Pyrimidine salvage in Trypanosoma brucei bloodstream forms and the trypanocidal action of halogenated pyrimidines

Abstract

African trypanosomes are capable of both pyrimidine biosynthesis and salvage of preformed pyrimidines from the host. However, uptake of pyrimidines in bloodstream form trypanosomes has not been investigated, making it difficult to judge the relative importance of salvage and synthesis or to design a pyrimidine-based chemotherapy. Detailed characterization of pyrimidine transport activities in bloodstream form Trypanosoma brucei brucei found that these cells express a high-affinity uracil transporter (designated TbU3) that is clearly distinct from the procyclic pyrimidine transporters. This transporter had low affinity for uridine and 2'deoxyuridine and was the sole pyrimidine transporter expressed in these cells. In addition, thymidine was taken up inefficiently through a P1-type nucleoside transporter. Of importance, the anticancer drug 5-fluorouracil was an excellent substrate for TbU3, and several 5-fluoropyrimidine analogs were investigated for uptake and trypanocidal activity; 5F-orotic acid, 5F-2'deoxyuridine displayed activity in the low micromolar range. The metabolism and mode of action of these analogs was determined using metabolomic assessments of T. brucei clonal lines adapted to high levels of these pyrimidine analogs, and of the sensitive parental strains. The analysis showed that 5-fluorouracil is incorporated into a large number of metabolites but likely exerts toxicity 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 as 5F-dUMP. We 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.

Fig. 1.

Timecourse of [³H]-uracil transport in T. b. brucei bloodstream forms over 120 seconds. Transport of 0.15 μM [³H]-uracil (■) was linear (r² = 0.99) and significantly different from zero (F test; P < 0.0001). In the presence of 1 mM unlabeled uracil (○), transport was reduced by >97% but still significantly different from zero (F-test, P = 0.03). Error bars are S.E.M. and, when not shown, fall inside the symbol. The experiment was performed in triplicate and one of several independent experiments with highly similar outcomes.

Fig. 2.

Characterization of [³H]-uracil transport in T. b. brucei bloodstream forms. (A) Inhibition of 0.15 μM [³H]-uracil uptake over 30 seconds by various concentrations of unlabeled uracil. Inset: conversion to Michaelis-Menten saturation plot. (B) Dose-dependent inhibition of 0.15 μM [³H]-uracil transport by uridine (■), 5-fluorouracil (●), and 5-bromouracil (□). Incubations (30 seconds) were terminated by the addition of 1 ml ice-cold 1 mM uracil in assay buffer and immediate centrifugation through oil. Error bars are S.E.M. of triplicate determinations.

Fig. 3.

Transport of pyrimidine nucleotides by T. b. brucei bloodstream forms. (A) Transport of 5 μM [³H]-2′deoxyuridine in the presence (○) or absence (■) of 2.5 mM unlabeled 2′-deoxyuridine. Lines were calculated by linear regression analysis, with correlation coefficients of 0.99 for both data sets. (B) Representative inhibition plot of 5 μM [³H]-2′-deoxyuridine transport, using a 180-second incubation time: ■, 2′-deoxyuridine; ○, uracil. The conversion of the 2′-dUrd inhibition plot to a Michaelis-Menten saturation plot for the determination of K_m and V_max is shown in the Supplementary data. (C) Transport of 10 μM [³H]-thymidine (■) was linear for up to 30 minutes and partly inhibited by the addition of 2.5mM unlabeled thymidine (○). (D) Transport of 5 μM [³H]-thymidine over 15 minutes in the presence of various concentrations of uracil (■), adenosine (○), or thymidine (■). The conversion of the thymidine inhibition plot to a saturation plot is shown in the Supplementary data. All error bars are S.E.M. of triplicate determinations; where not visible, error bars fall within the symbol. Experiments shown are representative of several similar and independent experiments.

Fig. 4.

Transport of uracil and 5-FU by bloodstream trypanosomes. Cells of wild-type (closed symbols) or 5-FURes (open symbols) were incubated with (A) [³H]-uracil or (B) [³H]-5FU in the presence (circles) or absence (squares) of 1 mM unlabeled permeant. Lines were calculated by linear regression. Error bars are S.E.M. of triplicate determinations. The graphs shown are representative of three similar experiments.

Fig. 5.

Uptake of orotic acid by T. b. brucei. Bloodstream trypanosomes were incubated with 0.2 μM [³H]-orotic acid in the presence (○) or absence (■) of 1 mM unlabeled orotic acid. Uptake was linear (r² was 0.97 and 0.98, respectively) over the 10-minute course of the experiment; 1 mM orotic acid did not significantly inhibit the rate of uptake. The experiment was performed in triplicate; error bars are S.E.M.

Fig. 6.

