A Lipid-modified Phosphoinositide-specific Phospholipase C (TcPI-PLC) Is Involved in Differentiation of Trypomastigotes to Amastigotes of Trypanosoma cruzi

Abstract

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...

Global Analysis of Protein Palmitoylation in African Trypanosomes

“…A number of proteins in kinetoplastids had been previously reported to undergo thioacylation, including SMP-1 (35) and hydrophilic acylated surface proteins (5) of Leishmania major, protein phosphatases with EF hand domains of L. major and T. brucei (24), phosphoinositide-specific phospholipase C (PLC) (12,26) and FCaBP of T. cruzi (13), and glycophosphatidylinositol-PLC (28), calflagins (8,10), and CAP5.5 (15) of T. brucei. Our previous finding that calflagin palmitoylation was mediated by a single DHHC-CRD protein, TbPAT7, established the conservation in T. brucei of PAT activity by DHHC-CRD proteins (10).…”

Identification of a palmitoyl acyltransferase required for protein sorting to the flagellar membrane

“…However, the full-length protein localizes to intracellular membranes by virtue of additional protein-protein interactions. Similarly, the T. brucei protein CAP5.5 (Hertz-Fowler et al, 2001) and the T. cruzi protein TcPI-PLC (Furuya et al, 2000;Okura et al, 2005) are dually acylated, yet localize to the pellicular membrane. For CAP5.5, this is believed to be mediated by a biochemical association with subpellicular microtubules.…”

Developmental Expression of a Trypanosoma cruzi Phosphoinositide-Specific Phospholipase C in Amastigotes and Stimulation of Host Phosphoinositide Hydrolysis

“…Only 1% radioactivity was found in these trypomastigotes. Control and infected cells were incubated for 6,12,18,21,24,30,48, and 72 h. Three hours before each extraction, LiCl (15 mM) was added to the culture medium to inhibit inositol phosphatase activities. Inositol phosphates were analyzed as described before (15) at the times indicated.…”

“…However, the T. cruzi enzyme possesses N myristoylation and palmitoylation consensus sequences that have not been described in any other PI-PLC and has been shown to be N myristoylated and palmitoylated in vivo (6). It has been shown that this lipid modification is important for its plasma membrane localization and for differentiation of trypomastigotes into amastigotes (18). The enzyme has been shown recently to localize to the outer surface of the plasma membrane of amastigotes (12).…”