ABSTRACT
Phosphatidylinositol (PtdIns) metabolism through phosphatidylinositol kinase (PIKs) activities plays a central role in different signaling pathways. In Trypanosoma cruzi, causative agent of Chagas disease, PIKs have been proposed as target for drug design in order to combat this pathogen. In this work, we studied the classes of PI4K, PIPK and PI3K that could participate in signaling pathways in T. cruzi epimastigote forms. For this reason, we analyzed their enzymatic parameters and detailed responses to avowed kinase inhibitors (adenosine, sodium deoxycholate, wortmannin and LY294002) and activators (Ca(2+), phosphatidic acid, spermine and heparin). Our results suggest the presence and activity of a class III PI4K, a class I PIPK, a class III PI3K previously described (TcVps34) and a class I PI3K. Class I PI3K enzyme, here named TcPI3K, was cloned and expressed in a bacterial system, and their product was tested for kinase activity. The possible participation of TcPI3K in central cellular events of the parasite is also discussed.
Subject(s)
Chagas Disease/parasitology , Phosphatidylinositol 3-Kinases/metabolism , Phosphatidylinositols/metabolism , Protozoan Proteins/metabolism , Trypanosoma cruzi/enzymology , Trypanosoma cruzi/growth & development , Cloning, Molecular , Drug Design , Enzyme Activators/pharmacology , Enzyme Inhibitors/pharmacology , Humans , Phosphatidylinositol 3-Kinases/classification , Phosphoinositide-3 Kinase Inhibitors , Phosphorylation , Phylogeny , Protozoan Proteins/antagonists & inhibitors , Protozoan Proteins/classification , Signal TransductionABSTRACT
Trypanosoma cruzi undergoes differentiation in the rectum of triatomine, where increased osmolarity is caused mainly by elevated content of NaCl from urine. Early biochemical events in response to high osmolarity in this parasite have not been totally elucidated. In order to clarify the relationship between these events and developmental stages of T. cruzi, epimastigotes were subjected to hyperosmotic stress, which caused activation of Na(+)/H(+) exchanger from acidic vacuoles and accumulation of inositol trisphosphate (InsP(3)). Suppression of InsP(3) levels was observed in presence of intracellular Ca(2+) chelator or pre-treatment with 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), which also inhibited the alkalinization of acidic vacuoles via a Na(+)/H(+) exchanger and the consequent increase in cytosolic calcium. These effects were activated and inhibited by PMA and Chelerythrine respectively, suggesting regulation by protein kinase C. The T. cruzi Na(+)/H(+) exchanger, TcNHE1, has 11 transmembrane domains and is localized in acidic vacuoles of epimastigotes. The analyzed biochemical changes were correlated with morphological changes, including an increase in the size of acidocalcisomes and subsequent differentiation to an intermediate form. Both processes were delayed when TcNHE1 was inhibited by EIPA, suggesting that these early biochemical events allow the parasite to adapt to conditions faced in the rectum of the insect vector.
