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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
2R01AI023682-02
Application #
3135971
Study Section
Tropical Medicine and Parasitology Study Section (TMP)
Project Start
1983-06-01
Project End
1989-05-31
Budget Start
1986-06-01
Budget End
1987-05-31
Support Year
2
Fiscal Year
1986
Total Cost
Indirect Cost
Name
Oregon Health and Science University
Department
Type
Schools of Medicine
DUNS #
009584210
City
Portland
State
OR
Country
United States
Zip Code
97239
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
Valdés, 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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