A novel lipid (phosphatidylthreonine)-regulated calcium homeostasis in Toxoplasma gondii

• Applicant: Dr. Nishith Gupta
• Subject Area: Medical Microbiology and Mycology, Hygiene, Molecular Infection Biology ; Metabolism, Biochemistry and Genetics of Microorganisms
• Term: from 2014 to 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 245364597

The major membrane glycerophospholipid classes, described thus far, include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine (PtdSer) and phosphatidylinositol. Our recent work has demonstrated the natural occurrence and genetic origin of an exclusive and rather abundant lipid phosphatidylthreonine (PtdThr) in a widespread and clinically relevant eukaryotic model parasite Toxoplasma gondii. Of note is the fact that PtdThr is a natural homolog of the otherwise-universal PtdSer, which is long known to control Ca2+ homeostasis in mammalian cells. Our earlier work showed that targeted genetic disruption of phosphatidylthreonine synthase (PTS) impairs the lytic cycle and virulence of the parasite due to unforeseen attenuation of the consecutive events of motility, egress and invasion. Using a calcium biosensor, we observed that loss of PtdThr causes a dysregulation of cytosolic calcium that in turn translates into a defective gliding motility. In this proposal, we aim to examine the mechanistic regulation of calcium homeostasis by PtdThr using an approach spanning across the disciplines of biochemistry, gene engineering, cell biology and synthetic chemistry. In brief, we will determine the levels of calcium in the cytosol and endoplasmic reticulum (i.e. site of PtdThr and PtdSer synthesis) of the PTS-mutant by expressing a gene-encoded sensor. Assays involving genetic or chemical modulation of calcium homeostasis, and genetic restoration of lipid perturbation in the PtdThr-mutant will be done to establish a role of lipids in calcium regulation. In parallel, we will identify PtdSer/PtdThr-binding proteins underlying Ca2+ regulation by click-chemistry and mass spectrometry, followed by making of selected parasite mutants and phenotyping. Equally, subcellular distribution of PtdThr and PtdSer will be detected using synthetic chemical probes and customized lipid biosensors via high-resolution imaging. Not least, physiological importance of the key PtdThr species in T. gondii will be investigated by chemical complementation of the parasite cultures. Upon completion, we shall understand the roles of PtdThr (and PtdSer) in calcium homeostasis during asexual reproduction of T. gondii, which can eventually be exploited to inhibit the parasite growth.

• DFG Programme: Research Grants
• Co-Investigator: Professor Dr. Christoph Arenz