Spermidine synthase is required for virulence of Leishmania donovani

Abstract

Genetic lesions in the polyamine biosynthetic pathway of Leishmania donovani, the causal agent of visceral leishmaniasis, are conditionally lethal mutations that render the insect vector form of the parasite auxotrophic for polyamines. Recently, we have demonstrated that a Δodc L. donovani null mutant lacking ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, was profoundly compromised in its ability to infect mice, indicating that ODC is essential for the infectious mammalian stage of the parasite and further validating the enzyme as a possible drug target. To assess whether other components of the polyamine biosynthetic pathway were also essential for parasite virulence, a cell line deficient in spermidine synthase (SPDSYN), the enzyme that converts putrescine to spermidine, was created by double-targeted gene replacement within a virulent L. donovani background. This Δspdsyn strain was auxotrophic for polyamines, required spermidine for growth in its insect vector form, and was adversely impacted in its ability to infect mice. These findings establish that SPDSYN, like ODC, is essential for maintaining a robust infection in mammals and indicate that pharmacologic inhibition of SPDSYN, and perhaps all components of the polyamine biosynthetic pathway, is a valid therapeutic strategy for the treatment of visceral and, potentially, other forms of leishmaniasis.

Fig. 1.

(A and B) Molecular characterization of the SPDSYN locus and SPDSYN expression in the Δspdsyn knockouts. Two micrograms of genomic DNA from wild-type, Δspdsyn1, or Δspdsyn2 parasites was digested with SalI and hybridized to a 1.0-kb fragment of the SPDSYN coding region (A) or XhoI and hybridized to a 1.2-kb fragment of the 5′ flanking region (B). Molecular size markers are indicated, and equal loading of DNA was verified via ethidium bromide staining. (C) For protein expression analysis, protein lysates from 1.0 × 10⁶ wild-type, Δspdsyn1, Δspdsyn2, and Δspdsyn1(pXG-BSD-SPDSYN) [Δspdsyn1(pSPDSYN)] promastigotes were fractionated by SDS-PAGE, blotted, and probed with polyclonal antibodies against L. donovani SPDSYN. Tubulin expression was analyzed as a loading control.

Fig. 2.

Growth phenotype of Δspdsyn promastigotes. Wild-type, Δspdsyn1, and Δspdsyn1(pXG-BSD-SPDSYN) [Δspdsyn1(pSPDSYN)] promastigotes were incubated in their respective growth media in the absence or presence of 100 μM spermidine or 100 μM putrescine. Δspdsyn1 parasites were starved of exogenous polyamines 2 days prior to the start of the growth assay. Parasites were inoculated at 5 × 10⁴ cells/ml, maintained at 26°C with 5% CO₂, and enumerated after 4 days. Reported cell densities represent final cell densities minus inoculation densities. Final cell densities of Δspdsyn1 parasites grown under nonpermissive conditions were significantly lower than those of the initial inocula, and thus, growth was zero.

Fig. 3.

Parasite burdens in mice. Three separate cohorts of BALB/c mice were infected with either wild-type, Δspdsyn1, or Δspdsyn1(pXG-BSD-SPDSYN) [Δspdsyn1(pSPDSYN)] stationary-phase promastigotes as described in Materials and Methods. Mice were killed after 4 weeks, and parasite loads in liver and spleen preparations were determined by limiting dilution. The experiment was repeated three times with similar conclusions.