Arginase Plays a Pivotal Role in Polyamine Precursor Metabolism in Leishmania

Abstract

The polyamine pathway of protozoan parasites has been successfully targeted in anti-parasitic therapies and is significantly different from that of the mammalian host. To gain knowledge into the metabolic routes by which parasites synthesize polyamines and their precursors, the arginase gene was cloned from Leishmania mexicana, and ⌬arg null mutants were created by double targeted gene replacement and characterized. The ARG sequence exhibited significant homology to ARG proteins from other organisms and predicted a peroxisomal targeting signal (PTS-1) that steers proteins to the glycosome, an organelle unique to Leishmania and related parasites. ARG was subsequently demonstrated to be present in the glycosome, whereas the polyamine biosynthetic enzymes, in contrast, were shown to be cytosolic. The ⌬arg knockouts expressed no ARG activity, lacked an intracellular ornithine pool, and were auxotrophic for ornithine or polyamines. The ability of the ⌬arg null mutants to proliferate could be restored by pharmacological supplementation, either with low putrescine or high ornithine or spermidine concentrations, or by complementation with an arginase episome. Transfection of an arg construct lacking the PTS-1 directed the synthesis of an arg that mislocalized to the cytosol and notably also complemented the genetic lesion and restored polyamine prototrophy to the ⌬arg parasites. This molecular, biochemical, and genetic dissection of ARG function in L. mexicana promastigotes establishes: (i) that the enzyme is essential for parasite viability; (ii) that Leishmania, unlike mammalian cells, expresses only one ARG activity; (iii) that the sole vital function of ARG is to provide polyamine precursors for the parasite; and (iv) that ARG is present in the glycosome, but this subcellular milieu is not essential for its role in polyamine biosynthesis.

An Effect of Parasite-Encoded Arginase on the Outcome of Murine Cutaneous Leishmaniasis

“…Although knockout organisms were unable to proliferate in unsupplemented growth medium, the growth of ⌬arg L. mexicana promastigotes (14) or amastigotes (data not shown) in axenic culture was restored by the addition of either 200 M putrescine or 200 M ornithine.…”

“…The creation and characterization of the ⌬arg knockout and the ⌬arg[pArg] cell lines has been reported (14). The ⌬arg strain was isolated after two rounds of targeted gene replacement, whereas the ⌬arg[pArg] parasites possess a ⌬arg chromosomal background and harbor a multicopy episomal plasmid encompassing the arginase gene (14). All L. mexicana lines were cultivated in DME-L, a completely defined DMEM (14).…”

“…The ⌬arg strain was isolated after two rounds of targeted gene replacement, whereas the ⌬arg[pArg] parasites possess a ⌬arg chromosomal background and harbor a multicopy episomal plasmid encompassing the arginase gene (14). All L. mexicana lines were cultivated in DME-L, a completely defined DMEM (14). The growth medium for the genetically manipulated L. mexicana contained the following additions: ⌬arg knockout parasites were supplemented with 50 g/ml phleomycin, 50 g/ml hygromycin, and 100 M putrescine, and episomally complemented ⌬arg [pArg] parasites were supplemented with 50 g/ml phleomycin, 50 g/ml hygromycin, and 20 g/ml G418.…”

“…Ornithine is a key intermediate in the synthesis of glutamine, proline, and polyamines in mammalian cells (13). However, in Leishmania promastigotes the sole role of ornithine is the production of polyamines, which are ubiquitous essential cations that play critical roles in a variety of cellular processes needed by proliferating cells (14,15).…”

“…However, because the host macrophage and the parasite cells each express functional arginase enzymes, it is not clear whether one or both arginases are needed for parasite survival. The generation of Leishmania mexicana gene deletion mutants deficient in parasite arginase activity enables a dissection of the relative contributions of parasite and host cell arginases to parasite survival both in vitro in the host macrophage and in vivo within the host (14). The results suggest that parasite-derived arginase presents a novel defense mechanism that enhances parasite survival through local depletion of the iNOS substrate arginine.…”