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Trypanosoma brucei encodes a relatively high number of genes of the equilibrative nucleoside transporter (ENT) family. We report here the cloning and in-depth characterization of one T. brucei brucei ENT member, TbNT9/AT-D. This transporter was expressed in Saccharomyces cerevisiae and displayed a uniquely high affinity for adenosine (Km = 0.068 +/- 0.013 microM), as well as broader selectivity for other purine nucleosides in the low micromolar range, but was not inhibited by nucleobases or pyrimidines. This selectivity profile is consistent with the P1 transport activity observed previously in procyclic and long-slender bloodstream T. brucei, apart from the 40-fold higher affinity for adenosine than for inosine. We found that, like the previously investigated P1 activity of long/slender bloodstream trypanosomes, the 3'-hydroxy, 5'-hydroxy, N3, and N7 functional groups contribute to transporter binding. In addition, we show that the 6-position amine group of adenosine, but not the inosine 6-keto group, makes a major contribution to binding (DeltaG0 = 12 kJ/mol), explaining the different Km values of the purine nucleosides. We further found that P1 activity in procyclic and long-slender trypanosomes is pharmacologically distinct, and we identified the main gene encoding this activity in procyclic cells as NT10/AT-B. The presence of multiple P1-type nucleoside transport activities in T. brucei brucei facilitates the development of nucleoside-based treatments for African trypanosomiasis and would delay the onset of uptake-related drug resistance to such therapy. We show that both TbNT9/AT-D and NT10/AT-B transport a range of potentially therapeutic nucleoside analogs.

Purine antimetabolites have been very successful therapeutic agents against a host of infectious diseases and malignancies. Success of the treatment relies as much on the efficient accumulation by the target cell or organism as it does on selective action on a vital biochemical pathway of the target cell. Here we compare the ability of a new class of tricyclic purine antimetabolites to interact with transporters from human erythrocytes or Trypanosoma brucei. We show that these compounds display a remarkable selectivity for the parasite's transporters. The adenine analogue showed greater trypanocidal activity than the hypoxanthine or guanine analogues in vitro. #This work was funded by the Wellcome Trust.

Resistance to diminazene aceturate (Berenil) is a severe problem in the control of African trypanosomiasis in domestic animals. It has been speculated that resistance may be the result of reduced diminazene uptake by the parasite. We describe here the mechanisms by which [ 3 H]diminazene is transported by Trypanosoma brucei brucei bloodstream forms. Diminazene was rapidly accumulated through a single transporter, with a K m of 0.45 ± 0.11 μM, which was dose dependently inhibited by pentamidine and adenosine. The K i values for these inhibitors were consistent with this transporter being the P2/TbAT1 adenosine transporter. Yeast expressing TbAT1 acquired the ability to take up [ 3 H]diminazene and [ 3 H]pentamidine. TbAT1 -null mutants had lost almost all capacity for [ 3 H]diminazene transport. However, this cell line still displayed a small but detectable rate of [ 3 H]diminazene accumulation, in a nonsaturable manner. We conclude that TbAT1 mediates [ 3 H]diminazene transport almost exclusively and that this explains the observed diminazene resistance phenotypes of TbAT1 -null mutants and field isolates.

Sleeping sickness, caused by Trypanosoma brucei spp., has become resurgent in sub-Saharan Africa. Moreover, there is an alarming increase in treatment failures with melarsoprol, the principal agent used against late-stage sleeping sickness. In T. brucei , the uptake of melarsoprol as well as diamidines is thought to be mediated by the P2 aminopurine transporter, and loss of P2 function has been implicated in resistance to these agents. The trypanosomal gene TbAT1 has been found to encode a P2-type transporter when expressed in yeast. Here we investigate the role of TbAT1 in drug uptake and drug resistance in T. brucei by genetic knockout of TbAT1. Tbat1 -null trypanosomes were deficient in P2-type adenosine transport and lacked adenosine-sensitive transport of pentamidine and melaminophenyl arsenicals. However, the null mutants were only slightly resistant to melaminophenyl arsenicals and pentamidine, while resistance to other diamidines such as diminazene was more pronounced. Nevertheless, the reduction in drug sensitivity might be of clinical significance, since mice infected with tbat1 -null trypanosomes could not be cured with 2 mg of melarsoprol/kg of body weight for four consecutive days, whereas mice infected with the parental line were all cured by using this protocol. Two additional pentamidine transporters, HAPT1 and LAPT1, were still present in the null mutant, and evidence is presented that HAPT1 may be responsible for the residual uptake of melaminophenyl arsenicals. High-level arsenical resistance therefore appears to involve the loss of more than one transporter.

