total lipids were isolated and analyzed by thin layer chromatography (TLC) (Fig 1)
We note that C. neoformans and C. albicans produce more DGTS than K. lactis during phosphate starvation, suggesting that phospholipid replacement by betaine lipids is more efficient in these pathogens than in K. lactis
Summary
Phosphorus in the form of phosphate (PO43–, Pi) is essential for cellular growth and function. Microorganisms, including fungi, are exposed to fluctuating levels of extracellular phosphate, depending on their environmental niche. In order to maintain a stable intracellular phosphate concentration, they have evolved tightly regulated mechanisms to sense, take up, store and utilize phosphate. In fungi, this process is dependent on the phosphate sensing and acquisition (PHO) pathway. When intracellular phosphate is low, activation of the pathway occurs and triggers expression of effector genes that encode proteins involved in the acquisition of phosphate from external sources and enzymes involved in recycling phosphate from internal sources (reviewed in [1,2])
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