A novel purine nucleoside transporter whose expression is up-regulated in the short stumpy form of the Trypanosoma brucei life cycle

Purine nucleoside and nucleobase transporters play a vital role in the metabolism and survival of Trypanosoma brucei because this parasitic protozoan is unable to synthesize purines de novo and thus must acquire preformed purines from its hosts. These parasites express a variety of nucleoside and nucleobase permeases with diverse substrate specificities and distinct patterns of expression during the trypanosome life cycle. We report here that expression of the newly characterized T. brucei nucleoside transporter 10 gene (TbNT10) is up-regulated in the short stumpy form of the life cycle, the bloodstream form of the parasite that is pre-adapted for infection of the tsetse fly vector. Functional expression of TbNT10 in Saccharomyces cerevisiae reveals that the TbNT10 gene encodes an adenosine/guanosine/inosine transporter with apparent Km values of approximately 1 microM and hence is a high affinity purine nucleoside transporter. The restricted expression of TbNT10 during the life cycle suggests that the functional properties of this permease may be specialized to support development and growth of the differentiated short stumpy form or to promote the transformation of short stumpy to procyclic forms within the insect vector.     

Two novel nucleobase/pentamidine transporters from Trypanosoma brucei

African trypanosomes are unable to synthesize purines de novo and must salvage preformed purine nucleosides and nucleobases from their hosts. The Trypanosoma brucei genome project has identified 12 members of the equilibrative nucleoside transporter family, most of which have been characterized previously as nucleoside and/or nucleobase transporters. Here the 11th member of this family, TbNT11.1, has been functionally expressed in null mutants of Leishmania that are deficient in purine nucleoside or nucleobase uptake and identified as a high-affinity purine nucleobase transporter. Expression of TbNT11.1 in Xenopus oocytes revealed that it is also a transporter for the diamidine drug pentamidine that is the principal drug employed to treat early stage human African trypanosomiasis and may thus contribute to the uptake of this therapeutically important compound. In addition, characterization of the 12th member of the family, TbNT12.1, reveals that it is an adenine/pentamidine transporter.     

The equilibrative nucleoside transporter family,SLC29

The human SLC29 family of proteins contains four members, designated equilibrative nucleoside transporters (ENTs) because of the properties of the first-characterised family member, hENT1. They belong to the widely-distributed eukaryotic ENT family of equilibrative and concentrative nucleoside/nucleobase transporters and are distantly related to a lysosomal membrane protein, CLN3, mutations in which cause neuronal ceroid lipofuscinosis. A predicted topology of 11 transmembrane helices with a cytoplasmic N-terminus and an extracellular C-terminus has been experimentally confirmed for hENT1. The best-characterised members of the family, hENT1 and hENT2, possess similar broad substrate specificities for purine and pyrimidine nucleosides, but hENT2 in addition efficiently transports nucleobases. The ENT3 and ENT4 isoforms have more recently also been shown to be genuine nucleoside transporters. All four isoforms are widely distributed in mammalian tissues, although their relative abundance varies: ENT2 is particularly abundant in skeletal muscle. In polarised cells ENT1 and ENT2 are found in the basolateral membrane and, in tandem with concentrative transporters of the SLC28 family, may play a role in transepithelial nucleoside transport. The transporters play key roles in nucleoside and nucleobase uptake for salvage pathways of nucleotide synthesis, and are also responsible for the cellular uptake of nucleoside analogues used in the treatment of cancers and viral diseases. In addition, by regulating the concentration of adenosine available to cell surface receptors, they influence many physiological processes ranging from cardiovascular activity to neurotransmission.     

Differential regulation of nucleoside and nucleobase transporters in Crithidia fasciculata and Trypanosoma brucei brucei

