Rapid kinetics of lipid second messengers controlled by a cGMP signalling network coordinates apical complex functions in Toxoplasma tachyzoites

Host cell invasion and subsequent egress by Toxoplasma parasites is regulated by a network of cGMP, cAMP, and calcium signalling proteins. Such eukaryotic signalling networks typically involve lipid second messengers including phosphatidylinositol phosphates (PIPs), diacylglycerol (DAG) and phosphatidic acid (PA). However, the lipid signalling network in Toxoplasma is poorly defined. Here we present lipidomic analysis of a mutant of central flippase/guanylate cyclase TgGC in Toxoplasma, which we show has disrupted turnover of signalling lipids impacting phospholipid metabolism and membrane stability. The turnover of signalling lipids is extremely rapid in extracellular parasites and we track changes in PA and DAG to within 5 seconds, which are variably defective upon disruption of TgGC and other signalling proteins. We then identify the position of each protein in the signal chain relative to the central cGMP signalling protein TgGC and map the lipid signal network coordinating conoid extrusion and microneme secretion for egress and invasion

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

Toxoplasma gondii is a unicellular parasite characterized by a unique extracellular and intracellular membrane compartments. 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. 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), Immunolabelling characterization supports that DGLE polar head is similar to that of DGDG. Mass spectrometry analyses pointed dihexosyl-lipids having various hydrophobic moieties (ceramide, diacylglycerol, acylalkylglycerol), that might react with anti-DGDG, but we cannot exclude that more complex dihexosyl-terminated lipids might also be immunolabelled. DGLE localization was analyzed by immunofluorescent and immunoelectron microscopy and confirmed by subcellular fractionation. No immunolabeling of the apicoplast could be observed. DGLE is scattered in pellicle membrane domains in extracellular tachyzoites and is relocalized to the anterior tip of the cell upon invasion, in an actin-dependent manner, providing insights on a possible role in pathogenetic process. DGLE was detected in other Apicomplexa, i.e. Neospora, Plasmodium, Babesia and Cryptosporidium

Rapid kinetics of lipid second messengers controlled by a cGMP signalling network coordinates apical complex functions in Toxoplasma tachyzoites

Host cell invasion and subsequent egress by Toxoplasma parasites is regulated by a network of cGMP, cAMP, and calcium signalling proteins. Such eukaryotic signalling networks typically involve lipid second messengers including phosphatidylinositol phosphates (PIPs), diacylglycerol (DAG) and phosphatidic acid (PA). However, the lipid signalling network in Toxoplasma is poorly defined. Here we present lipidomic analysis of a mutant of central flippase/guanylate cyclase TgGC in Toxoplasma , which we show has disrupted turnover of signalling lipids impacting phospholipid metabolism and membrane stability. The turnover of signalling lipids is extremely rapid in extracellular parasites and we track changes in PA and DAG to within 5 seconds, which are variably defective upon disruption of TgGC and other signalling proteins. We then identify the position of each protein in the signal chain relative to the central cGMP signalling protein TgGC and map the lipid signal network coordinating conoid extrusion and microneme secretion for egress and invasion