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.

The phosphoinositide-specific phospholipase C (PI-PLC) is an important component of the inositol phosphate/diacylglycerol signaling pathway. A newly discovered Trypanosoma cruzi PI-PLC (TcPI-PLC) is lipid modified in its N terminus, targeted to its plasma membrane, and believed to play a role in differentiation of the parasite because its expression increases during the differentiation of trypomastigote to amastigote stages. To determine whether TcPI-PLC is involved in this differentiation step, antisense inhibition using phosphorothioate-modified oligonucleotides, and overexpression of the gene were performed. Antisense oligonucleotidetreated parasites showed a reduced rate of differentiation in comparison to controls, as well as accumulation of intermediate forms. Overexpression of TcPI-PLC led to a faster differentiation rate. In contrast, overexpression of a mutant TcPI-PLC that lacked the lipid modification at its N terminus did not affect the differentiation rate. Therefore, TcPI-PLC is involved, when expressed in the plasma membrane, in the differentiation of trypomastigotes to amastigotes, an essential step for the intracellular replication of these parasites.Phosphatidylinositol-specific phospholipase C (PI-PLC) 1 catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP 2 ) to the second messengers diacylglycerol and inositol 1,4,5-trisphosphate (IP 3 ) (1). Diacylglycerol is the physiological activator of protein kinase C (2), and IP 3 induces the release of Ca 2ϩ from internal stores (3). Together, these second messengers cause an increase in phosphorylation of proteins that result in a cellular response. This pathway, also known as the inositol phosphate/diacylglycerol pathway, is known to regulate a large array of cellular processes, including metabolism, secretion, contraction, neural activity, and proliferation (1).Previous research has indicated that some of the signaling components of the inositol phosphate/diacylglycerol signal pathway are present in Trypanosoma cruzi, the etiologic agent of Chagas disease, and seem to be associated with cell differentiation. T. cruzi has three main developmental stages, the epimastigote that is found in the insect vector and can be grown in axenic culture, the amastigote or intracellular form, which lives in the cytosol of nucleated cells, and the trypomastigote, which is the terminal differentiation stage in the vector (metacyclic form) or is found in the bloodstream of mammalian hosts (bloodstream form). Ca 2ϩ stimulated IP 3 and diacylglycerol formation in digitonin-permeabilized epimastigotes of T. cruzi, thus suggesting the presence of a PI-PLC (4). Protein kinase C activities were characterized in T. cruzi epimastigotes (5, 6) and could be resolved into three subtypes in hydroxylapatite columns (6). The enzyme isoforms require phosphatidylserine and Ca 2ϩ for activity and are stimulated by diacylglycerol (5, 6). The presence of significant amounts of inositol phosphates in amastigotes (7) and much lower amounts in trypomastigotes (8) was al...

The protozoan parasite Trypanosoma cruzi, the agent of Chagas disease, has a large number of mucin molecules on its surface, whose expression is regulated during the life cycle. These mucins are the main acceptors of sialic acid, a monosaccharide that is required by the parasite to infect and survive in the mammalian host. A large mucin-like gene family named TcMUC containing about 500 members has been identified previously in T. cruzi. TcMUC can be divided into two subfamilies according to the presence or absence of tandem repeats in the central region of the genes. In this work, T. cruzi parasites were transfected with one tagged member of each subfamily. Only the product from the gene with repeats was highly O-glycosylated in vivo. The O-linked oligosaccharides consisted mainly of beta-d-Galp(1-->4)GlcNAc and beta-d-Galp(1-->4)[beta-d-Galp(1-->6)]-d-GlcNAc. The same glycosyl moieties were found in endogenous mucins. The mature product was anchored by glycosylphosphatidylinositol to the plasma membrane and exposed to the medium. Sera from infected mice recognized the recombinant product of one repeats-containing gene thus showing that they are expressed during the infection. TcMUC genes encode a hypervariable region at the N terminus. We now show that the hypervariable region is indeed present in the exposed mature N termini of the mucins because sera from infected hosts recognized peptides having sequences from this region. The results are discussed in comparison with the mucins from the insect stages of the parasite (Di Noia, J. M., D'Orso, I., Sánchez, D. O., and Frasch, A. C. C. (2000) J. Biol. Chem. 275, 10218-10227) which do not have variable regions.