The regulation of the activity of purine transporters in two protozoan species, Crithidia fasciculata and Trypanosoma brucei brucei, was investigated in relation to purine availability and growth cycle. In C. fasciculata, two high-affinity purine nucleoside transporters were identified. The first, designated CfNT1, displayed a K(m) of 9.4 +/- 2.8 microM for adenosine and was inhibited by pyrimidine nucleosides as well as adenosine analogues; a second C. fasciculata nucleoside transporter (CfNT2) recognized inosine (K(m) = 0.38 +/- 0.06 microM) and guanosine but not adenosine. The activity of both transporters increased in cells at mid-logarithmic growth, as compared to cells in the stationary phase, and was also stimulated 5-15-fold following growth in purine-depleted medium. These increased rates were due to increased Vmax values (K(m) remained unchanged) and inhibited by cycloheximide (10 microM). In the procyclic forms of T. b. brucei, adenosine transport by the P1 transporter was upregulated by purine starvation but only after 48 h, whereas hypoxanthine transport was maximally increased after 24 h. The latter effect was due to the expression of an additional hypoxanthine transporter, H2, that is normally absent from procyclic forms of T. b. brucei and was characterised by its high affinity for hypoxanthine (K(m) approximately 0.2 microM) and its sensitivity to inhibition by guanosine. The activity of the H1 hypoxanthine transporter (K(m) approximately 10 microM) was unchanged. These results show that regulation of the capacity of the purine transporters is common in different protozoa, and that, in T. b. brucei, various purine transporters are under differential control.     

Differences in glucose transport between blood stream and procyclic forms of Trypanosoma brucei rhodesiense

In African trypanosomes the requirements for glucose and its metabolism vary in different stages of the life cycle. Here we present evidence that cultured procyclic trypanosomes of Trypanosoma brucei rhodesiense uptake glucose against a concentration gradient in a time and dose-dependent manner. Moreover, glucose transport is completely inhibited by the sulphydryl inhibitor N-ethylmaleimide, suggesting the presence of a protein moiety as the carrier molecule. Comparison of glucose uptake in bloodstream and procyclic trypanosomes point to the possibility that different transporters may function in the 2 developmental stages. Glucose uptake by bloodstream trypanosomes requires Na+ ions and is inhibited by phlorizin, an inhibitor of Na(+)-dependent glucose transporters in mammalian cells. Conversely, procyclic trypanosomes transport glucose in a Na(+)-dependent manner, and transport is not affected by phlorizin. Finally, the putative procyclic glucose transporter has a higher affinity for glucose (apparent Km 23 microM) than the bloodstream carrier (apparent Km 237 microM).     

Purine salvage and metabolism inBabesia bovis

Studies of the incorporation of radio-labelled purine precursors into the erythrocytic forms ofBabesia bovis under tissue-culture conditions have confirmed the presence in the parasite of enzymatic activities responsible for the salvage of preformed purines. The results also revealed that the parasite was capable of a variety of nucleotide interconversions, such that exogenous hypoxanthine and adenosine were incorporated into both adenine and guanine nucleotides followed by the incorporation of these nucleotides into the adenine and guanine moieties of RNA and DNA. No evidence was found for salvage of preformed pyrimidines. Evidence was also obtained for the insertion of a parasite-specific nucleoside/nucleobase transporter into the membrane of the bovine (host) red cell. Thus, whereas normal (non-parasitised) bovine red cells are essentially incapable of transporting nucleosides across their membranes, the invasion of these cells byB. bovis introduces a transporter that can be inhibited by classic nucleoside transport inhibitors.     

Identification of Genes Encoding Amino Acid Permeases by Inactivation of Selected ORFs from the Synechocystis Genomic Sequence

Genes encoding elements of four amino acid permeases were identified by insertional inactivation of ORFs from the genomic sequence of the cyanobacterium Synechocystis sp. strain PCC 6803 whose putative products are homologous to amino acid permease proteins from other bacteria. A transport system for neutral amino acids and histidine and a transport system for basic amino acids and glutamine were identified as ABC-type transporters, whereas Na(+)-dependent transport of glutamate was found to be mediated by at least two systems, the secondary permease GltS and a TRAP-type transporter. Except for GltS, substrate specificities of the identified permeases do not match those of previously characterized systems homologous to these permeases.     

Transport of methionine in Trypanosoma brucei brucei

African trypanosomes live free in the bloodstream and central nervous system of mammalian hosts and also within the midgut of the tsetse fly vectors which transmit them. The parasite plasma membrane represents the interface between both hosts and parasite, and trypanosomes accumulate many essential metabolites via specific transport processes. L-Methionine uptake by procyclic and bloodstream forms of Trypanosoma brucei has been measured and shown to be mediated by a transporter presenting similar characteristics in both forms of the parasite. The carrier shows, in both forms, a relatively high affinity for methionine (Km ca. 30 microM). The effect of inhibitors of ion gradients across the membrane indicated that the uptake process is likely to be dependent upon a proton motive force. Various methionine analogues were tested against the transporter and these have demonstrated that the recognition depends on the motif common to all amino acids, and an electronegative group at the position of the sulphur atom separated from the alpha-carbon atom by a two carbon spacer.     

