S-Adenosylmethionine Decarboxylase fromLeishmania donovani

Abstract

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...

Arginase Plays a Pivotal Role in Polyamine Precursor Metabolism in Leishmania

“…No equivalent PTS-1 sequence is observed among 31 ARG family members that have been analyzed in detail (58,59). Moreover, the L. donovani ODC (61), ADOMETDC (20), and SPDSYN (19) polypeptides do not accommodate a PTS-1, and all three enzymes have now been immunolocalized to the cytosol (Fig. 2).…”

“…Putrescine, Spermidine, S-Adenosylmethionine, Decarboxy-S-adenosylmethionine (dAdoMet), Ornithine, and Arginine Pool AnalysesPolyamine, AdoMet, and dAdoMet pools were measured as previously reported (19,20,38). Ornithine and arginine were separated using standard ion-exchange high-performance liquid chromatography with Li/citrate elution and quantification by post-column ninhydrin derivatization (39).…”

Centrin Gene Disruption Impairs Stage-specific Basal Body Duplication and Cell Cycle Progression in Leishmania

“…Such a defect in survivability in macrophages is an indication of the lack of parasite virulence, and therefore these LdCENdeficient parasites could be further tested as a potential vaccine against leishmaniasis. Studies of gene knockouts and their effect on Leishmania virulence have also been performed by several other investigators (16,17,(35)(36)(37)(38), although such alterations did not differentiate between the promastigote and amastigote forms of Leishmania. The present report describes for the first time a gene knockout for a cytoskeletal structural protein in Leishmania and the importance of such a gene for the growth of the parasite.…”

Allosteric regulation of an essential trypanosome polyamine biosynthetic enzyme by a catalytically dead homolog

“…Knockout of the AdoMetDC gene in the trypanosomatid Leishmania donovani led to spermidine auxotropy (11). The suicide inhibitor of AdoMetDC, MDL73811, has demonstrated efficacy in animal models of both T. brucei (12) and a related parasite Trypanosoma cruzi (13), which is the causative agent of Chagas disease.…”