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.
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