Parasitic protozoa are the etiological agents for a variety of devastating and lethal diseases in humans and animals. The rational design of chemotherapeutic regimens for treating these diseases requires knowledge of metabolic differences between the parasite and the host. Virtually all the metabolic pathways in protozoan parasites and mammals are similar with one exception. All the parasitic protozoa studied thus far are incapable of synthesizing purines and are therefore auxotrophic for purines. They have evolved a unique series of purine salvage enzymes which enable them to scavenge host purines. This proposal offers a genetic approach to study three important components of the purine salvage system of Leishmania donovani; the nucleoside transport functions, adenine phosphoribosyltransferase(APRTase), and hypoxanthine-guanine phosphoribosyltransferase (GHPRTase). The multiplicity, substrate specificities and kinetic parameters of the nucleoside transport systems will be determined using both mutants deficient in transport and a rapid sampling kinetic assay. These mutants will be exploited to identify the transport proteins in wildtype cells by cell surface iodination and photoaffinity and affinity labelling techniques. The role of APRTase in adenine salvage by the intracellular stage of the parasite will be studied using wildtype and APRTase-deficient organisms. Polyclonal antisera and monoclonal antibodies directed against the leishmanial APRTase will be prepared. These immunological reagents will be used to determine whether APRTase-deficient cells synthesize immunoprecipitable APRTase protein. DNA probes corresponding to a portion or all of the APRTase gene will be isolated by screening Lambdagtll libraries with antisera and by functional complementation of APRTase-deficient yeast. These DNA probes will be used to examine the in vivo transcription products of the gene(s) coding for APRTase in wildtype and APRTase-deficient Leishmania. Finally, the postualted critical role of HGPRTase to purine salvage in Leishmania will be assessed by isolating and characterizing HGPRTase-deficient mutants and by generating immunological reagents directed against the HGPRTase protein.
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