Amalgamating the tools and techniques of molecular biology, genetics, biochemistry, and cell biology, this competing continuation application offers an interdisciplinary dissection of the purine salvage pathway of Leishmania donovani, the etiologic agent of visceral leishmaniasis. L. donovani, unlike the mammalian host, cannot synthesize purines de novo and consequently accommodates unique purine acquisition mechanisms to fulfill its nutritional needs. Inhibition of purine salvage, therefore, presents a selective therapeutic paradigm for treating visceral leishmaniasis. Over the last funding cycle, we have developed a model of purine salvage based largely on the conditionally lethal phenotype of an L. donovani strain (?hgprt/?xprt) that is deficient in both hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and xanthine phosphoribosyltransferase (XPRT). This model reveals a purine pathway that is multifaceted, functionally redundant, convergent, and largely compartmentalized within the glycosome. Furthermore, the model predicts that adenylosuccinate synthetase (ADSS) and adenylosuccinate lyase (ASL) are nutritionally indispensable enzymes for L. donovani and that adenine aminohydrolase (AAH), an enzyme unique to the parasite, plays a central role in purine interconversion within the parasite. The three Specific Aims of this proposal will test our model.
Specific Aim I entails a genetic and biochemical validation of the ADSS and ASL enzymes and has two components. First, we will create ?adss and ?asl knockouts by targeted gene replacement and characterize the consequent growth, metabolic, and infectivity phenotypes. Second, we will produce, purify, and characterize the recombinant ADSS and ASL enzymes.
Specific Aim II proposes a genetic and biochemical characterization of AAH and has three parts. First, we will introduce a ?aah null mutation into adenine phosphoribosyltransferase- deficient (?aprt) parasites and examine the resultant phenotypes of the ?aprt/?aah double knockout and a ?aah line that was recently created in a wild type genetic background. Second, we will establish the importance of AAH in our model of purine salvage by inserting a ?aah lesion into the ?hgprt/?xprt background and determining whether the conditionally lethal phenotype of the ?hgprt/?xprt mutant is altered. Finally, a biochemical and physico-chemical investigation on AAH will be carried out. The final Specific Aim of this proposal is to ascertain the intracellular milieu of components of the purine salvage pathway that have yet to be localized employing cell biological and/or immunocytochemical approaches. These proteins include: ADSS, ASL, GMP synthetase, the three phosphoribosylpyrophosphate synthetases, and the peroxisomal-like nucleotide translocase homolog.
The overall purpose of this proposal is to investigate and validate components of the purine salvage pathway of Leishmania donovani, the causative agent of visceral leishmaniasis, a parasitic disease that is invariably fatal if untreated. There is currently no vaccine for visceral leishmaniasis and no consistently effective chemotherapy, so there is an urgent need for new drugs and new drug targets. Because purine salvage is essential for Leishmania donovani and not for humans, our studies address the vital issue of identifying potential new targets.
|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|
|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|
|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|
|Boitz, Jan M; Ullman, Buddy (2013) Adenine and adenosine salvage in Leishmania donovani. Mol Biochem Parasitol 190:51-5|
|Soysa, Radika; Wilson, Zachary N; Elferich, Johannes et al. (2013) Substrate inhibition of uracil phosphoribosyltransferase by uracil can account for the uracil growth sensitivity of Leishmania donovani pyrimidine auxotrophs. J Biol Chem 288:29954-64|
|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|
|Arendt, Cassandra S (2013) Crithidia fasciculata adenosine transporter 1 (CfAT1), a novel high-affinity equilibrative nucleoside transporter specific for adenosine. Mol Biochem Parasitol 191:75-9|
|Boitz, Jan M; Strasser, Rona; Hartman, Charles U et al. (2012) Adenine aminohydrolase from Leishmania donovani: unique enzyme in parasite purine metabolism. J Biol Chem 287:7626-39|
|Wilson, Zachary N; Gilroy, Caslin A; Boitz, Jan M et al. (2012) Genetic dissection of pyrimidine biosynthesis and salvage in Leishmania donovani. J Biol Chem 287:12759-70|
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