Pyrimidine salvage and metabolism in kinetoplastid parasites

Pyrimidine uptake has previously been investigated in Trypanosoma brucei procyclics and partly investigated in promastigotes of Leishmania major; however, no such study has been performed using bloodstream forms of Trypanosoma or promastigotes of Leishmania. Here we report a comprehensive study of pyrimidine salvage and metabolism in bloodstream forms of Trypanosoma and promastigotes of Leishmania species. In T. b. brucei bloodstream forms, the uptake of 3H-uracil and 3H-tymidine each appeared to be mediated by a single transporter, designated TbU3 and TbT1, respectively. The procyclic uracil transporter,TbU1, has a high affinity for uracil, with a Km value of 0.46 ± 0.09 μM and Vmax of 0.65 ± 0.008 pmol (107cell)-1 s-1. These values were similar for TbU3 (Km = 0.54 ± 0.11 µM; Vmax = 0.14 ± 0.03), but the main differences between TbU1 and TbU3 are their sensitivity to uridine and 4-thiouracil. Thymidine uptake is detectable at 10 μM over a period from 5 to 30 minutes. This uptake was not inhibited by uracil which indicates that TbT1 is a novel thymidine transporter. The uptake of other pyrimidines, including uridine and 2’-deoxyuridine, by BSF are investigated here but these substrates were also transported by TbU3, and no additional pyrimidine transport activities were found. In L. mexicana and L. major, the uptake of 3H-uracil and 3H-uridine was mediated by separate transporters, designated as follows; for uracil uptake LmexU1, LmajU1; and for uridine uptake LmexNT1, LmajNT1 and LmajNT2, respectively. LmexU1 is a uracil transporter with high affinity to uridine and 2’deoxyuridine, and the LmexNT1 is a nucleoside transporter with broad specificity for purine and pyrimidine nucleosides. L. major uracil transporter (LmajU1) has already been reported by others; and here we report that there are also two distinct uridine transporters expressed in L. major. LmajNT1 is a high affinity uridine transporter which is also inhibited by uracil, inosine and adenine; LmajNT2 is low affinity uridine transporter, with very poor affinity for uracil, inosine and adenine. However, both transporters are inhibited by 2’-deoxyuridine, thymidine and adenosine. Several fluorinated pyrimidine analogues were assessed against kinetoplastid cells, the most effective compounds, which displayed EC50 values at micromolar level, are 5-FU, 5F-2’dUrd, 5-FOA (only against T. brucei BSF) and 5F-Urd (only against L. major). We induced resistance to 5-FU, 5-F2’dUrd and 5-FOA by in vitro exposure of Tbb-BSF and promastigotes of L. mexicana and L. major. The resistance was performed by stepwise increase concentration of the drugs. For T. b. brucei BSF, the resistance factors of clonal lines were 131, 825, and 83-fold, respectively. For L. mexicana and L. major, the resistance factor for 5-FU were 147 and 17-fold, and for 5F-2’dUrd were >3500 and 381-fold, respectively. We also measured 3H–pyrimidine uptake in these cell lines; the resistant bloodstream form strains showed no changes in pyrimidine uptake, with one exception, which is a 76% reduction in 5-FU uptake. In contrast, each resistant strain of Leishmania spp had lost its natural pyrimidine transporter. For example, Leishmania cells resistant to 5-FU had lost uracil transport activity, and cells that were resistant to 5F-2’dUrd had lost uridine transport activity. In addition, we identified kinetoplastid genes that appeared to be associated with resistance to fluorinated pyrimidines. Based on these findings, metabolomic analysis of fluorinated pyrimidines in T. b. brucei resistant cell lines was performed in comparison with parental wild-type; for Leishmania species we only investigated the metabolism of fluorinated pyrimidine in wild type cells, as the fluorinated analogues were simply not taken up in the resistant clones. The metabolomic analysis data showed that, in T. b. brucei, 5-fluorouracil and 5-fluoro orotate are incorporated into a large number of metabolites, but likely act through incorporation into RNA. 5F-2’dUrd and 5F-2’dCtd are not incorporated into nucleic acids but act as prodrugs by inhibiting thymidylate synthase after conversion to 5F-dUMP. Cells treated with 5-fluoro-2’deoxyuridine showed an increase of dUMP, which suggest a block in thymidylate synthase or possibly thymidylate kinase. We also present the most complete model of pyrimidine salvage in T. brucei to date, supported by genome-wide profiling of the predicted pyrimidine biosynthesis and conversion enzymes. The effect of fluorinated pyrimidine analogues in the two Leishmania species was almost identical. Each of the tested drugs (5-FU, 5F-2’dUrd and 5F-Urd) produced a limited number of fluorinated metabolites, and their common mode of action was inhibition in thymidylate synthase by 5F-dUMP and thymidine kinase by 5F-2’dUrd. Interestingly, we found that the cause of L. mexicana resistance to 5F-Urd was due to the absence of the 5F-2’dUrd metabolite, as a result of the rapid conversion of 5F-2’dUrd to 5F-dUMP; also we suggest that, in L. mexicana, but not in L. major the high affinity salvage of thymidine is sufficient to provide the cells with thymidine deoxynucleotides. It has been found that pyrimidine salvage is not an essential function for Leishmania cells in vitro conditions. However, it is not known whether either, pyrimidine salvage or biosynthesis, or both of these systems are essential to the trypanosomes in vitro and in vivo study. As T. b. brucei bloodstream forms grew unimpeded in vitro in the complete absence of pyrimidines, uptake is clearly not essential. Disruption of the pyrimidine biosynthesis pathway by deletion of the OMPDCase/OPRTase gene resulted in pyrimidine auxotrophic trypanosomes that were unable to grow in the absence of added pyrimidines. The phenotype was rescued by addition of uracil, and to a lesser extent by some pyrimidine nucleosides. Pyrimidine starvation led rapidly to DNA fragmentation. Adaptations to low pyrimidine availability included upregulation of uracil transport capacity and of uridine phosphorylase expression. However, pyrimidine auxotrophic T. brucei were able to establish a high parasitemia in mice. We therefore conclude that pyrimidine salvage was not an essential function for bloodstream T. b. brucei. However, trypanosomes lacking de novo pyrimidine biosynthesis are completely dependent on an extracellular pyrimidine source, strongly preferring uracil, and display reduced infectivity and strongly increased sensitivity to fluorinated pyrimidines. As T. brucei are able to salvage sufficient pyrimidines from the host environment, the pyrimidine biosynthesis pathway is not a viable drug target, although any interruption of pyrimidine supply was lethal.