Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is a key enzyme in the purine salvage pathway of many protozoan parasites. The predicted amino acid sequences of certain HGPRT proteins from parasites of the Trypanosomatidae family reveal a COOH-terminal tripeptide signal that is consistent with the degenerate topogenic signal targeting proteins to the glycosome, a fuel-metabolizing microbody unique to these parasites. To determine definitively the intracellular milieu of HG-PRT in these pathogens, polyclonal antiserum to the purified recombinant HGPRT from Leishmania donovani was generated in rabbits, and confocal and immunoelectron microscopy were employed to establish that the L. donovani HGPRT is localized exclusively to the glycosome. No HGPRT protein was detected in ⌬hgprt null mutants in which both alleles of the HGPRT locus had been replaced by a drug-resistance cassette. Transfectants of the ⌬hgprt knockout strain in which a wildtype HGPRT was amplified on an expression plasmid contained augmented amounts of HGPRT, all of which was localized to the glycosome. ⌬hgprt transfectants containing amplified copies of a mutated HGPRT construct in which the Ser-Lys-Val COOH-terminal targeting signal had been deleted expressed HGPRT throughout the parasite, including subcellular organelles such as the nucleus and flagellum. These data demonstrate that the L. donovani HGPRT is compartmentalized exclusively within the glycosome and that the COOH-terminal tripeptide of the protein is necessary to achieve targeting to this organelle.

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Homozygous null mutants of the hypoxanthine-guanine phosphoribosyltransferase (hgprt) and adenine phosphoribosyltransferase (aprt) loci were created in Leishmania donovani in which both alleles were eliminated using only a single targeting construct. Functional heterozygotes were first generated by homologous recombination after transfection with vectors containing 5-and 3-flanking regions of either the hgprt or the aprt gene circumscribing drug resistance markers. Homozygous null mutants were then isolated from the heterozygotes by negative selection in media containing subversive substrates of the encoded proteins, i.e. allopurinol for HGPRT and 4-aminopyrazolopyrimidine for APRT. The novel alleles created by homologous recombination were verified by Southern blotting, and the effects of gene replacement upon gene expression in intact parasites were evaluated by direct enzymatic assay and by immunoblotting. All mutant strains were viable under the selection conditions and exhibited appropriate drug resistance phenotypes. The ability to generate homozygous knockouts with single targeting constructs greatly facilitates the genetic dissection and subsequent biochemical investigations of the purine pathway in Leishmania and has important general implications for the genetic manipulation and analysis of the leishmanial genome.

To dissect the contributions of hypoxanthine-guanine phosphoribosyltransferase (HGPRT), adenine phosphoribosyltransferase (APRT), and adenosine kinase (AK) to purine salvage in Leishmania donovani, null mutants genetically deficient in HGPRT and/or APRT were generated by targeted gene replacement in wild type cells and preexisting mutant strains lacking either APRT or AK activity. These knockouts were obtained either by double targeted gene replacement or by single gene replacement followed by negative selection for loss-of-heterozygosity. Genotypes were confirmed by Southern blotting and the resultant phenotypes evaluated by enzymatic assay, resistance to cytotoxic drugs, ability to incorporate radiolabeled purine bases, and growth on various purine sources. All mutant strains could propagate in defined growth medium containing any single purine source and could metabolize exogenous Protozoan parasites cause a variety of devastating and often fatal diseases in humans and their domestic animals. The treatment and control of parasitic diseases, however, is severely compromised by the dearth of effective and selective antiparasitic therapies. Many of the current antiparasitic drugs cause severe toxicity in the host, a predicament that can be attributed to lack of target specificity. Moreover, these drugs are potentially mutagenic and/or carcinogenic, they often require protracted courses with multiple drug administrations, and therapeutic unresponsiveness and drug resistance have exacerbated the necessity for new and improved antiparasitic agents.The institution of a rational therapeutic regimen for the treatment and prevention of parasitic diseases hinges upon exploitation of fundamental biochemical disparities between parasite and host. Perhaps the most striking metabolic discrepancy between parasites and humans is the purine pathway.Whereas mammalian cells can synthesize the purine heterocycle de novo, all protozoan parasites studied thus far are auxotrophic for purines (1). As a consequence, each genus of parasite has evolved a unique complement of purine salvage enzymes that enable the organism to scavenge host purines. Unique features of the purine salvage pathway of Leishmania and Trypanosoma constitute the basis for the susceptibility of these genera to several pyrazolopyrimidine analogs of naturally occurring purine bases and nucleosides (2, 3). The intact parasites efficiently metabolize these analogs to the nucleotide level, whereas mammalian cells are essentially incapable of these metabolic transformations. One of these pyrazolopyrimidines, allopurinol (4-hydroxypyrazolo [3,4]pyrimidine, HPP), 1 a drug that is nontoxic to humans and is widely used in the treatment of hyperuricemia and gout (4), has demonstrated significant therapeutic efficacy in patients with either cutaneous leishmaniasis (5) or chronic Chagas disease (6).Leishmania donovani, the causative agent of visceral leishmaniasis, is a digenetic parasite that exists as an extracellular promastigote within the insect vector, members of the ph...

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