While multiple nucleoside transporters, some of which can also transport nucleobases, have been cloned in recent years from many different organisms, no sequence information is available for the high affinity, nucleobase-selective transporters of metazoa, parazoa, or protozoa. We have identified a gene, TbNBT1, from Trypanosoma brucei brucei that encodes a 435-residue protein of the equilibrative nucleoside transporter superfamily. The gene was expressed in both the procyclic and bloodstream forms of the organism. Expression of TbNBT1 in a Saccharomyces cerevisiae strain lacking an endogenous purine transporter allowed growth on adenine as sole purine source and introduced a high affinity transport activity for adenine and hypoxanthine, with Km values of 2.1 +/- 0.6 and 0.66 +/- 0.22 microm, respectively, as well as high affinity for xanthine, guanine, guanosine, and allopurinol and moderate affinity for inosine. A transporter with an indistinguishable kinetic profile was identified in T. b. brucei procyclics and designated H4. RNA interference of TbNBT1 in procyclics reduced cognate mRNA levels by approximately 80% and H4 transport activity by approximately 90%. Expression of TbNBT1 in Xenopus oocytes further confirmed that this gene encodes the first high affinity nucleobase transporter from protozoa or animals to be identified at the molecular level.

The therapeutic index of antimetabolites such as purine analogues is in large part determined by the extent to which it is selectively accumulated by the target cell. In the current study we have compared the transport of purine nucleobase analogues by the H2 transporter of bloodstream form Trypanosoma brucei brucei and the equilibrative nucleobase transporter of human erythrocytes. The H2 transporter forms hydrogen bonds with hypoxanthine at positions N3, N7, N(1)H, and N(9)H of the purine ring, with apparent Delta G(0) of 7.7-12.6 kJ/mol. The transporter also appears to H-bond with the amine group of adenine. The human transporter forms hydrogen bonds that form to (6)NH(2) and N1 of adenine. An H-bond is also formed with N3 and the 6-keto and amine groups of guanine but not with the protonated N1, thus explaining the low affinity for hypoxanthine. N7 and N9 do not directly interact with the human transporter in the form of H-bonds, and it is proposed that pi-pi stacking interactions contribute significantly to permeant binding. The potential for selective uptake of antimetabolites by the parasite transporter was demonstrated.

In an effort to study the effects of flexibility on enzyme recognition and activity, we have developed several different series of flexible nucleoside analogues in which the purine base is split into its respective imidazole and pyrimidine components. The focus of this particular study was to synthesize the truncated neplanocin A fleximers to investigate their potential anti-protozoan activities by inhibition of S-adenosylhomocysteine hydrolase (SAHase). The three fleximers tested displayed poor anti-trypanocidal activities, with EC50 values around 200 μM. Further studies of the corresponding ribose fleximers, most closely related to the natural nucleoside substrates, revealed low affinity for the known T. brucei nucleoside transporters P1 and P2, which may be the reason for the lack of trypanocidal activity observed.

Lipoic acid (LA) is an essential cofactor of α-keto acid dehydrogenase complexes (KADHs) and the glycine cleavage system. In Plasmodium, LA is attached to the KADHs by organelle-specific lipoylation pathways. Biosynthesis of LA exclusively occurs in the apicoplast, comprising octanoyl-[acyl carrier protein]: protein N-octanoyltransferase (LipB) and LA synthase. Salvage of LA is mitochondrial and scavenged LA is ligated to the KADHs by LA protein ligase 1 (LplA1). Both pathways are entirely independent, suggesting that both are likely to be essential for parasite survival. However, disruption of the LipB gene did not negatively affect parasite growth despite a drastic loss of LA (>90%). Surprisingly, the sole, apicoplast-located pyruvate dehydrogenase still showed lipoylation, suggesting that an alternative lipoylation pathway exists in this organelle. We provide evidence that this residual lipoylation is attributable to the dual targeted, functional lipoate protein ligase 2 (LplA2). Localisation studies show that LplA2 is present in both mitochondrion and apicoplast suggesting redundancy between the lipoic acid protein ligases in the erythrocytic stages of P. falciparum., Author Summary Plasmodium falciparum is the causative agent of severe malaria. The parasites possess two organelles that are integral to their metabolism—the mitochondrion and the apicoplast, a remnant plastid. Both organelles contain enzymes that depend on the attachment of the cofactor lipoic acid for their catalytic activity. These are the α-keto acid dehydrogenase complexes and the glycine cleavage system (GCS). The pyruvate dehydrogenase (PDH) is solely found in the apicoplast of the parasites whereas α-keto glutarate and branched chain α-keto acid dehydrogenase as well as the GCS are mitochondrial. Both organelles possess specific and independent mechanisms that guarantee the posttranslational lipoylation of these enzyme complexes. In this study we show that the apicoplast located lipoic acid protein ligase, octanoyl-[acyl carrier protein]: protein N-octanoyltransferase (LipB), is not essential for parasite survival by disrupting the LipB gene locus. Despite a drastic loss of total lipoic acid, the parasites progress through their intraerythrocytic development unperturbed although the apicoplast-located PDH shows a reduced level of lipoylation. This phenotype is attributable to the presence of the recently described lipoic acid protein ligase 2, LplA2, which we show to be dually targeted to mitochondrion and apicoplast.

