Ornithine Decarboxylase Gene Deletion Mutants of Leishmania donovani

Abstract

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...

Arginase Plays a Pivotal Role in Polyamine Precursor Metabolism in Leishmania

“…Recently, AGM has also been identified in mammalian cells (67,68), although the existence of an arginine decarboxylase is still unresolved. The AGM pathway does not appear to contribute to the putrescine pool in Leishmania promastigotes, because ⌬odc (18) and ⌬arg promastigotes are putrescine auxotrophs. Furthermore, although ARG and AGM catalyze similar reactions and belong to the same enzyme superfamily (about 24% identity), the putative AGM does not appear to be a functional ARG, because the ⌬arg parasites displayed no residual ARG activity.…”

“…The resulting expression plasmid was designated pXG-GFPϩ2Ј-arginase. This plasmid was then transfected into wild type L. mexicana using standard electroporation conditions (17,18). Transfected parasites were selected in 20 g/ml G418 and used for localization studies.…”

“…Genetic Complementation of the ⌬arg Knockouts-The pXG-GFPϩ2Ј-arginase and pXG-GFPϩ-arg⌬skl constructs were separately transfected into ⌬arg parasites as reported (17,18). Transfected parasites were then selected in 20 g/ml G418 in DME-L-CS medium supplemented with 200 M putrescine and 200 M ornithine.…”

“…pX63-HYG-⌬arg and pX63-PHLEO⌬arg were digested with HindIII and BglII to liberate linear fragments, designated HYG-⌬arg and PHLEO-⌬arg, respectively, containing the drug resistance marker and the arginase flanking regions. HYG-⌬arg and PHLEO-⌬arg were isolated from agarose gels and then transfected into parasites using standard electroporation conditions for transfection of Leishmania promastigotes (17,18). First, HYG-⌬arg was transfected into wild type L. mexicana to create the arginase/arg heterozygote, and colonies were isolated by selection on plates of semi-solid DME-L-CS medium containing 50 g/ml hygromycin.…”

Manipulación genética y el estudio del parásito protozoario Leishmania.

“…Por ejemplo, la eliminación de los genes de enzimas clave en la síntesis de poliaminas, como la espermidina sintasa (SPDSYN) y la ornitina decarboxilasa (ODC), producen promastigotes auxótrofos para poliaminas (37,46), los cuales deben importar espermidina exógena para sobrevivir. Además, el nivel de tripanotión, tiol único en tripanosomátidos que contiene espermidina y que, además, es el componente principal de la defensa antioxidante, también se reduce.…”