A knockout strain of Leishmania donovani lacking both ornithine decarboxylase (ODC) alleles has been created by targeted gene replacement. Growth of ⌬odc cells in polyamine-deficient medium resulted in a rapid and profound depletion of cellular putrescine pools, although levels of spermidine were relatively unaffected. Concentrations of trypanothione, a spermidine conjugate, were also reduced, whereas glutathione concentrations were augmented. The ⌬odc L. donovani exhibited an auxotrophy for polyamines that could be circumvented by the addition of the naturally occurring polyamines, putrescine or spermidine, to the culture medium. Whereas putrescine supplementation restored intracellular pools of both putrescine and spermidine, exogenous spermidine was not converted back to putrescine, indicating that spermidine alone is sufficient to meet the polyamine requirement, and that L. donovani does not express the enzymatic machinery for polyamine degradation. The lack of a polyamine catabolic pathway in intact parasites was confirmed radiometrically. In addition, the ⌬odc strain could grow in medium supplemented with either 1,3-diaminopropane or 1,5-diaminopentane (cadaverine), but polyamine auxotrophy could not be overcome by other aliphatic diamines or spermine. These data establish genetically that ODC is an essential gene in L. donovani, define the polyamine requirements of the parasite, and reveal the absence of a polyamine-degradative pathway.Polyamines are cationic compounds that play essential roles in cell proliferation, differentiation, and macromolecular synthesis (1-3). Ornithine decarboxylase (ODC) 1 catalyzes the conversion of ornithine to putrescine (1,4-diaminobutane) and is the initial and rate-limiting enzyme in polyamine biosynthesis in most organisms (4). The ODC enzyme of protozoan parasites is a novel therapeutic target, because D,L-␣-difluoromethylornithine (DFMO; eflornithine), an irreversible inhibitor of ODC (5), exhibits notable efficacy against the central nervous system phase of African sleeping sickness caused by Trypanosoma brucei gambiense (3, 6). DFMO is also active against T. b. rhodesiense and T. congolense in murine models and has proven effective against other genera of protozoan parasites in vivo and in vitro, including Plasmodia (7), Giardia (8), and Leishmania (9). DFMO has been shown to induce a lethal polyamine depletion in both T. brucei (10) and L. donovani (9), the etiologic agent of visceral leishmaniasis, and toxicity to both species is ameliorated by polyamine addition (3, 9).The ability of trypanosomatids to undergo a very high frequency of homologous recombination allows the disruption of chromosomal loci with transfected drug resistance cassettes (11,12) and permits a direct test of gene function. This enables the creation of conditionally lethal parasite strains whose survival and ability to propagate are dependent upon the provision of compounds that can ameliorate the consequences of the genetic lesion. This genetic approach is predicated on the availability of c...

The polyamine biosynthetic enzyme, S-adenosylmethionine decarboxylase (ADOMETDC) has been advanced as a potential target for antiparasitic chemotherapy. To investigate the importance of this protein in a model parasite, the gene encoding ADOMETDC has been cloned and sequenced from Leishmania donovani. The ⌬adometdc null mutants were created in the insect vector form of the parasite by double targeted gene replacement. The ⌬adometdc strains were incapable of growth in medium without polyamines; however, auxotrophy could be rescued by spermidine but not by putrescine, spermine, or methylthioadenosine. Incubation of ⌬adometdc parasites in medium lacking polyamines resulted in a drastic increase of putrescine and glutathione levels with a concomitant decrease in the amounts of spermidine and the spermidine-containing thiol trypanothione. Parasites transfected with an episomal ADOMETDC construct were created in both wild type and ⌬adometdc parasites. ADOMETDC overexpression abrogated polyamine auxotrophy in the ⌬adometdc L. donovani. In addition, ADOMETDC overproduction in wild type parasites alleviated the toxic effects of 5-(((Z)-4-amino-2-butenyl)methylamino)-5-deoxyadenosine (MDL 73811), but not pentamidine, berenil, or methylglyoxyl bis(guanylhydrazone), all inhibitors of ADOMETDC activities in vitro. The molecular, biochemical, and genetic characterization of ADOMETDC establishes that it is essential in L. donovani promastigotes and a potential target for therapeutic validation.The protozoan