Leishmania donovani ornithine decarboxylase is indispensable for parasite survival in the mammalian host

Abstract

Mutations within the polyamine biosynthetic pathway of Leishmania donovani, the etiological agent of visceral leishmaniasis, confer polyamine auxotrophy to the insect vector or promastigote form of the parasite. However, whether the infectious or amastigote form of the parasite requires an intact polyamine pathway has remained an open question. To address this issue, conditionally lethal Deltaodc mutants lacking ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, were created by double targeted gene replacement within a virulent strain of L. donovani. ODC-deficient promastigotes and axenic amastigotes were auxotrophic for polyamines and capable of robust growth only when exogenous putrescine was supplied in the culture medium, confirming that polyamine biosynthesis is an essential nutritional pathway for L. donovani promastigotes. To assess whether the Deltaodc lesion also affected the ability of amastigotes to sustain a robust infection, macrophage and mouse infectivity experiments were performed. Parasite loads in murine macrophages infected with each of two independent Deltaodc knockout lines were decreased approximately 80% compared to their wild-type counterpart. Furthermore, alpha-difluoromethylornithine, a suicide inhibitor of ODC, inhibited growth of wild-type L. donovani amastigotes and effectively cured macrophages of parasites, thereby preventing host cell destruction. Strikingly, however, parasitemias of both Deltaodc null mutants were reduced by 6 and 3 orders of magnitude, respectively, in livers and spleens of BALB/c mice. The compromised infectivity phenotypes of the Deltaodc knockouts in both macrophages and mice were rescued by episomal complementation of the genetic lesion. These genetic and pharmacological studies strongly implicate ODC as an essential cellular determinant that is necessary for the viability and growth of both L. donovani promastigotes and amastigotes and intimate that pharmacological inhibition of ODC is a promising therapeutic paradigm for the treatment of visceral and perhaps other forms of leishmaniasis.

FIG. 1.

Expression of polyamine biosynthetic pathway proteins in L. donovani promastigotes and amastigotes. Lysates of wild-type L. donovani promastigotes (Pro) and axenic amastigotes (Am) were prepared from exponentially growing parasites and equal amounts of protein from both extracts fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After blotting onto nitrocellulose filters was done, the blots were probed with antibodies to ODC (A), ADOMETDC (B), SPDSYN (C), A2, an amastigote-specific protein (59) (D), or tubulin (E). The sources of these antibodies have been reported (46, 59). Molecular mass markers are indicated to the left of each blot.

FIG. 2.

Molecular characterization of the ODC locus and ODC expression in the Δodc knockouts. Genomic DNA from wild-type, Δodc1, and Δodc2 parasites and protein lysates from wild-type, Δodc1, Δodc2, Δodc1[pXG-BSD-ODC], and Δodc2[pXG-BSD-ODC] parasites were obtained by standard protocols. (A) 2 μg of genomic DNA from wild-type, Δodc1, or Δodc2 parasites was digested with SalI and hybridized to a 1.0-kb fragment of the ODC coding region that was prepared by PCR. Molecular size markers are indicated to the left. (B) Ethidium bromide-stained gel that was employed in the Southern blot in panel A. (C) Cell extracts prepared from 1.0 × 10⁷ wild-type, Δodc1, Δodc2, Δodc1[pXG-BSD-ODC] (Δodc1[pODC]), or Δodc[pXG-BSD-ODC] (Δodc2[pODC]) promastigotes were fractionated by sodium dodecyl sulfate electrophoresis and the blots probed with polyclonal antibodies against L. donovani ODC and a commercial monoclonal antibody that recognizes tubulin. The tubulin antibody was employed to verify equivalent loading of protein on all lanes.

FIG. 3.

Growth phenotypes of Δodc promastigotes and axenic amastigotes. Wild-type, Δodc1, Δodc1[pXG-BSD-ODC] (Δodc1[pODC]), Δodc2, and Δodc2[pXG-BSD-ODC] (Δodc2[pODC]) promastigotes (A) and axenic amastigotes (B) were each incubated in their respective growth media in the absence or presence of 200 μM putrescine. Parasites were enumerated after 5 days by using a hemocytometer. Promastigotes were maintained at pH 7.4 and 28°C, while axenic amastigotes were cultured at pH 5.5 and 37°C.

FIG. 4.

Survival of Δodc L. donovani in peritoneal murine macrophages. Peritoneal macrophages were infected with either wild-type, Δodc1, Δodc1[pXG-BSD-ODC] (Δodc1[pODC]), Δodc2, or Δodc2[pXG-BSD-ODC] (Δodc2[pODC]) stationary-phase promastigotes at a parasite/macrophage ratio of 10:1 as described in Materials and Methods. Cells were stained after 72 h and macrophages and amastigotes enumerated visually. The results are the averages and ranges of two experiments that were each performed in triplicate (n = 6).

FIG. 5.

Effects of DFMO on L. donovani axenic and tissue culture amastigotes. (A) Wild-type axenic amastigotes were seeded at 5 × 10⁴/100 μl in various concentrations of DFMO in the absence (▪) or presence (▴) of 200 μM putrescine. Parasites were counted after 96 h by using a hemacytometer and plotted as a percentage of control growth in the absence of DFMO as a function of DFMO concentration. (B) Culture of peritoneal macrophages 96 h after initial exposure to wild-type L. donovani at a ratio of 10 promastigotes per macrophage. (C) Peritoneal macrophages infected with parasites as for panel B but treated with 50 μM DFMO. (D) Uninfected macrophages treated with 50 μM DFMO. Cells were stained and examined under the microscope after 96 h as described in Materials and Methods. These experiments were repeated one additional time with equivalent results.

FIG. 6.

Parasite burdens in livers and spleens of mice infected with wild-type, Δodc, and “add-back” parasites. (A and B) Five separate groups of BALB/c mice were infected with either wild-type, Δodc1, Δodc1[pXG-BSD-ODC] (Δodc1[pODC]), Δodc2, or Δodc2[pXG-BSD-ODC] (Δodc2[pODC]) stationary-phase promastigotes as described in Materials and Methods. Mice were sacrificed after 4 weeks and parasite loads in liver (A) or spleen (B) preparations determined by limiting dilution.