Biogenesis of Phosphatidylinositol and CDP-diacylglycerol in Toxoplasma gondii
Pengfei Kong, Richard Lucius and Nishith Gupta, Department of Molecular Parasitology, Humboldt-University Berlin, Philippstr. 13, House 14, 10115 Berlin, Germany

Toxoplasma gondii is an intracellular apicomplexan parasite that replicates virtually in all nucleated vertebrate cells. The parasite replication is dependent on either de novo biosynthesis or scavenging of host-derived nutrients. Phosphatidylinositol (PI) is an essential phospholipid required for the biogenesis of parasite organelles, as well as for lipid-mediated signalling. Previous study have indicated that the acquisition of PI in T. gondii depends on the de novo biosynthesis rather than import of pre-synthesied lipid from the host. PI biogenesisis is catalyzed by a PI synthase (PIS) using myo-inositol and cytidine diphosphate-diacylglycerol (CDP-DAG) in the endoplasmic reticulum (ER) and/or Golgi bodies. CDP-DAG is a central intermediate precursor lipid for PI and cardiolipin syntheses. It is synthesized from phosphatidic acid (PA) and cytidine triphosphate (CTP) via a CDP-DAG synthase (CDS). The mechanisms of PI and CDP-DAG syntheses and their physiological importance in T. gondii remain to be understood. 

We have identified and characterized the PIS and CDS enzymes in T. gondii. TgPIS and TgCDS localize in the Golgi bodies and ER, respectively. Both enzymes contain a unique N-terminal extension, which is not required for their localization or catalysis. TgPIS is a functional enzyme in E. coli and produces PI in a myo-inositol-dependent manner. Knockdown of TgCDS in a conditional mutant exerts a ~70% growth defect and a ~50% decline in the PI content, indicating the requirement of this enzyme for optimal lipid synthesis and parasite growth. Our current studies aim to generate the mutants of TgPIS and functionally characterize the impact of TgCDS knockdown on cardiolipin and minor inositol-related lipids .