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.
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