The human malaria parasite Plasmodium falciparum possesses a single mitochondrion and a plastid-like organelle called the apicoplast. Both organelles contain members of the KADH (alpha-keto acid dehydrogenase) complexes--multienzyme complexes that are involved in intermediate metabolism. In the asexual blood stage forms of the parasites, the alpha-ketoglutarate dehydrogenase and branched chain KADH complexes are both located in the mitochondrion, whereas the pyruvate dehydrogenase is exclusively found in the apicoplast. In agreement with this distribution, Plasmodium parasites have two separate and organelle-specific pathways that guarantee lipoylation of the KADH complexes in both organelles. A biosynthetic pathway comprised of lipoic acid synthase and lipoyl (octanoyl)-ACP:protein Nepsilon-lipoyltransferase B is present in the apicoplast, whereas the mitochondrion is supplied with exogenous lipoic acid, and ligation of the metabolite to the KADH complexes is accomplished by a lipoate protein ligase A similar to that of bacteria and plants. Both pathways are excellent potential targets for the design of new antimalarial drugs.

Purine antimetabolites have been very successful therapeutic agents against a host of infectious diseases and malignancies. Success of the treatment relies as much on the efficient accumulation by the target cell or organism as it does on selective action on a vital biochemical pathway of the target cell. Here we compare the ability of a new class of tricyclic purine antimetabolites to interact with transporters from human erythrocytes or Trypanosoma brucei. We show that these compounds display a remarkable selectivity for the parasite's transporters. The adenine analogue showed greater trypanocidal activity than the hypoxanthine or guanine analogues in vitro. #This work was funded by the Wellcome Trust.

Allopurinol is a hypoxanthine analogue used to treat Leishmania infections that also displays activity against the related parasite Trypanosoma brucei. We have investigated the ease by which resistance to this drug is established in Trypanosoma brucei brucei and correlated this to the mechanisms by which it is accumulated by the parasite. Long-term exposure of procyclic T. b. brucei to 3mM allopurinol did not induce resistance. This appears to be related to the fact that allopurinol was taken up through two distinct nucleobase transporters, H1 and H4, both with high affinity for the drug. The apparent Km for [3H]allopurinol transport by H4 (2.1+/-0.4 microM) was determined by expressing the encoding gene in Saccharomyces cerevisiae. Long-term allopurinol exposure did not change Km (hypoxanthine), Ki (allopurinol), or Vmax values of either H1 or H4 transporters and the cells retained their ability to proliferate with hypoxanthine as sole purine source. This study shows that transport-related resistance to purine antimetabolites is not easily induced in Trypanosoma spp. as long as uptake is mediated by multiple transporters.

Nucleobase transporters play an important role in the physiology of protozoan parasites, because these organisms are purine auxotrophs and rely entirely on salvage of these vital compounds. Purine transporters have also been shown to mediate the uptake of important antiparasitic drugs. In the current study, we investigated the uptake of [(3)H]adenine, [(3)H]hypoxanthine, and [(3)H]allopurinol, an antileishmanial hypoxanthine analog, by Leishmania major. These compounds were all taken up by a single high-affinity transporter, LmNBT1, with K(m) values of 4.6 +/- 0.9, 0.71 +/- 0.07, and 54 +/- 3 microM, respectively. Guanine and xanthine fully inhibited [(3)H]adenine transport, with K(i) values of 2.8 +/- 0.7 and 23 +/- 8 microM. Using purine analogs, an inhibitor profile for LmNBT1 was obtained, which allowed the construction of a quantitative model for the interactions between the transporter binding site and the permeant. The model predicts that hypoxanthine was bound through hydrogen bonds to N(1)H, N3, N7, and N(9)H of the purine ring, with a total Gibbs free energy of -39.5 kJ/mol. The interactions with adenine were similar, except for a weak hydrogen bond to N1 (unprotonated in adenine). The predicted mode of substrate binding for LmNBT1 was almost identical to that for the Trypanosoma brucei H2 (TbH2) transporter. It is proposed that the architecture of their respective binding sites is very similar and that LmNBT1 can be named a functional homolog of TbH2.