Differentiation of Trypanosoma cruzi trypomastigotes to amastigotes inside myoblasts or in vitro, at low extracellular pH, in the presence of [3 H]palmitic acid or [ 3 H]inositol revealed differential labeling of inositolphosphoceramide and phosphatidylinositol, suggesting that a remodeling process takes place in both lipids. Using 3 H-labeled inositolphosphoceramide and phosphatidylinositol as substrates, we demonstrated the association of at least five enzymatic activities with the membranes of amastigotes and trypomastigotes. These included phospholipase A 1 , phospholipase A 2 , inositolphosphoceramide-fatty acid hydrolase, acyltransferase, and a phospholipase C releasing either ceramide or a glycerolipid from the inositolphospholipids. These enzymes may be acting in remodeling reactions leading to the anchor of mature glycoproteins or glycoinositolphospholipids and helping in the transformation of the plasma membrane, a necessary step in the differentiation of slender trypomastigotes to round amastigotes. Synthesis of inositolphosphoceramide and particularly of glycoinositolphospholipids was inhibited by aureobasidin A, a known inhibitor of fungal inositolphosphoceramide synthases. The antibiotic impaired the differentiation of trypomastigotes at acidic pH, as indicated by an increased appearance of intermediate forms and a decreased expression of the Ssp4 glycoprotein, a characteristic marker of amastigote forms. Aureobasidin A was also toxic to differentiating trypomastigotes at acidic pH but not to trypomastigotes maintained at neutral pH. Our data suggest that inositolphosphoceramide is implicated in T. cruzi differentiation and that its metabolism could provide important targets for the development of antiparasitic therapies.

Inositol phospholipids (IPL) from epimastigote forms of Trypanosoma cruzi have been investigated by metabolic labelling with [3H]palmitic acid and by GLC-MS analysis of the lipids obtained from non-labelled parasites. The IPL fraction was separated into phosphatidylinositol (PI) and inositol-phosphoceramide subfractions, the latter accounting for 80-85% of the total IPL. The neutral lipids released from the IPLs by PI-specific phospholipase C (PI-PLC) from Bacillus thuringiensis were analysed by silica-gel and reverse-phase TLC for the radioactive lipids and by GLC-MS for the non-radioactive samples. Ceramides containing dihydrosphingosine and sphingosine with C16:0 and C18:0 fatty acids were identified. The main component in the [3H]palmitic acid-labelled ceramides was palmitoyldihydrospingosine, while in the non-labelled sample the ceramides contained mainly sphingosine. This could reflect partial uptake of phospholipid from the medium. The PI contain both alkylacyl- and diacyl-glycerol lipids, with the ether lipid being more abundant. The latter was identified as 1-O-hexadecylglycerol esterified by C18:2 and C18:1 fatty acids. Interestingly, the same lipid had been identified in the anchor of the 1G7 glycoprotein of T. cruzi metacyclic forms.

The most important glycoproteins of trypanosomatids are anchored by glycoinositolphospholipids (GIPLs) to their plasma membrane. In addition, free GIPLs have been described, for instance the lipopeptidophosphoglycan (LPPG) which is a major component of the surface of T. cruzi epimastigotes. An inositolphosphoceramide (IPC) is part of the LPPG and of glycoproteins present in different stages of T. cruzi. Ceramide was not found in mammal GIPL-anchors. The lipid moieties in T. cruzi anchors can be quite variable. However, no diacylglycerol (DAG) was found in contrast with the African trypanosomes. In GIPLs of epimastigotes collected at the logarithmic phase of growth both, 1-O-hexadecyl-2-O-palmitoylglycerol and ceramide were identified. Lignoceroylsphinganine is the major ceramide, however, no lignoceric acid was detected when analysing the candidate precursors IPCs, in any of the stages of T. cruzi. An alkylglycerol has been found either as a lyso species in the Tc85 glycoprotein of trypomastigotes or acylated as in the 1G7 anchor of metacyclic forms and in the mucins of epimastigote forms. The lipid in the mucins is replaced by ceramide when the parasite differentiates to metacyclic forms. Also, in the Ssp-4 glycoprotein characteristic of amastigotes, a ceramide was identified as the anchor lipid. These variations suggest that a remodelling mechanism is working in T. cruzi. On the other hand, the oligosaccharide core in the GIPLs of T. cruzi is substituted with galactofuranose. This monosaccharide is found only in the pyranose configuration in mammalian glycoproteins and glycolipids. Thus, the biosynthetic steps for the introduction of galactofuranose and ceramide in the anchors of T. cruzi are good targets for the development of therapeutic agents.

