Growth/Development of T. cruzi:An Emerging AIDS Pathogen

Yeshiva University, New York NY

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Abstract

DESCRIPTION (provided by applicant): Chagas' disease is one of the leading causes of cardiac and gastrointestinal disease in Latin America and has become an AIDS-defining opportunistic infection in the endemic areas. The long-term goals of these proposed studies are to investigate the role of reversible serine/threonine phosphorylation on the growth and differentiation of Trypanosoma cruzi, a hemoflagellate that causes Chagas' disease. Knowledge of the mechanisms underlying control of growth and differentiation of T. cruzi is essential for identifying unique targets for chemotherapeutic intervention. Reversible phosphorylation on serine/threonine residues is an important component in the control of a wide variety of cellular processes in higher eukaryotes. Evidence is emerging to show that similar pathways exist in T. cruzi and other lower protozoan parasites. The availability of potent and highly specific natural product inhibitors towards specific members of the PPP phosphatase gene family have allowed investigation of their role in the regulation of cellular processes. In T. cruzi, calyculin A, but not okadaic acid, prevents the completion of cytokinesis and disrupts the morphology of epimastigotes. Others have shown that calyculin A, but not okadaic acid, stimulated the differentiation of trypomastigotes into amastigotes. The differential sensitivities between calyculin A and okadaic acid observed in these studies suggest that PP1-related phosphatases are important to the growth and development of the parasite. In higher eukaryotes, PP1-like enzymes are involved in the regulation of a diverse range of cellular functions including metabolism, cell motility, cell cycle progression and the control of cell morphology. The investigators have cloned two PP1-related protein phosphatase genes (TcPP1alpha and TcPPabeta) from T. cruzi by homology cloning using degenerate primers against conserved domains in mammalian PPP-type phosphatases. Importantly, the deduced amino acid sequence of both genes contains the motif NYCGEG in their C-terminal region. This sequence motif is a major determinant in the differential sensitivity of mammalian PP1 towards calyculin A and okadaic acid. The overall goal of this proposal is to demonstrate that the inhibition of cytokinesis and morphological defects induced in T. cruzi epimastigotes by the phosphatase inhibitor calyculin A are mediated through the inhibition of these PP1-related protein phosphatases. The specific aims are: 1) Identify and characterize specific regulatory/targeting proteins for TcPP1alpha and TcPP1beta in T. cruzi, 2) Determine the domains responsible for targeting the trypanosomal phosphatases to specific location in the cell, 3) Determine the subcellular localization and the mRNA/protein expression levels of TcPP1alpha and TcPP1beta and their respective regulatory/targeting proteins in the various developmental forms, and 4) Characterize the role of these phosphatases in the biology of T. cruzi by overexpression of mutant phosphatases and by targeted gene disruption.