Identification and characterization of trypanocides by functional expression of an adenosine transporter from Trypanosoma brucei in yeast

The causative agents of sleeping sickness, Trypanosoma brucei rhodesiense and T. brucei gambiense, do not synthesize purines de novo but salvage purine bases and nucleosides from their hosts. We used yeast as an expression system for functional characterization of the trypanosomal adenosine transporter TbAT1. A selection of purine analogs and flavonoids were tested for their ability to interfere with adenosine transport, with the aims of identifying (a) trypanocidal TbAT1 substrates, and (b) inhibitors of trypanosomal purine transport. Cordycepin (3'-deoxyadenosine) was a TbAT1 substrate of high activity against T. brucei rhodesiense (IC50 0.2 nM). Inhibitors of mammalian nucleoside transport were not active, while the flavonol silibinin was a potent, noncompetitive inhibitor of TbAT1-mediated adenosine transport in yeast. Silibinin also inhibited melarsen-induced lysis of bloodstream form trypanosomes. IC50 values to T. brucei rhodesiense and to human carcinoma cells were 0.6 and 140 microM, respectively, indicating a good selectivity towards the parasites. Further studies are necessary to elucidate the effects of flavonoids on trypanosomal purine transport and their potential as trypanocides.     

Alteration to one of three adenosine transporters is associated with resistance to Cymelarsan in Trypanosoma evansi

 Continuous exposure of Trypanosoma evansi bloodstream forms to Cymelarsan in vitro resulted in the induction of resistance to the drug over a period of 4.5 months. Induction of resistance to Cymelarsan was accompanied by increased resistance to both Arsobal and Berenil, but Cymelarsan-resistant trypanosomes remained sensitive to Suramin and Antrycide. Since resistance to arsenical drugs in trypanosomes has been linked to changes in adenosine uptake, the adenosine metabolism in drug-sensitive and drug-resistant clones was measured. The initial rate of [³H]-adenosine uptake was much higher in sensitive trypanosomes than in drug-resistant parasites, but the amount of radiolabel accumulated by each population was similar. Both adenine and inosine inhibited incorporation of [³H]-adenosine in each population, but in quite different ways. Adenine inhibited more than 80% of adenosine incorporation in drug-sensitive trypanosomes but suppressed less than half of this process in the resistant population. In contrast, inosine inhibited only 10–15% of adenosine incorporation in the sensitive parasites but was inhibitory to a much greater extent in resistant trypanosomes. Lysis of drug-sensitive trypanosomes by 1 μM Cymelarsan was inhibited by adenosine, adenine and Berenil but not by inosine. Furthermore, in drug-sensitive trypanosomes, adenosine uptake could be reduced in three stages by the sequential addition of inosine, Berenil and adenine, whereas in drug-resistant parasites a reduction in adenosine uptake was caused only by the addition of inosine and adenine. These observations provide evidence that the acquisition of resistance to Cymelarsan is accompanied by the loss of one of three adenosine transporters in T. evansi. In drug-sensitive trypanosomes, this transporter mediates the entry of adenine and also of Cymelarsan and Berenil into the parasites. Received: 10 July 1995 / Accepted: 16 September 1995     

Two high affinity nucleoside transporters in Leishmania donovani

A rapid sampling kinetic technique has been used to evaluate the nucleoside transport functions of Leishmania donovani. The results indicated that L. donovani promastigotes possessed two independent purine nucleoside transporters with nonoverlapping substrate specificity. The first transported inosine, guanosine, and their analogs, while the second carried adenosine, analogs of adenosine, and the pyrimidine nucleosides, uridine, cytidine, and thymidine. The apparent Km values of the two nucleoside permeases for their purine nucleoside substrates were extraordinarily low, in the micromolar range. The organisms were capable of concentrating purine nucleosides from the medium and converting them to the nucleotide level with great efficiency and rapidity. Inosine and adenosine transport could be distinguished by different sensitivities to sulfhydryl reagents, suggesting structural differences between the two transporters. Finally, the two nucleoside transport systems of L. donovani were virtually refractory to inhibition by 4-nitrobenzylthioinosine and dipyridamole, two potent inhibitors of nucleoside entry into mammalian cells.     