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Modifications in content and lipid composition induced by fasting were examined in fat bodies from adults of Triatominae, Dipetalogaster maximus, Triatoma infestans and Panstrongylus megistus. With fasting, total lipid stores dropped approximately 50% for T. infestans and more than 70% for P. megistus. Total lipids analyzed by thin layer chromatography and fractionated by column chromatography on Unisil showed triacylglycerols as the main component in the three species, although P. megistus showed high levels of diacylglycerols (31-46%). Cholesterol amounted to 8-15%. In diacylglycerol fractions, C16:0, C18:1 and C18:0 fatty acids were detected; their ratio varied with species but it was not dependent on nutritional status. In triacylglycerol fractions C18:1 fatty acid was the major component at different times (48-68%); the ratio of monounsaturated to saturated in this fraction was 1.3, 2.6 and 1.2 for D. maximus, T. infestans and P. megistus respectively. The remarkable drop in lipid stores without noticeable changes in their relative composition would suggest that all types of lipid are used at similar rates. The higher content of diacylglycerols in P. megistus may be associated with the better flight performance of this species.

The lipid moiety in the glycosylphosphatidylinositol anchors of glycoproteins of Trypanosoma cruzi consists of an alkylacylglycerol, a lysoalkylglycerol or a ceramide. Previously, we showed that the inositolphosphoceramides (IPCs) are the major components in the precursor inositolphospholipids of epimastigote and trypomastigote forms. Using (3)H-labelled subfractions of IPC, phosphatidylinositol (PI) and glycoinositolphospholipids (GIPLs) as substrates with a cell-free system, we now demonstrate the association of at least five enzyme activities with the trypanosomal membranous particulate material. These include: phospholipase A(1) and phospholipase A(2), enzymes that release free fatty acid from the PI and GIPLs; an acyltransferase responsible for the acylation of the generated monoacyl or monoalkylglycerolipids with endogenous unlabelled fatty acid; two activities of phospholipase C, one releasing ceramide from IPC and the other alkylacylglycerol, alkylglycerol or diacylglycerol from PI. The neutral lipids were also generated on incubation of the GIPLs. The phospholipase C activities were inhibited by p-chloromercuriphenylsulphonic acid, as reported for other PI phospholipases C. An IPC-fatty-acid hydrolase, releasing fatty acid from the labelled IPC, was also observed. The enzyme activities reported in the present study may be acting in remodelling reactions leading to the anchor of the mature glycoproteins of T. cruzi.

A simple method is presented to esterify 1-O-hexadecyl-rac-glycerol using lipases in different organic solvents. The following fatty acids were used: C14:0, C16:0, C18:0, C18:1, and C18:2. Monoesterification was achieved by using a limiting amount of fatty acid. Both the 1-O-hexadecyl-3-O-acylglycerol and the 2-O-acylglycerol were obtained in a total yield of 75% and a ratio 7:1 in dichloromethane after 3 d. Chromatographic data for the monoesters, useful for the identification of the natural products, are given (gas-liquid chromatography, thin-layer chromatography, reverse-phase thin-layer chromatography). The structure was confirmed by a chemical synthesis of 1-O-hexadecyl-2-O-hexadecanoylglycerol. The 3-O-glyceride was also formed by acyl migration, as the minor component. The monoesters were separated by column chromatography and characterized by 1H and 13C nuclear magnetic resonance spectra.