Toxoplasma gondii grown in human cells uses GalNAc-containing glycosylphosphatidylinositol precursors to anchor surface antigens while the immunogenic Glc–GalNAc-containing precursors remain free at the parasite cell surface

Toxoplasma gondii is a unicellular parasite characterized by unique extracellular and intracellular membrane compartments. The lipid composition of subcellular membranes has not been determined, limiting our understanding of lipid homeostasis, control, and trafficking, a series of processes involved in pathogenesis. In addition to a mitochondrion, Toxoplasma contains a plastid called the apicoplast. The occurrence of a plastid raised the question of the presence of chloroplast galactolipids. Using three independent rabbit and rat antibodies against digalactosyldiacylglycerol (DGDG) from plant chloroplasts, we detected a class of Toxoplasma lipids harboring a digalactolipid-like epitope (DGLE). Immunolabeling characterization supports the notion that the DGLE polar head is similar to that of DGDG. Mass spectrometry analyses indicated that dihexosyl lipids having various hydrophobic moieties (ceramide, diacylglycerol, and acylalkylglycerol) might react with anti-DGDG, but we cannot exclude the possibility that more complex dihexosyl-terminated lipids might also be immunolabeled. DGLE localization was analyzed by immunofluorescence and immunoelectron microscopy and confirmed by subcellular fractionation. No immunolabeling of the apicoplast could be observed. DGLE was scattered in pellicle membrane domains in extracellular tachyzoites and was relocalized to the anterior tip of the cell upon invasion in an actin-dependent manner, providing insights on a possible role in pathogenetic processes. DGLE was detected in other Apicomplexa (i.e

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“…Detergent-resistant membranes (DRMs) were isolated from Toxoplasma tachyzoites according to the procedure described previously (48).…”

Section: Purification and Analysis Of Toxoplasma Detergent-resistant mentioning

confidence: 99%

Subcellular localization and dynamics of a digalactolipid-like epitope in Toxoplasma gondii

Botté

Saïdani

Mondragón

et al. 2008

Journal of Lipid Research

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“…Parasites and Cell Culture-RH strain T. gondii tachyzoites were grown in Vero cells (20). Confluent cell cultures (175 cm 2 ) were infected with 5 ϫ 10 7 tachyzoites in Dulbecco's modified Eagle's medium supplemented with 1% (v/v) fetal calf serum.…”

Section: Materials-d-[6-mentioning

confidence: 99%

“…Extraction of Glycolipids-All in vivo and in vitro labeled glycolipid products were extracted three times in chloroform/ methanol/water (10:10:3, v/v), dried, and recovered from a butan-1-ol partitioning, as previously described (20).…”

Section: Materials-d-[6-mentioning

confidence: 99%

“…Identification of the T. gondii genes for the GPI biosynthetic pathway using homologues from P. falciparum, human, and yeast has been observed in the GPI pathways of other organisms (20,21,33). Thus, the formation of lyso-mature GPI intermediates (Step 11, Fig.…”

Section: Tablementioning

confidence: 99%

“…In the case of T. gondii a side chain consisting of glucose ␣1-4N-acetylgalactosamine linked to the first mannose has been identified (15). It has been shown that free GPIs (the so called "low molecular weight antigen" (16,17)) present in large amounts on the T. gondii surface are highly immunogenic in humans (18), some able to induce tumor necrosis factor-␣ production in macrophages (19,20).…”

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confidence: 99%

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The Role of Inositol Acylation and Inositol Deacylation in the Toxoplasma gondii Glycosylphosphatidylinositol Biosynthetic Pathway

Smith

Kimmel

Azzouz

et al. 2007

Journal of Biological Chemistry

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Toxoplasma gondii is a ubiquitous parasitic protozoan that invades nucleated cells in a process thought to be in part due to several surface glycosylphosphatidylinositol (GPI)-anchored proteins, like the major surface antigen SAG1 (P30), which dominates the plasma membrane. The serine protease inhibitors phenylmethylsulfonyl fluoride and diisopropyl fluoride were found to have a profound effect on the T. gondii GPI biosynthetic pathway, leading to the observation and characterization of novel inositol-acylated mannosylated GPI intermediates. This inositol acylation is acyl-CoA-dependent and takes place before mannosylation, but uniquely for this class of inositol-acyltransferase, it is inhibited by phenylmethylsulfonyl fluoride. The subsequent inositol deacylation of fully mannosylated GPI intermediates is inhibited by both phenylmethylsulfonyl fluoride and diisopropyl fluoride. The use of these serine protease inhibitors allows observations as to the timing of inositol acylation and subsequent inositol deacylation of the GPI intermediates. Inositol acylation of the non-mannosylated GPI intermediate D-GlcN␣1-6-D-myo-inositol-1-HPO 4 -sn-lipid precedes mannosylation. Inositol deacylation of the fully mannosylated GPI intermediate allows further processing, i.e. addition of GalNAc side chain to the first mannose. Characterization of the phosphatidylinositol moieties present on both free GPIs and GPI-anchored proteins shows the presence of a diacylglycerol lipid, whose sn-2 position contains almost exclusively an C18:1 acyl chain. The data presented here identify key novel inositol-acylated mannosylated intermediates, allowing the formulation of an updated T. gondii GPI biosynthetic pathway along with identification of the putative genes involved.

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Chemical Synthesis of GPI Anchors and GPI‐Anchored Molecules

Vilotijević

Götze

Seeberger

et al. 2013

Modern Synthetic Methods in Carbohydrate Chemistry

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Toxoplasma gondii grown in human cells uses GalNAc-containing glycosylphosphatidylinositol precursors to anchor surface antigens while the immunogenic Glc–GalNAc-containing precursors remain free at the parasite cell surface

Cited by 30 publications

References 45 publications

Subcellular localization and dynamics of a digalactolipid-like epitope in Toxoplasma gondii

Subcellular localization and dynamics of a digalactolipid-like epitope in Toxoplasma gondii

The Role of Inositol Acylation and Inositol Deacylation in the Toxoplasma gondii Glycosylphosphatidylinositol Biosynthetic Pathway

Chemical Synthesis of GPI Anchors and GPI‐Anchored Molecules

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