Scheme of pyrimidine biosynthesis and metabolism in T. b. brucei. The double curved line represents the plasma membrane and arrows across its (potential) transport activities. Dotted lines indicate transport or conversions that probably do not take place in bloodstream trypanosomes. Red boxes indicate metabolites, of which fluorinated analogs were detected by metabolomic techniques; black boxes indicate metabolites not detected in fluorinated form. Numbers above arrows indicate the following enzymes, listed here with EC numbers. 1, carbamoyl phosphate synthase (6.3.5.5); 2, aspartate carbamoyl transferase (2.1.3.2); 3, dihydroorotase (3.5.2.3); 4, dihydroorotate dehydrogenase (1.3.3.1); 5, orotate phosphoribosyltransferase (2.4.2.10); 6, orotidine 5-phosphate decarboxylase (4.1.1.23); 7, uracil phosphoribosyltransferase (2.4.2.9); 8, nucleoside diphosphatase (3.6.1.6). 9 nucleoside diphosphate kinase (2.7.4.6). 10, cytidine triphosphate synthase (6.3.4.2); 11, ribonucleoside-diphosphate reductase (1.17.4.1); 12, uridine phosphorylase (2.4.2.3); 13, dUTPase (3.6.1.23); 14, thymidylate kinase (2.7.4.9); 15, thymidine kinase (2.7.1.21); 16, thymidylate synthase (2.1.1.45); 17 cytidine deaminase (3.5.4.5); 18, UDP-glucose pyrophosphorylase (2.7.7.9); 19, UDP-glucose epimerase (5.1.3.2); 20, adenylate kinase G (2.7.4.10); 21 phosphatidate cytidylyltransferase (2.7.7.41); 22, ethanolamine-phosphate cytidylyltransferase (2.7.7.14); 23, choline-phosphate cytidylyltransferase (2.7.7.15); 24, orotate reductase (1.3.1.14, not present); 25, dihydroorotate dehydrogenase (1.3.5.2, not present); 26, pseudouridylate synthase (4.2.1.70); 27, UTP:N-acetyl-α-D-glucosamine-1-phosphate uridylyltransferase (2.7.7.23); 28, α-1,6-N-acetylglucosaminyltransferase. Abbreviations: Gln, glutamine; Carb-P carbamoyl phosphate; Asp, aspartate; Carb-Asp, N-carbamoyl-L-aspartate; DHO, dihydroorotate; OMP, orotidine-5-phosphate; Urd, uridine; Tmd, thymidine; 2’dCtd, 2’-deoxycytidine; Glc, glucose; Gal, galactose; GlcNAc, N-acetylglucosamine. Lipid metabolism refers to formation of CDP-diacylglycerol (EC 2.7.7.41), CDP-ethanolamine (EC 2.7.7.14) and CDP-choline (EC 2.7.7.15).

Fig. 7.

Metabolomic profiles of wild-type and resistant cells treated with fluoro-pyrimidine nucleobases. Relative levels of (A) 2′-deoxyuridine and (B,C) fluorinated pyrimidines in trypanosomes exposed to (A,B) 100 μM 5-FU or (C) 5-FOA. Cultures of T. b. brucei bloodstream forms (50 ml of 2 × 10⁶ cells/ml) in normal HMI-9 medium with 10% fetal calf serum (FCS) were incubated with 100 μM 5-FU for 8 hours. Extracts from cell pellets collected at the end of the experiment were subjected to metabolomic analysis, and the intensity of the mass spectrometer signal is plotted here for the metabolites observed. *P < 0.05; **P < 0.02; ***P < 0.01 by unpaired two-tailed Student’s t test comparing intensity of a particular metabolite in wild-type and resistant lines; n = 3. Hatched bars, wild-type; solid bars, resistant clones, FURes (A,B) or FOARes (C).

Fig. 8.

Metabolomic profiles of wild-type and 5F-dURes cells exposed or not (control) to 100 μM 5F-2′-dUrd for 8 hours. The level of 5F-2′dUrd in wild-type and 5F-dURes cells was not significantly different (A). After treatment with 5F-2′dUrd, the level of dUMP was much higher in wild-type (hatched bars) than in 5F-dURes cells (solid bars) (B). The abundance of TMP (C), TDP and TTP (not shown) was statistically identical in control and 5F-dURes cells, whether 5F-2′dUrd-treated or not. Experimental conditions as described in the legend to Figure 7. Hatched bars, wild-type; solid bars, 5F-dURes.

Fig. 9.

Effect of fluorinated pyrimidines on T. b. brucei bloodstream forms in the presence and absence of 100 μM thymidine. Left panel, wild-type427; Right panel, 5F-2′dURes trypanosomes. Cultures were grown in a minimal version of HMI-9 without pyrimidines and with dialyzed fetal calf serum (FCS) (blue bars), to which 100 μM thymidine was added (green bars), or in standard HMI-9 (brown bars). Diminazene was used as an internal control (not significantly different between conditions; not shown). The results shown are the mean of three independent experiments; error bars are S.E.M. *P < 0.05; **P < 0.02; ***P < 0.01 by unpaired two-tailed Student’s t test. For 5F-2′dCtd on the 5F-2′dURes cells, the test compound did not sufficiently inhibit trypanosome growth at the highest concentration tested, 5 mM; IC₅₀ values of 5000 μM were added for each of the three independent experiments for the purpose of this graph.

Fig. 10.

Analysis of pyrimidine metabolic enzymes in major protozoan pathogens and two reference mammalian genomes. Profiles specific for the known pyrimidine metabolic enzymes were constructed as described in Methods. The profiles were scanned against selected eukaryote proteomes. (A) Heat map of the best scores obtained by each proteome against profiles for enzymes of pyrimidine synthesis (1–6), salvage (7–17), sugar (18–19), and lipid metabolism (20–23). Enzyme numbers are the same as in Figure 6. (B) Hierarchical clustering of the pyrimidine metabolic vectors (top) based on Canberra distance (scale bar); the red numbers are approximately unbiased confidence (au), where p = (100-au)/100.