A Novel Phosphatidylinositol-Phospholipase C of Trypanosoma cruzi That Is Lipid Modified and Activated during Trypomastigote to Amastigote Differentiation

Abstract

The phosphoinositide (PI)-specific phospholipase C gene (TcPI-PLC) of the protozoan parasite Trypanosoma cruzi was cloned, sequenced, expressed in Escherichia coli, and the protein product (TcPI-PLC) was shown to have enzymatic characteristics similar to those of mammalian ␦-type PI-PLCs. The TcPI-PLC gene is expressed at high levels in the epimastigote and amastigote stages of the parasite, and its expression is induced during the differentiation of trypomastigotes into amastigotes, where TcPI-PLC associates with the plasma membrane and increases its catalytic activity. In contrast to other PI-PLCs described so far, the deduced amino acid sequence of TcPI-PLC revealed some unique features such as an N-myristoylation consensus sequence at its aminoterminal end, lack of an apparent pleckstrin homology domain and a highly charged linker region between the catalytic X and Y domains. Many pathogenic parasites have developed the ability to live in two distinct hosts, one vertebrate and the other invertebrate. Such parasites include Trypanosoma cruzi, the etiologic agent of Chagas' disease or American trypanosomiasis. During its life cycle, T. cruzi has to adapt to environments of different temperature, osmolarity, ionic composition, and pH, and some of these adaptation processes are paralleled by morphological and functional changes. Very litttle is known about the signaling mechanisms involved in these processes.Phosphoinositide-specific phospholipases C (PI-PLCs) 1 catalyze the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP 2 ) to D-myo-inositol-1,4,5-trisphosphate (IP 3 ) and sn-1,2-diacylglycerol (DAG) (1, 2). Both products of this reaction function as second messengers in eukaryotic signal transduction cascades. The soluble IP 3 triggers release of calcium from intracellular stores (1). The membrane-resident DAG controls cellular protein phosphorylation states by activating various protein kinase C isozymes (2). Three classes of mammalian PI-PLCs with 10 different isozymes have been characterized (␤1-␤4, ␥1-␥2, and ␦1-␦4) (3). The activity of ␤-and ␥-isozymes (145-150 kDa) is regulated by G protein-coupled and tyrosine kinase-linked receptors, respectively. These isozymes are related to the much smaller ␦-isozymes (ϳ85 kDa) (3). It seems very likely that PI-PLC-␦ evolved first, because every PI-PLC cloned so far from a non-mammalian species (for example, Dictyostelium, yeast, higher plants, and Chlamydomonas) is clearly a ␦-isoform (3). It is currently not known how ␦-isozymes are regulated in vivo (4). It is possible that they are regulated only by calcium ions (3) although the idea of PI-PLC-␦ being regulated by GTP-binding proteins is one which has increasing support (3, 5). Results from several laboratories (6 -8) have suggested that, at least in yeasts, PI-PLC-␦ is required for a number of nutritional and stress-related responses. It has also been postulated that PI-PLC-␦ could have a role in differentiation of Dictyostelium discoideum (9). Transcription of this PI-PLC-␦ appears to be enhanced dur...

A Functional Aquaporin Co-Localizes with the Vacuolar Proton Pyrophosphatase to Acidocalcisomes and the Contractile Vacuole Complex of Trypanosoma cruzi

“…Densitometric analyses of Northern blots were done with an ISI-1000 digital imaging system (Alpha Inotech Corp.). Comparison of levels of TcAQP transcription between the different stages was performed by taking as a reference the densitometric values obtained with the TcP0 transcripts and assuming a similar level of expression of this gene in all stages (20). Similar results were obtained when the densitometric values were compared by taking into account the amount of RNA added to each lane.…”

“…All Southern and Northern blots were visualized by autoradiography. The TcP0 (T. cruzi ribosomal protein 1) fragment used as a control in Northern blots was obtained as described before (20). Densitometric analyses of Northern blots were done with an ISI-1000 digital imaging system (Alpha Inotech Corp.).…”

Toxoplasma gondii Microneme Secretion Involves Intracellular Ca2+ Release from Inositol 1,4,5-Triphosphate (IP3)/Ryanodine-sensitive Stores

“…Consequently, parasites likely contain only the ancestral PLC-␦ isoform. Consistent with this idea, T. cruzi contains a PLC-␦ isoform (41) and PLC-␦ candidate sequences are present in the Plasmodium falciparum and Plasmodium vivax genomes (42). While not conventionally associated with Ca 2ϩ signaling, PLC-␦ has recently been linked to Ca 2ϩ transients associated with fertilization in mice (43).…”

Trypanosoma brucei Vacuolar Transporter Chaperone 4 (TbVtc4) Is an Acidocalcisome Polyphosphate Kinase Required for in Vivo Infection

“…Both TbVtc1 and TbVtc4 were reported to be palmitoylated. However, our attempts to demonstrate palmitoylation of TbVtc4 in PCF following established protocols (48,49) were unsuccessful (data not shown). In summary, the essentiality of TbVtc4 for growth and establishment of an efficient infection suggests that this enzyme is a potential drug target and that it would be possible to develop inhibitors.…”