Purine nucleotides function in a variety of vital cellular and metabolic processes including energy production, cell signaling, synthesis of vitamin-derived cofactors and nucleic acids, and as determinants of cell fate. Unlike their mammalian and insect hosts, Leishmania cannot synthesize the purine ring de novo and are absolutely dependent upon them to meet their purine requirements. The obligatory nature of purine salvage in these parasites, therefore, offers an attractive paradigm for drug targeting, and consequently, the delineation of the pathway has been under scientific investigation for over 30 years. Here we review recent developments that reveal how purines flux in Leishmania and offer a potential ‘Achilles’ heel’ for future validation.

Mutations within the polyamine biosynthetic pathway of Leishmania donovani, the etiological agent of visceral leishmaniasis, confer polyamine auxotrophy to the insect vector or promastigote form of the parasite. However, whether the infectious or amastigote form of the parasite requires an intact polyamine pathway has remained an open question. To address this issue, conditionally lethal ⌬odc mutants lacking ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, were created by double targeted gene replacement within a virulent strain of L. donovani. ODC-deficient promastigotes and axenic amastigotes were auxotrophic for polyamines and capable of robust growth only when exogenous putrescine was supplied in the culture medium, confirming that polyamine biosynthesis is an essential nutritional pathway for L. donovani promastigotes. To assess whether the ⌬odc lesion also affected the ability of amastigotes to sustain a robust infection, macrophage and mouse infectivity experiments were performed. Parasite loads in murine macrophages infected with each of two independent ⌬odc knockout lines were decreased ϳ80% compared to their wild-type counterpart. Furthermore, ␣-difluoromethylornithine, a suicide inhibitor of ODC, inhibited growth of wild-type L. donovani amastigotes and effectively cured macrophages of parasites, thereby preventing host cell destruction. Strikingly, however, parasitemias of both ⌬odc null mutants were reduced by 6 and 3 orders of magnitude, respectively, in livers and spleens of BALB/c mice. The compromised infectivity phenotypes of the ⌬odc knockouts in both macrophages and mice were rescued by episomal complementation of the genetic lesion. These genetic and pharmacological studies strongly implicate ODC as an essential cellular determinant that is necessary for the viability and growth of both L. donovani promastigotes and amastigotes and intimate that pharmacological inhibition of ODC is a promising therapeutic paradigm for the treatment of visceral and perhaps other forms of leishmaniasis.

Studies of Leishmania donovani have shown that both ornithine decarboxylase and spermidine synthase, two enzymes of the polyamine biosynthetic pathway, are critical for promastigote proliferation and required for maximum infection in mice. However, the importance of arginase (ARG), the first enzyme of the polyamine pathway in Leishmania, has not been analyzed in L. donovani. To test ARG function in intact parasites, we generated Δarg null mutants in L. donovani and evaluated their ability to proliferate in vitro and trigger infections in mice. The Δarg knockout was incapable of growth in the absence of polyamine supplementation, but the auxotrophic phenotype could be bypassed by addition of either millimolar concentrations of ornithine or micromolar concentrations of putrescine or by complementation with either glycosomal or cytosolic versions of ARG. Spermidine supplementation of the medium did not circumvent the polyamine auxotrophy of the Δarg line. Although ARG was found to be essential for ornithine and polyamine synthesis, ornithine decarboxylase appeared to be the rate-limiting enzyme for polyamine production. Mouse infectivity studies revealed that the Δarg lesion reduced parasite burdens in livers by an order of magnitude but had little impact on the numbers of parasites recovered from spleens. Thus, ARG is essential for proliferation of promastigotes but not intracellular amastigotes. Coupled with previous studies, these data support a model in which L. donovani amastigotes readily salvage ornithine and have some access to host spermidine pools, while host putrescine appears to be unavailable for salvage by the parasite.

Purines and pyrimidines are indispensable to all life, performing many vital functions for cells: ATP serves as the universal currency of cellular energy, cAMP and cGMP are key second messenger molecules, purine and pyrimidine nucleotides are precursors for activated forms of both carbohydrates and lipids, nucleotide derivatives of vitamins are essential cofactors in metabolic processes, and nucleoside triphosphates are the immediate precursors for DNA and RNA synthesis. Unlike their mammalian and insect hosts, Leishmania lack the metabolic machinery to make purine nucleotides de novo and must rely on their host for preformed purines. The obligatory nature of purine salvage offers, therefore, a plethora of potential targets for drug targeting, and the pathway has consequently been the focus of considerable scientific investigation. In contrast, Leishmania are prototrophic for pyrimidines and also express a small complement of pyrimidine salvage enzymes. Because the pyrimidine nucleotide biosynthetic pathways of Leishmania and humans are similar, pyrimidine metabolism in Leishmania has generally been considered less amenable to therapeutic manipulation than the purine salvage pathway. However, evidence garnered from a variety of parasitic protozoa suggests that the selective inhibition of pyrimidine biosynthetic enzymes offers a rational therapeutic paradigm. In this chapter, we present an overview of the purine and pyrimidine pathways in Leishmania, make comparisons to the equivalent pathways in their mammalian host, and explore how these pathways might be amenable to selective therapeutic targeting.

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