Leishmania parasites are protozoa that cause devastating diseases in much of the tropical and subtropical world. Current drug treatments for these parasites are deficient in many respects and improved therapies founded upon the basic biochemistry of the parasites are urgently required. In this proposal we will study the role of three membrane transporters, those for glucose, myo-inositol and nucleosides, in the biology of Leishmania parasites. 1) Glucose is a major source of metabolic energy in the promastigote stage of the parasite that lives in the insect vector. In one set of experiments, the function of the Pro-1 glucose transporter genes in vivo will be studied by knocking out these genes by targeted gene replacement and testing the phenotype of the resulting Pro- 1 null mutants. In a second set of experiments, a subcellular targeting signal that exists within the flagellar isoform of Pro-1 but not within the plasma membrane isoform of Pro-1 will be identified. This will be done by mutagenizing the one domain of the flagellar isoform that differs from the plasma membrane isoform and determining which amino acids are required to target the protein to the flagellum. These experiments will begin to define a novel pathway for subcellular targeting of membrane proteins. 2) Myo-inositol is an important building block for many of the cell surface components of Leishmania parasites. In one aim of this proposal, site-directed mutagenesis will be used to identify -amino acids in the myo-inositol transporter that allow this permease to concentrate myo-inositol inside the parasite by co-transporting a proton down its electrochemical gradient. These experiments will begin a detailed structure-function analysis of this model proton symporter. 3) Purines are essential nutrients that can not be synthesized by Leishmania parasites. The final specific aim of this proposal is to clone a gene encoding a Leishmania nucleoside transporter, a membrane permease responsible for salvaging purine nucleosides from the host. This nucleoside transporter gene will be functionally expressed, and its DNA sequence will be determined to predict the amino acid sequence of the transporter. Nucleoside transporters are of considerable pharmacological importance, because they mediate the uptake of cytotoxic nucleoside analogs that are candidates for novel anti-parasite drug therapies. These experiments will initiate a detailed molecular study of these biochemically and pharmacologically important transporters in these purine auxotrophic parasites.
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