Developmental expression of a Trypanosoma cruzi phosphoinositide-specific phospholipase C in amastigotes and stimulation of host phosphoinositide hydrolysis.

Phosphoinositide phospholipase C (PI-PLC) plays an essential role in cell signaling. A unique Trypanosoma cruzi PI-PLC (TcPI-PLC) is lipid modified in its N terminus and localizes to the outer surface of the plasma membrane of amastigotes. We show here that TcPI-PLC is developmentally regulated in amastigotes and shows two peaks of surface expression during the developmental cycle of T. cruzi, the first immediately after differentiation of trypomastigotes into amastigotes and the second before differentiation of amastigotes into trypomastigotes. Surface expression of TcPI-PLC coincides with phosphatidylinositol 4,5-bisphosphate (PIP(2)) depletion in the host cell membrane and with an increase in the levels of its product, inositol 1,4,5-trisphosphate. During extracellular differentiation, PI-PLC is secreted into the incubation medium. Maximal early expression of TcPI-PLC on the surface of amastigotes and PIP(2) depletion coincide with host cytoskeletal changes, Ca(2+) signaling, and transcriptional responses described previously. The presence of TcPI-PLC on the outer surface of the plasma membrane of the parasite and the capacity to be secreted and to alter host phospholipids are novel mechanisms of the host-parasite interaction.

Phospholipid and glycolipid composition of acidocalcisomes of Trypanosoma cruzi.

Highly purified acidocalcisomes from Trypanosoma cruzi epimastigotes were obtained by differential centrifugation and iodixanol gradient ultracentrifugation. Lipid analysis of acidocalcisomes revealed the presence of low amounts of 3beta-hydroxysterols and predominance of phospholipids. Alkylacyl phosphatidylinositol (16:0/18:2), diacyl phosphatidylinositol (18:0/18:2), diacyl phosphatidylcholine (16:0/18:2; 16:1/18:2; 16:2/18:2; 18:1/18:2 and 18:2/18:2), and diacyl phosphatidylethanolamine (16:0/18:2 and 16:1/18:2) were the only phospholipids characterized by electrospray ionization-mass spectrometry (ESI-MS). Incubation of epimastigotes with [(3)H]-mannose and isolation of acidocalcisomes allowed the detection of a glycoinositolphospholipid (GIPL) in these organelles. The sugar content of the acidocalcisomal GIPL was similar to that of the GIPL present in a microsomal fraction but the amount of galactofuranose and inositol with respect to the other monosaccharides was lower, suggesting a different chemical structure. Taken together, these results indicate that acidocalcisomes of T. cruzi have a distinct lipid and carbohydrate composition.

A lipid-modified phosphoinositide-specific phospholipase C (TcPI-PLC) is involved in differentiation of trypomastigotes to amastigotes of Trypanosoma cruzi.

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 oligonucleotide-treated 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.

Formation and remodeling of inositolphosphoceramide during differentiation of Trypanosoma cruzi from trypomastigote to amastigote.

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.