Selective disruption of phosphatidylcholine metabolism of the intracellular parasite Toxoplasma gondii arrests its growth.

Toxoplasma gondii is an intracellular protozoan parasite capable of causing devastating infections in immunocompromised and immunologically immature individuals. In this report, we demonstrate the relative independence of T. gondii from its host cell for aminoglycerophospholipid synthesis. The parasite can acquire the lipid precursors serine, ethanolamine, and choline from its environment and use them for the synthesis of its major lipids, phosphatidylserine (PtdSer), phosphatidylethanolamine (PtdEtn), and phosphatidylcholine (PtdCho), respectively. Dimethylethanolamine (Etn(Me)(2)), a choline analog, dramatically interfered with the PtdCho metabolism of T. gondii and caused a marked inhibition of its growth within human foreskin fibroblasts. In tissue culture medium supplemented with 2 mm Etn(Me)(2), the parasite-induced lysis of the host cells was dramatically attenuated, and the production of parasites was inhibited by more than 99%. The disruption of parasite growth was paralleled by structural abnormalities in its membranes. In contrast, no negative effect on host cell growth and morphology was observed. The data also reveal that the Etn(Me)(2)-supplemented parasite had a time-dependent decrease in its PtdCho content and an equivalent increase in phosphatidyldimethylethanolamine, whereas other major lipids, PtdSer, PtdEtn, and PtdIns, remained largely unchanged. Relative to host cells, the parasites incorporated more than 7 times as much Etn(Me)(2) into their phospholipid. These findings reveal that Etn(Me)(2) selectively alters parasite lipid metabolism and demonstrate how selective inhibition of PtdCho synthesis is a powerful approach to arresting parasite growth.

On the biogenesis of lipid bodies in ancient eukaryotes: synthesis of triacylglycerols by a Toxoplasma DGAT1-related enzyme.

In mammalian cells, the main stored neutral lipids are triacylglycerol and cholesteryl esters, which are produced by two related enzymes, acyl-CoA:diacylglycerol acyltransferase (DGAT) and acyl-CoA:cholesterol acyltransferase (ACAT), respectively. Very little is known about the metabolism, intracellular storage and function of neutral lipids in many pathogenic lower eukaryotes. In this paper, we have characterized the activity of an important triacylglycerol synthetic enzyme in the protozoan Toxoplasma gondii. A full-length cDNA and gene encoding a T. gondii DGAT1-related enzyme were identified and designated TgDGAT1. The gene is composed of 15 exons and 14 introns, and encodes a protein with a predicted M(r) 63.5kDa, containing signature motifs characteristic of the DGAT1 family. The native protein migrates at 44kDa under reducing conditions. TgDGAT1 is an integral membrane protein localized to the parasite cortical and perinuclear endoplasmic reticulum, with the C-terminus oriented to the lumen of the organelle. When a Saccharomyces cerevisiae mutant strain lacking neutral lipid production is transformed with TgDGAT1 cDNA, a significant DGAT activity is reconstituted, resulting in triacylglycerol synthesis and biogenesis of cytosolic lipid inclusions, resembling lipid bodies in T. gondii. No production of steryl esters is observed upon TgDGAT1 expression in yeast. In contrast to human DGAT1 lacking fatty acid specificity, TgDGAT1 preferentially incorporates palmitate. Our results indicate that parasitic protozoa are also neutral lipid accumulators and illustrate the first example of the existence of a functional DGAT gene in an ancient eukaryote, demonstrating that diacylglycerol esterification is evolutionarily conserved.