Arginase Is Essential for Survival of Leishmania donovani Promastigotes but Not Intracellular Amastigotes

Abstract

Studies of Leishmania donovani have shown that both ornithine decarboxylase and spermidine synthase, two enzymes of the polyamine biosynthetic pathway, are critical for promastigote proliferation and required for maximum infection in mice. However, the importance of arginase (ARG), the first enzyme of the polyamine pathway in Leishmania, has not been analyzed in L. donovani To test ARG function in intact parasites, we generated Δarg null mutants in L. donovani and evaluated their ability to proliferate in vitro and trigger infections in mice. The Δarg knockout was incapable of growth in the absence of polyamine supplementation, but the auxotrophic phenotype could be bypassed by addition of either millimolar concentrations of ornithine or micromolar concentrations of putrescine or by complementation with either glycosomal or cytosolic versions of ARG. Spermidine supplementation of the medium did not circumvent the polyamine auxotrophy of the Δarg line. Although ARG was found to be essential for ornithine and polyamine synthesis, ornithine decarboxylase appeared to be the rate-limiting enzyme for polyamine production. Mouse infectivity studies revealed that the Δarg lesion reduced parasite burdens in livers by an order of magnitude but had little impact on the numbers of parasites recovered from spleens. Thus, ARG is essential for proliferation of promastigotes but not intracellular amastigotes. Coupled with previous studies, these data support a model in which L. donovani amastigotes readily salvage ornithine and have some access to host spermidine pools, while host putrescine appears to be unavailable for salvage by the parasite.

Keywords: Leishmania; arginase; polyamines.

FIG 1

Genotypic and phenotypic analysis of genetically manipulated parasites. (A, B, and C) Genomic DNA from the following parasite strains was used as the template: lanes 1, wild type; lanes 2, ARG/arg heterozygote; lanes 3, Δarg mutant 1; lanes 4, Δarg mutant 2; lanes 5, Δarg[ARG] mutant; lanes 6, Δarg[argΔAKL] mutant. (A) Primers designed to encompass the coding region were used to amplify the ARG gene. (B) The forward primer sequence was located in a region upstream of the 5′ flanking sequence (upstream of the targeting construct), and the reverse primer sequence was located within the hygromycin resistance gene. (C) The same sense primer as that used for panel B was used, and the antisense primer sequence was located within the phleomycin resistance gene. (D) Western blot analyses were performed with cell extracts prepared from wild-type, ARG/arg heterozygote, Δarg mutant 1, Δarg mutant 2, Δarg[ARG], and Δarg[argΔAKL] parasites. Parasites were fractioned by SDS-PAGE and the blot probed with polyclonal antibodies against L. mexicana ARG and a commercial monoclonal antibody that recognizes tubulin. The tubulin antibody was employed to verify equal loading of protein on all lanes.

FIG 2

Localization of ARG and argΔAKL. Wild-type (A to C), Δarg (D to F), Δarg[ARG] (G to I), or Δarg[argΔAKL] (J to L) L. donovani promastigotes were subjected to immunofluorescence analysis using rabbit anti-ARG polyclonal antibodies (A, D, G, and J). Goat anti-rabbit Oregon green-conjugated secondary antibody was used to detect the ARG primary antibodies. Parasites were also stained with DAPI (B, E, H, and K) and photographed using differential interference contrast (DIC) (C, F, I, and L).

FIG 3

Growth phenotypes of genetically modified promastigotes. (A) Growth phenotypes of wild-type, ARG/arg, Δarg mutant 1, Δarg mutant 2, Δarg[ARG], and Δarg[argΔAKL] promastigotes in unsupplemented medium (black bars) or medium supplemented with 100 μM putrescine (gray bars) or 100 μM ornithine (white bars) are shown. (B) Growth phenotypes of the Δarg parasites in unsupplemented medium and medium supplemented with three different concentrations (100 μM, 500 μM, and 1,000 μM) of ornithine (orn.), putrescine (put.), or spermidine (spd.) are depicted. Parasites were incubated at a density of 5 × 10⁵ parasites/ml, and cell viability was evaluated after 5 days by resazurin-to-resorufin conversion. The capacity of wild-type parasites to metabolize resazurin in unsupplemented medium was equated to 100% proliferation. The experiments were set up in duplicate and repeated three times with similar results.

FIG 4

Requirements for ornithine and putrescine in Δarg promastigotes and axenic amastigotes. Δarg promastigotes (A) and Δarg axenic amastigotes (B) were incubated in serial dilutions of putrescine (black circles) or ornithine (gray squares). Supplement concentrations ranged from 1 to 2,000 μM. Promastigotes were seeded at 5 × 10⁵/ml in 96-well plates and incubated in DME-L at 27°C, while axenic amastigotes were seeded at 5 × 10⁵/ml in 96-well plates and incubated in a modified acidic medium at 37°C. After 5 days, the ability of parasites to metabolize resazurin was assessed by fluorescence, and readings obtained with the highest supplement concentrations equated to 100% proliferation. The experiments were set up in duplicate and repeated three times with similar results.

FIG 5

Ornithine requirement for Δarg[ODC] promastigotes. (A) Western blot analysis was performed with cell lysates prepared from wild-type parasites, the parental Δarg line, and the Δarg[ODC] ODC overproducer strain. Parasite lysates were fractioned by SDS-PAGE and the blot probed with polyclonal antibodies against L. donovani ODC and an anti-tubulin antibody as a loading control. (B) Growth phenotypes of Δarg (gray squares) and Δarg[ODC] (black triangles) promastigotes were established in increasing concentrations of ornithine. Parasites were incubated at 5 × 10⁵ parasites/ml, and percent proliferation was evaluated after 5 days via the ability of parasites to convert resazurin to resorufin as assessed by fluorescence, and readings obtained with the highest supplement concentrations were equated with 100% proliferation. The experiments were set up in duplicate and repeated three times with similar results.

FIG 6

Parasite burden in mice infected with wild-type and genetically modified parasites. Five separate groups of five BALB/c mice were infected with either wild-type, Δarg mutant 1, Δarg mutant 2, Δarg[ARG], or Δarg[argΔAKL] stationary-phase promastigotes via tail vein inoculation. Mice were sacrificed after 4 weeks, and parasite loads in liver (A) or spleen (B) preparations were determined by limiting dilution. Statistical analyses were performed using the paired t test and revealed significant differences in liver infectivity between wild-type and Δarg mutant 1 parasites, as well as between the wild type and the Δarg mutant 2 strain (denoted with an asterisk).

FIG 7

Polyamine salvage model for promastigotes and intracellular amastigotes. (A) L. donovani promastigotes efficiently transport arginine, ornithine, and polyamines. Arginine is an essential amino acid (88, 89), while promastigotes are able to synthesize ornithine and polyamines endogenously. Putrescine and spermidine, but not ornithine, are essential for parasite growth and survival, and gene deletion mutants depend on polyamine supplementation of the media. (B) L. donovani amastigotes reside inside the phagolysosome (shaded gray) of host macrophages. Our data support a model wherein ornithine salvage is hypothesized to be efficient, while spermidine and especially putrescine salvage pools are too limited to support robust infections. Although amastigotes have putrescine and spermidine transporters (50, 51), both polyamines have been shown to be rapidly metabolized to spermine in macrophages (69) and thus are not accessible to the intracellular parasite. Spermine cannot meet the polyamine requirements of Leishmania parasites (33).