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

African trypanosomes are capable of both pyrimidine biosynthesis and salvage of pre-formed 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. Importantly, the anticancer drug 5-fluorouracil was an excellent substrate for TbU3 and several 5-fluoropyrimidine analogues were investigated for uptake and trypanocidal activity; 5F-orotic acid, 5F-2'deoxyuridine displayed activity in the low muM range. The metabolism and mode of action of these analogues was determined using metabolomic assessments of T. brucei clonal lines adapted to high levels of these pyrimidine analogues, as well as 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 Trypanosoma brucei to date, supported by genome-wide profiling of the predicted pyrimidine biosynthesis and conversion enzymes