Genetic evidence for the essential role of PfNT1 in the transport and utilization of xanthine, guanine, guanosine and adenine by Plasmodium falciparum

The malaria parasite, Plasmodium falciparum, is unable to synthesize the purine ring de novo and is therefore wholly dependent upon purine salvage from the host for survival. Previous studies have indicated that a P. falciparum strain in which the purine transporter PfNT1 had been disrupted was unable to grow on physiological concentrations of adenosine, inosine and hypoxanthine. We have now used an episomally complemented pfnt1Delta knockout parasite strain to confirm genetically the functional role of PfNT1 in P. falciparum purine uptake and utilization. Episomal complementation by PfNT1 restored the ability of pfnt1Delta parasites to transport and utilize adenosine, inosine and hypoxanthine as purine sources. The ability of wild-type and pfnt1Delta knockout parasites to transport and utilize the other physiologically relevant purines adenine, guanine, guanosine and xanthine was also examined. Unlike wild-type and complemented P. falciparum parasites, pfnt1Delta parasites could not proliferate on guanine, guanosine or xanthine as purine sources, and no significant transport of these substrates could be detected in isolated parasites. Interestingly, whereas isolated pfnt1Delta parasites were still capable of adenine transport, these parasites grew only when adenine was provided at high, non-physiological concentrations. Taken together these results demonstrate that, in addition to hypoxanthine, inosine and adenosine, PfNT1 is essential for the transport and utilization of xanthine, guanine and guanosine.     

The major cell surface glycoprotein procyclin is a receptor for induction of a novel form of cell death in African trypanosomes in vitro

Bloodstream forms (BSF) and procyclic culture forms (PCF) of African trypanosomes were incubated with a variety of lectins in vitro. Cessation of cell division and profound morphological changes were seen in procyclic forms but not in BSF after incubation with concanavalin A (Con A), wheat germ agglutinin and Ricinus communis agglutinin. These lectins caused the trypanosomes to cease division, become round and increase dramatically in size, the latter being partially attributable to the formation of what appeared to be a large 'vacuole-like structure' or an expanded flagellar pocket. Con A was used in all further experiments. Spectrophotometric quantitation of extracted DNA and flow cytometry using the DNA intercalating dye propidium iodide showed that the DNA content of Con A-treated trypanosomes increased dramatically when compared to untreated parasites. Examination of these cells by fluorescence microscopy showed that many of the Con A-treated cells were multinucleate whereas the kinetoplasts were mostly present as single copies, indicating a disequilibrium between nuclear and kinetoplast replication. Immunofluorescence experiments using monoclonal antibodies (mAb) specific for paraflagellar rod proteins and for kinetoplastid membrane protein-11 (KMP-11), showed that the Con A-treated parasites had begun to duplicate the flagellum but that this had only proceeded along part of the length of the cells, suggesting that the cell division process was initiated but that cytokinesis was subsequently inhibited. Tunicamycin-treated wild-type trypanosomes and mutant trypanosomes expressing both high levels of non-glycosylated procyclins and procyclin isoforms with truncated N-linked sugars were resistant to the effects of Con A, suggesting that N-linked carbohydrates on the procyclin surface coat were the ligands for Con A binding. This was supported by data obtained using mutant parasites created by deletion of all three EP procyclin isoforms, two of which contain N-glycosylation sites, by homologous recombination. The knockout mutants showed reduced binding of fluorescein-labelled Con A as determined by flow cytometry and were resistant to the effects of Con A. Taken together the results show that Con A induces multinucleation, a disequilibrium between nuclear and kinetoplast replication and a unique form of cell death in procyclic African trypanosomes and that the ligands for Con A binding are carbohydrates on the EP forms of procyclin. The possible significance of these findings for the life cycle of the trypanosomes in the tsetse fly vector is discussed.     