parasite Leishmania donovani is the causative agent of visceral leishmaniasis, a devastating and often fatal disease in humans. The parasite exhibits a digenetic life cycle with the extracellular promastigote residing in the phlebotomine sandfly vector and the intracellular amastigote propagating within the phagolysosome of mammalian macrophages. Because no effective vaccine for leishmaniasis is available, chemotherapy offers the only means of disease treatment. However, the current arsenal of drugs for treating leishmaniasis is far from ideal because these compounds are moderately to highly toxic, the result of their lack of target specificity. Recently, the emergence of drug-resistant strains has exacerbated the need for more selective and efficacious drugs to treat or prevent leishmaniasis or, for that matter, many other parasitic diseases.One pathway that has been exploited successfully in antiparasitic drug regimens is that for the synthesis of polyamines, organic cations that play indispensable roles in key cellular processes such as growth, differentiation, and macromolecular biosynthesis (1, 2). D,L-␣-Difluoromethylornithine (DFMO), 1 an irreversible inhibitor of ornithine decarboxylase (ODC), the first enzyme in the polyamine biosynthetic pathway, can eradicate Trypanosoma brucei infections in both mice (3) and patients with late stage African sleeping sickness (4, 5). The selective window for drug efficacy is not achieved, however, by dissimilar DFMO binding affinities for the T. brucei and human ODC enzymes but r...

A number of anticancer and antiparasitic drugs are postulated to target the polyamine biosynthetic pathway and polyamine function, but the exact mode of action of these compounds is still being elucidated. To establish whether polyamine analogs specifically target enzymes of the polyamine pathway, a model was developed using strains of the protozoan parasite Leishmania donovani that overproduce each of the polyamine biosynthetic enzymes. Promastigotes overexpressing episomal constructs encoding ornithine decarboxylase (ODC), Sadenosylmethionine decarboxylase (ADOMETDC), or spermidine synthase (SPDSYN) revealed robust overproduction of the corresponding polyamine biosynthetic enzyme. Polyamine pools, however, were either unchanged or only marginally affected, implying that regulatory mechanisms must exist. The ODC, ADOMETDC, and SPDSYN overproducer strains exhibited a high level of resistance to difluoromethylornithine, 5-{[(Z)-4-amino-2-butenyl]methylamino}-5-deoxyadenosine, and n-butylamine, respectively, confirming previous observations that these agents specifically target polyamine enzymes. Conversely, augmented levels of polyamine biosynthetic enzymes did not affect the sensitivity of L. donovani promastigotes to pentamidine, berenil, and mitoguazone, drugs that were postulated to target the polyamine pathway, implying alternative and/or additional targets for these agents. The sensitivities of wild-type and overproducing parasites to a variety of polyamine analogs were also tested. The polyamine enzyme-overproducing lines offer a rapid cell-based screen for assessing whether synthetic polyamine analogs exert their mechanism of action predominantly on the polyamine biosynthetic pathway in L. donovani. Furthermore, the drug resistance engendered by the amplification of target genes and the overproduction of the encoded protein offers a general strategy for evaluating and developing therapeutic agents that target specific proteins in Leishmania.Polyamines are ubiquitous organic cations that play critical but still not completely defined roles in key cellular processes such as cell proliferation, differentiation, and nucleic acid synthesis (6,17,19,34,35,59,65). Since polyamines are especially important to rapidly growing cells, the polyamine pathway has been targeted in a multiplicity of antineoplastic and antiparasitic drug regimens. Most notably, DL-␣-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase (ODC), the first enzyme in the polyamine biosynthesis pathway, is effectively curative against late-stage African sleeping sickness caused by the protozoan parasite Trypanosoma brucei gambiense (3,14,57). Interestingly, the selectivity of DFMO for the metabolic machinery of the parasite is not brought about by differential sensitivities of the parasite and human ODC enzymes to inactivation by DFMO but is rather due to a novel mechanism involving disparities in ODC turnover rates between T. brucei and the mammalian host (22,23). DFMO is also active against other trypanosome spe...