Title

Understanding phospholipid biosynthesis in the human malaria parasite Plasmodium falciparum using Saccharomyces cerevisiae as a surrogate system

Date of Completion

January 2005

Keywords

Biology, Cell

Degree

Ph.D.

Abstract

Malaria is an infectious disease caused by protozoan parasites that invade and multiply within erythrocytes. Among several species that infect humans, Plasmodium falciparum is the most infectious and causes most deaths. Resistance of P. falciparum to major antimalarial drugs stresses the need for novel compounds that target metabolic pathways essential for parasite survival. During malarial infection, infected erythrocytes exhibit up to 5-fold increase in lipid content, mainly due to the production of malarial membranes. Phosphatidylcholine (PC) is the most abundant phospholipid in P. falciparum membranes. Synthesis of PC from choline transported from the host plasma and metabolized by the parasite enzymatic machinery has been suggested to play an important role in parasite physiology. Thus, pathways that transport choline into the parasite and incorporate it into the parasite membranes are important for parasite growth and development, making them attractive targets for antimalarial chemotherapy. Accordingly, choline analogs have been shown to block choline transport into the parasite and inhibit parasite growth. Genetic studies have identified a gene from Saccharomyces cerevisiae, GAT2, and a gene from Torpedo marmorata, tCTL1, which can complement a choline transport-defective mutant. Here, I present my work on the identification and characterization of homologs of GAT2 and tCTL1 in the P. falciparum genome called PfGAT and PfCTL, respectively. These studies demonstrated that PfGAT encodes a yeast-like glycerol-3-phosphate (GPAT) enzyme that catalyzes the initial step of phospholipid synthesis. Expression and localization studies revealed that PfGatp is an endoplasmic reticulum membrane protein expressed throughout the intraerythrocytic cycle of the parasite but mainly induced during the trophozoite stage. Assays in better transport systems allowed us to reexamine the role of choline transporter-like (CTL) proteins in choline transport. These studies demonstrated that tCtl1p and its yeast homolog Pns1p, are not choline transporters. We performed initial characterization of PfCTL, including the identification of its full-length cDNA and creation of a PfctlΔ knockout. These studies demonstrated that PfCTL undergoes alternative splicing and that its disruption results in a viable mutant. We also performed an analysis of the global response of yeast cells to inositol and choline and attempted to use it as a tool for understanding transcriptional regulation of Plasmodium genes. Taken together, our studies generated valuable information that increases our understanding of phospholipid biosynthesis in P. falciparum. Furthermore, these studies provide evidence for gene regulation in P. falciparum occurring at the post-transcriptional level. ^