Toxoplasma gondii acyl-lipid metabolism: de novo synthesis from apicoplast-generated fatty acids versus scavenging of host cell precursors

Abstract

Toxoplasma gondii is an obligate intracellular parasite that contains a relic plastid, called the apicoplast, deriving from a secondary endosymbiosis with an ancestral alga. Metabolic labelling experiments using [14C]acetate led to a substantial production of numerous glycero- and sphingo-lipid classes in extracellular tachyzoites. Syntheses of all these lipids were affected by the herbicide haloxyfop, demonstrating that their de novo syntheses necessarily required a functional apicoplast fatty acid synthase II. The complex metabolic profiles obtained and a census of glycerolipid metabolism gene candidates indicate that synthesis is probably scattered in the apicoplast membranes [possibly for PA (phosphatidic acid), DGDG (digalactosyldiacylglycerol) and PG (phosphatidylglycerol)], the endoplasmic reticulum (for major phospholipid classes and ceramides) and mitochondria (for PA, PG and cardiolipid). Based on a bioinformatic analysis, it is proposed that apicoplast produced acyl-ACP (where ACP is acyl-carrier protein) is transferred to glycerol-3-phosphate for apicoplast glycerolipid synthesis. Acyl-ACP is also probably transported outside the apicoplast stroma and irreversibly converted into acyl-CoA. In the endoplasmic reticulum, acyl-CoA may not be transferred to a three-carbon backbone by an enzyme similar to the cytosolic plant glycerol-3-phosphate acyltransferase, but rather by a dual glycerol-3-phosphate/dihydroxyacetone-3-phosphate acyltransferase like in animal and yeast cells. We further showed that intracellular parasites could also synthesize most of their lipids from scavenged host cell precursors. The observed appearance of glycerolipids specific to either the de novo pathway in extracellular parasites (unknown glycerolipid 1 and the plant like DGDG), or the intracellular stages (unknown glycerolipid 8), may explain the necessary coexistence of both de novo parasitic acyl-lipid synthesis and recycling of host cell compounds.

Figure 1. Scheme for metabolic labelling of T. gondii extra- and intra-cellular tachyzoites

Extracellular Toxoplasma cells were labelled for 4–6 h in the presence of [¹⁴C]acetate (left panel) and the lipid profile was subsequently analysed. When required, a 1 h treatment with the aryloxyphenoxypropionate herbicide haloxyfop (fop) preceded the metabolic labelling. HFF cells were labelled in the presence of [¹⁴C]acetate und subsequently infected by Toxoplasma tachyzoites (right panel). Lipid analyses of intracellular parasites were then carried out to detect diversion of labelled carbons from host cell compounds for the parasite acyl-lipid metabolism.

Figure 2. Identification of de novo synthesized lipids by T. gondii tachyzoites

Parasites were labelled with the FA precursor [¹⁴C]acetate and extracted lipids were analysed by 2D-TLC along with total A. thaliana lipids as markers for identification. Lipids extracted from the same number of parasites (6×10⁸) were deposited on the chromatography plates. (A) Parasites were labelled for 4 h extracellularly prior to lipid extraction and TLC analysis. (B) Same labelling conditions as in (A), but radiolabelled lipids were subjected to an alkaline treatment (KOH in methanolic phase) to identify sphingolipids amongst the parasite-synthesized lipids. (C) Same conditions as in (A), but the labelling was preceded by a 2 h treatment with the herbicide haloxyfop; spots represent the radiolabelled lipids de novo synthesized by the parasite, broken line circled positions correspond to the positions of the co-migrated Arabidopsis lipids identified by coloration with ANS, and solid line circled spots indicate positions of lipids not found in the Arabidopsis lipid mixture. TAG, triacylglycerol; U₁–U₇, unknown lipids in the Arabidopsis lipid mixture. U₃ was tentatively identified as PA; +, deposit point of lipids. FFA, NEFA.

Figure 3. Purity control experiment of the intracellular parasite fraction

After an overnight intracellular growth within HFF cells, parasites were collected by scraping the monolayer and were mechanically released from host cells by sequential passage through 20, 23, 25 and 27G needles. Parasites were further purified through a column of silicon-treated glass wool. (A) Western-blot analysis of the parasite fraction before (−) and after (+) filtration; detection of host cell membranes was done using the anti-HsTfR (α-HsTfR) mAb H68.4. Protein extracts correspond to 10⁷ (1), 10⁶ (2) and 10⁵ (3) parasites. The α-TgSAG1 antibody that is directed against the T. gondii major surface protein SAG1 was used as an internal control of protein load in each lane. (B) IF microscopy of the PV membrane protein GRA5 (α-TgGRA5) in the same fractions before and after filtration.

Figure 4. Lipid acquisition from the host cell

HFF host cells were labelled with [¹⁴C]acetate prior to infection with unlabelled parasites. Intracellular tachyzoites were purified and their radiolabelled lipid content was analysed by 2D-TLC along with total Arabidopsis lipids as markers for identification. (A) HFF radiolabelled lipids. (B) Toxoplasma radiolabelled lipids produced from ¹⁴C-labelled host cell components. U₂–U₈, unknown lipids in the Arabidopsis lipid mixture. U₃ was tentatively identified as PA; +, deposit point of lipids. FFA, NEFA.