This grant application offers an interdisciplinary approach to the characterization of key enzymes of the polyamine biosynthetic pathway of Leishmania and Trypanosoma brucei. Amalgamating techniques from genetics, molecular biology, biochemistry, and structural biology, this proposal provides a blueprint for validating polyamine enzymes as antiparasitic targets. This application will focus on the ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (ADOMETDC), and spermidine synthase (SPDSYN) proteins from Leishmania donovani and the ADOMETDC from T. brucei. My laboratory has cloned and sequenced the genes encoding these proteins, and these molecular reagents will provide the foundation for the genetic manipulations and biochemical analyses described in this proposal. In addition, we have created and characterized homozygous deltaODC null mutants of L. donovani by targeted gene replacement, and constructs of ODC and ADOMET have been overexpressed in E. coli. There are four specific aims. The first specific aim will involve tests of polyamine gene function employing targeted gene replacement strategies. Homozygous null mutants of L. donovani, L. mexicana, and T. brucei with generic lesions in polyamine loci will be created by homologous recombination and characterized with respect to growth phenotype, polyamine metabolic capacity, and when relevant, infectivity.
Specific Aim II will verify genetically whether polyamine enzymes are the primary cellular targets of a spectrum of polyamine antimetabolites in intact parasites. Parasites that overexpress each of the polyamine genes will be generated and examined for their growth sensitivities to a spectrum of well characterized polyamine antimetabolites. The penultimate specific aim of this application is to evaluate potentially novel mechanisms of drug resistance in L. donovani promastigotes for their refractoriness to polyamine antimetabolites. Finally, we will perform a thorough biochemical characterization of the L. donovani and T. brucei ADOMETDC and L. donovani SPDSYN proteins. These genes will be overexpressed in bacteria and the recombinant proteins purified to homogeneity for kinetic, physicochemical, and preliminary crystallization studies.
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