Parasitic protozoa are the causative agents of a plethora of devastating and often fatal diseases to humans and their domestic animals. This proposal addresses two critical issues for controlling parasitic diseases - effective chemotherapy and drug resistance. The rational design of chemotherapeutic regimens for treating, preventing, and eradicating these insidious diseases depends on a thorough understanding of the basic biochemical and metabolic machinery of the parasite, as well as on an elucidation of the molecular mechanisms by which these organisms become refractory to drugs. Most of the major metabolic pathways are similar between protozoan parasites and mammalian cells with one major exception. All parasitic protozoa studied, thus far, are auxotrophic for purines, and each genus has evolved a series of salvage enzymes which enable them to scavenge host purines. Recent evidence has also indicated that Leishmania donovani and mammalian cells differ in their pterin-folate pathway and in their polyamine content. This application will exploit genetic, biochemical, and molecular techniques to analyze four important and unique components of these metabolic pathways in Leishmania donovani; the nucleoside transporters, the purine phosphoribosyltransferases, the pterin-folate pathway, and the polyamine pathway and ornithine decarboxylase. An affinity labeling protocol will be developed further to identify the two leishmanial nucleoside transporters, and purification of the membrane permeases on affinity columns will be attempted. The genes encoding the phosphoribosyltransferases for adenine (APRT) and for hypoxanthine and guanine (HGPRT) will be cloned. The cloned genes will be used as probes to analyze the size, structure, and amount of corresponding mRNA, gene copy number, and the nature of mutations in APRT-deficient cells. The folate transporter will be purified and sequenced, and the ability of wild type and mutant parasites to interconvert pterins and folates assessed. The leishmanial ornithine decarboxylase (ODC) gene will be cloned in order to examine the sizes and amounts of mRNA and gene copy number in wild type and mutant cells that overexpress ODC. Finally, the stability of ODC protein and ODC mRNA will be assessed, and the striking drug supersensitivity of the ODC overproducing cells examined.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
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Tropical Medicine and Parasitology Study Section (TMP)
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Oregon Health and Science University
Schools of Medicine
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Boitz, Jan M; Jardim, Armando; Ullman, Buddy (2016) GMP reductase and genetic uncoupling of adenylate and guanylate metabolism in Leishmania donovani parasites. Mol Biochem Parasitol 208:74-83
Ortiz, Diana; Forquer, Isaac; Boitz, Jan et al. (2016) Targeting the Cytochrome bc1 Complex of Leishmania Parasites for Discovery of Novel Drugs. Antimicrob Agents Chemother 60:4972-82
Smith, Sabrina; Boitz, Jan; Chidambaram, Ehzilan Subramanian et al. (2016) The cystathionine-?-synthase domains on the guanosine 5''-monophosphate reductase and inosine 5'-monophosphate dehydrogenase enzymes from Leishmania regulate enzymatic activity in response to guanylate and adenylate nucleotide levels. Mol Microbiol 100:824-40
Soysa, Radika; Tran, Khoa D; Ullman, Buddy et al. (2015) Integrating ribosomal promoter vectors that offer a choice of constitutive expression profiles in Leishmania donovani. Mol Biochem Parasitol 204:89-92
Rodriguez-Contreras, Dayana; Aslan, Hamide; Feng, Xiuhong et al. (2015) Regulation and biological function of a flagellar glucose transporter in Leishmania mexicana: a potential glucose sensor. FASEB J 29:11-24
Martin, Jessica L; Yates, Phillip A; Soysa, Radika et al. (2014) Metabolic reprogramming during purine stress in the protozoan pathogen Leishmania donovani. PLoS Pathog 10:e1003938
Vald├ęs, Raquel; Elferich, Johannes; Shinde, Ujwal et al. (2014) Identification of the intracellular gate for a member of the equilibrative nucleoside transporter (ENT) family. J Biol Chem 289:8799-809
Soysa, Radika; Carter, Nicola S; Yates, Phillip A (2014) A dual luciferase system for analysis of post-transcriptional regulation of gene expression in Leishmania. Mol Biochem Parasitol 195:1-5
Boitz, Jan M; Strasser, Rona; Yates, Phillip A et al. (2013) Adenylosuccinate synthetase and adenylosuccinate lyase deficiencies trigger growth and infectivity deficits in Leishmania donovani. J Biol Chem 288:8977-90
Tran, Khoa D; Rodriguez-Contreras, Dayana; Vieira, Danielle P et al. (2013) KHARON1 mediates flagellar targeting of a glucose transporter in Leishmania mexicana and is critical for viability of infectious intracellular amastigotes. J Biol Chem 288:22721-33

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