Genetic dissection of pyrimidine biosynthesis and salvage in Leishmania donovani

Abstract

Protozoan parasites of the Leishmania genus express the metabolic machinery to synthesize pyrimidine nucleotides via both de novo and salvage pathways. To evaluate the relative contributions of pyrimidine biosynthesis and salvage to pyrimidine homeostasis in both life cycle stages of Leishmania donovani, individual mutant lines deficient in either carbamoyl phosphate synthetase (CPS), the first enzyme in pyrimidine biosynthesis, uracil phosphoribosyltransferase (UPRT), a salvage enzyme, or both CPS and UPRT were constructed. The Δcps lesion conferred pyrimidine auxotrophy and a growth requirement for medium supplementation with one of a plethora of pyrimidine nucleosides or nucleobases, although only dihydroorotate or orotate could circumvent the pyrimidine auxotrophy of the Δcps/Δuprt double knockout. The Δuprt null mutant was prototrophic for pyrimidines but could not salvage uracil or any pyrimidine nucleoside. The capability of the Δcps parasites to infect mice was somewhat diminished but still robust, indicating active pyrimidine salvage by the amastigote form of the parasite, but the Δcps/Δuprt mutant was completely attenuated with no persistent parasites detected after a 4-week infection. Complementation of the Δcps/Δuprt clone with either CPS or UPRT restored infectivity. These data establish that an intact pyrimidine biosynthesis pathway is essential for the growth of the promastigote form of L. donovani in culture, that all uracil and pyrimidine nucleoside salvage in the parasite is mediated by UPRT, and that both the biosynthetic and salvage pathways contribute to a robust infection of the mammalian host by the amastigote. These findings impact potential therapeutic design and vaccine strategies for visceral leishmaniasis.

FIGURE 1.

Schematic of pyrimidine metabolism in L. donovani. The double curved line represents the parasite plasma membrane, whereas arrows indicate the biochemical function or transport activity of the following: CPS (1), ACT (2), DHO (3), DHODH (4), UMPS (5 and 6), nucleoside transporter 1 (7), uracil transporter (8), cytidine deaminase (9); uridine phosphorylase (10), nucleoside hydrolase (11), and UPRT (12).

FIGURE 2.

Southern blot analysis of Δcps, Δuprt, and Δcps/Δuprt knockouts. Genomic DNA from wild type (lane 1), Δcps (lane 2), Δuprt (lane 3), and Δcps/Δuprt (lane 4) parasites was digested with either SacI (A and C) or XhoI (B and D), fractionated on a 1% agarose gel, and blotted onto nylon membranes. Blots were hybridized under high stringency conditions with probes for the CPS ORF (A), CPS 5′-flank (B), UPRT ORF (C), or UPRT 3′-flank (D). The arrows denote band positions corresponding to wild type CPS (A and B) and UPRT (C and D) alleles or to alleles in which the wild type genes have been replaced by HYG (B and D, lane 3), PHLEO (B), bsd (D), or NEO (D, lane 4) drug resistance markers.

FIGURE 3.

Growth of wild type, Δcps, and Δcps/Δuprt promastigotes in various pyrimidine supplements. Wild type (A), Δcps (B), and Δcps/Δuprt (C) promastigotes were tested for their ability to grow in medium with or without supplementation by the indicated pyrimidines at concentrations of 100 μm (black bars), 500 μm (white bars), or 2 mm (hashed bars). The percentage of maximum growth of wild type and mutant promastigotes was determined by normalizing growth of each cell line in the indicated pyrimidine supplement to its growth in the supplement that results in the highest cell density, 100 μm uridine (wild type and Δcps) or 2 mm orotate (Δcps/Δuprt). None, no pyrimidine supplementation. Results depicted are the averages and S.E. (error bars) of three technical replicates.

FIGURE 4.

Growth rates of wild type (■), Δcps (△), Δuprt (♦), and Δcps/Δuprt (○) promastigotes. Logarithmic phase promastigotes were seeded at a density of 1 × 10⁶ parasites/ml in medium supplemented with either 250 μm cytidine for Δcps parasites or 2 mm orotate for Δcps/Δuprt parasites. Parasites were enumerated at 8–16-h intervals for a period of 48 h to determine growth rates. Results depicted are the averages and S.E. (error bars) of three technical replicates.

FIGURE 5.

Uracil hypersensitivity in de novo pyrimidine biosynthesis mutants. A, wild type (■), Δcps (△), and Δumps (○) promastigotes were tested for their ability to grow in media supplemented with various concentrations of uracil. The growth of wild type and mutant promastigotes in each concentration of uracil was normalized to the maximum growth achieved within the assay by that cell line and is represented as the percentage of maximum growth. B, growth rates of Δcps promastigotes in media supplemented with various concentrations of uracil. Logarithmic phase promastigotes were seeded a density of 1 × 10⁶ parasites/ml in medium containing either 100 μm (▴), 500 μm (▾), 2 mm (△), or 4 mm (▿) uracil, and growth of the culture was determined as in Fig. 4. Results depicted are the averages and S.E. (error bars) of three technical replicates.

FIGURE 6.

Uracil and uridine incorporation by wild type or Δuprt promastigotes. 5 × 10⁷ wild type (■) or Δuprt (♦) parasites were incubated with either 26.3 nm [5,6-³H]uracil (A) or 22.9 nm [5,6-³H]uridine (B) for 1 h. At various time points, 5 × 10⁶ parasites were removed, and incorporation of either [5,6-³H]uracil or [5,6-³H]uridine into the pyrimidine nucleotide pool was determined via a DE81 filter binding assay. The results shown represent the averages and S.E. (error bars) of three technical replicates.

FIGURE 7.

Infectivity of L. donovani pyrimidine pathway mutants in a mouse model. Groups of five BALB/c mice were inoculated with 5 × 10⁶ parasites via tail vein injection. After 4 weeks, the liver and spleen of each mouse were harvested, and parasitemias were determined via a limiting dilution assay. A, parasitemias of the livers (black bars) and spleens (gray bars) of BALB/c mice infected with either wild type, Δcps, or Δcps[pCPS] parasites. B, parasitemias of the livers (black bars) and spleens (gray bars) of BALB/c mice infected with either wild type, Δuprt, Δcps/Δuprt, Δcps/Δuprt[RP-CPS], or Δcps/Δuprt[RP-UPRT] parasites. Error bars, S.E.

FIGURE 8.

Short term in vitro and in vivo infections with wild type and Δcps/Δuprt parasites. A, images of stained murine macrophages after 6 h of infection with either wild type or Δcps/Δuprt parasites. The arrows indicate intracellular amastigotes. Amastigotes within infected macrophages in 50 fields of view were enumerated visually, and the average number of parasites per infected macrophage is depicted in B. C, groups of three BALB/c mice were inoculated with either wild type or Δcps/Δuprt parasites as described in the legend to Fig. 7. After 1 week, the liver and spleen of each mouse was harvested, and the parasitemias in the livers (black bars) and spleens (gray bars) were determined via a limiting dilution assay. Error bars, S.E.