Membrane transport of sepiapterin and dihydrobiopterin by equilibrative nucleoside transporters: a plausible gateway for the salvage pathway of tetrahydrobiopterin biosynthesis

Tetrahydrobiopterin (BH(4)) is synthesized de novo in particular cells, but in the case of a systemic or local BH(4) deficiency, BH(4) supplementation therapy is applied. BH(4)-responsive PKU has also been effectively treated with BH(4) supplementation. However, the rapid clearance of the supplemented BH(4) has prevented the therapy from being widely accepted. Deposition of BH(4) after supplementation involves oxidation of BH(4) to dihydrobiopterin (BH(2)) and subsequent conversion to BH(4) by the salvage pathway. This pathway is known to be almost ubiquitous in the body. However, the mechanism for the redistribution and exclusion of BH(4) across the plasma membrane remains unclear. The aim of this work was to search for the key transporter of the uptake precursor of the salvage pathway. Based on the observed sensitivity of pterin transport to nitrobenzylthioinosine (NBMPR), we examined the ability of ENT1 and ENT2, representative equilibrative nucleoside transporters, to transport sepiapterin (SP), BH(2) or BH(4) using HeLa cell and Xenopus oocyte expression systems. hENT2 was capable of transporting the pterins with an efficiency of SP>BH(2)>BH(4). hENT1 could also transport the pterins but less efficiently. Non-transfected HeLa cells and rat aortic endothelial cells were able to incorporate the pterins and accumulate BH(4) via uptake that is likely mediated by ENT2 (SP>BH(2)>BH(4)). When exogenous BH(2) was given to mice, it was efficiently converted to BH(4) and its tissue deposition was similar to that of sepiapterin as reported (Sawabe et al., 2004). BH(4) deposition after BH(2) administration was influenced by prior treatment with NBMPR, suggesting that the distribution of the administered BH(2) was largely mediated by ENT2, although urinary excretion appeared to be managed by other mechanisms. The molecular basis of the transport of SP, BH(2), and BH(4) across the plasma membrane has now been described for the first time: ENT2 is a transporter of these pterins and is a plausible gateway to the salvage pathway of BH(4) biosynthesis, at least under conditions of exogenous pterin supplementation. The significance of the gateway was discussed in terms of BH(2) uptake for BH(4) accumulation and the release for modifying the intracellular BH(2)/BH(4) ratio.     

Culture form and tsetse fly midgut form procyclic Trypanosoma brucei express common proteins

Proteins expressed by culture form and tsetse fly midgut form procyclic trypanosomes were examined by polyacrylamide gel electrophoretic techniques. Analysis of the proteins of the two forms of procyclic organisms was performed by comparison of autoradiographs of high resolution two-dimensional polyacrylamide gels prepared using [35S]methionine-labelled parasites. Only eight spots were found to differ between autoradiographs of culture form and tsetse fly midgut form parasites. Seven of these differences were attributable to 35S-labelled non-trypanosomal proteins from the tsetse midgut. The other single spot difference was seen in one of two experiments and was present only in the autoradiograph of the material from trypanosome-infected tsetse fly midgut. Thus the cultivated procyclic organisms did not differ significantly from their tsetse-derived counterparts in protein composition and therefore their use as models for the natural stage is probably justified for most studies.     

GNP1, the high-affinity glutamine permease of S. cerevisiae

  Glutamine uptake in S. cerevisiae is mediated by at least three transporters: high- and low-affinity glutamine permeases and the general amino-acid permease. We have isolated the gene encoding the high-affinity glutamine permease and named it GNP1. The amino-acid sequence of GNP1, and its hydropathy profile of 12 transmembrane domains, closely resemble those of known amino-acid permeases. The Km of GNP1 for glutamine uptake was determined to be 0.59 mM. Cells lacking GNP1 exhibit reduced levels of glutamine transport, and are resistant to a toxic analog of glutamine, L-glutamic acid γ-monohydroxamate. Unlike other amino-acid permeases, whose expression is nitrogen-source limited, GNP1 is expressed on both rich and poor nitrogen sources. Received: 27 February / 30 March 1996     

Stage-specific development of a novel adenosine transporter in Leishmania donovani amastigotes

Leishmania donovani, like all other kinetoplastida, is a purine auxotroph. Comparative studies of adenosine transport in L. donovani amastigotes and promastigotes revealed that, unlike the promastigote stage, the amastigote possesses two distinct adenosine transporters (T(1) and T(2)) both with high affinities (K(m), 1.14+/-0.05 and 2. 09+/-0.13 microM, respectively). One of these transporters (T(1)) appears to be identical with the adenosine/pyrimidine nucleoside transporter of the promastigote reported earlier. The other transporter (T(2)) is specific for the amastigote stage and transports only purine nucleosides. The biological significance of this stage-specific development of the second adenosine transporter has been briefly discussed.