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...

Intracellular Ca2؉ in Trypanosoma cruzi is mainly located in an acidic compartment named the acidocalcisome, which among other pumps and exchangers possesses a plasma membrane-type Ca

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 plasma membrane of amastigotes. Here, we show that TcPI-PLC is located onto the extracellular phase of the plasma membrane of amastigotes and that its N-terminal 20 amino acids are necessary and sufficient to target the fused GFP to the outer surface of the parasite. Mutagenesis of the predicted acylated residues confirmed that myristoylation of a glycine residue in the 2nd position and acyl modification of a cysteine in the 4th but not in the 8th or 15th position of the coding sequence are required for correct plasma membrane localization in T. cruzi epimastigotes or amastigotes. Interestingly, mutagenesis of the cysteine at the 8th position increased its flagellar localization. When expressed as fusion constructs with GFP, the N-terminal 6 and 10 amino acids fused to GFP are predominantly located in the cytosol and concentrated in a compartment that co-localizes with a Golgi complex marker. The N-terminal 20 amino acids of TcPI-PLC associate with lipid rafts when dually acylated. Taken together, these results indicate that N-terminal acyl modifications serve as a molecular addressing system for sending TcPI-PLC to the outer surface of the cell.