Genetic Dissection of Pyrimidine Biosynthesis and Salvage in Leishmania donovani

Abstract

Background: Leishmania synthesize pyrimidine nucleotides via biosynthetic and salvage pathways. Results: Genetic blocks in pyrimidine biosynthesis and/or salvage can impact both life cycle stages of Leishmania. Conclusion: Pyrimidine biosynthesis and salvage both contribute to Leishmania homeostasis in animal infections. Significance: Functional characterization of pyrimidine pathways is crucial for understanding pyrimidine metabolism and validation of potential drug targets and vaccine strains.

Substrate Inhibition of Uracil Phosphoribosyltransferase by Uracil Can Account for the Uracil Growth Sensitivity of Leishmania donovani Pyrimidine Auxotrophs

“…Uracil is not, however, growth inhibitory to wild type L. donovani (5). Similarly, T. gondii (11), T. cruzi (12), and T. brucei (13) with genetic defects in pyrimidine biosynthesis pathway are susceptible to uracil-mediated growth inhibition, whereas their wild type counterparts are not.…”

“…5 and data not shown). These findings imply that the null mutants within the macrophage phagolysosome can access a source of host pyrimidines that can satisfy the pyrimidine nucleotide requirements of the parasite.…”

“…Expression and Purification of LdUPRT and T. gondii UPRT (TgUPRT) in Escherichia coli-The cloning of LdUPRT into the pET 200/D-TOPO E. coli expression vector has been previously reported (5). The full-length TgUPRT cDNA was amplified by PCR using the forward primer 5Ј-GAGGCCAC-CTGGGCCATGGCGCAGGTCCCAGCGAG-3Ј and reverse primer 5Ј-GAGGCCAGCCCGGCCCTACATGGTTCCAAA-GTACCGGTCACCGAA-3Ј (SfiI restriction sites are in bold, and unique triads are underlined) from a previously reported TgUPRT cDNA construct (18).…”

“…Gene sequencing, supported by biochemical studies, has revealed a number of significant differences between the pyrimidine biosynthetic pathways of Leishmania and mammals: 1) the genes encoding the pyrimidine pathway of Leishmania are syntenic (2-5), whereas the mammalian pyrimidine genes are not; 2) the genes encoding the first three enzymes in Leishmania are dis-crete, unlike the mammalian pathway in which there is a single gene encoding a trifunctional protein (6, 7); 3) the last two enzymes of the pyrimidine biosynthetic pathway are expressed as a single bifunctional protein, designated UMP synthase (UMPS), although the domain order in Leishmania and mammalian cells is reversed (3,8); and 4) the UMP synthase of Leishmania is localized within the glycosome (3), a unique peroxisomal-like organelle that is found uniquely among Leishmania and related parasites (9,10). Genetic ablation of either carbamoyl phosphate synthetase (CPS), the first enzyme of pyrimidine biosynthesis, or UMPS in L. donovani confer pyrimidine auxotrophy that can be circumvented by supplementation of the defined growth medium with uracil, uridine, deoxyuridine, cytidine, or deoxycytidine (3,5). Furthermore, both the ⌬cps and the ⌬umps null mutants exhibit a striking collateral supersensitivity to uracil, which is innocuous to wild type parasites, that is not observed with any of the ribonucleosides (5).…”

“…Parasite Cell Culture-The creation and characterization of ⌬uprt, ⌬cps, and ⌬umps L. donovani lines have been reported previously (3,5). Wild type and null mutant promastigotes were continuously cultured in 26°C in pH 7.4 DME-L medium that was supplemented with 10% Serum Plus (SAFC Biosciences, Lenexa, KS), 1 mg/ml hemin, and 100 M hypoxanthine as a purine source.…”