Pyrimidine transport activities in trypanosomes

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TRENDS in Parasitology

Therefore, the limited repertoire of stevor transcripts that is observed in the asexual and sexual blood stages, which is cited by Duffy and Tham as evidence that exclusive transcription of a subset of stevor might occur, could also reflect some specialization of STEVOR variants for particular life-cycle compartments.

References 1 Duffy, M. and Tham, W-H. (2007) Transcription and coregulation of multigene families in Plasmodium falciparum. Trends Parasitol. 23, 183–186

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2 Sharp, S. et al. (2006) Programmed transcription of the var gene family, but not of stevor, in Plasmodium falciparum gametocytes. Eukaryot. Cell 5, 1206–1214 3 Kaviratne, M. et al. (2002) Small variant STEVOR antigen is uniquely located within Maurer’s clefts in Plasmodium falciparum-infected red blood cells. Eukaryot. Cell 1, 926–935 4 McRobert, L. et al. (2004) Distinct trafficking and localization of STEVOR proteins in three stages of the Plasmodium falciparum life cycle. Infect. Immun. 72, 6597–6602 5 Florens, L. et al. (2002) A proteomic view of the Plasmodium falciparum life cycle. Nature 419, 520–526 1471-4922/$ – see front matter ß 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.pt.2007.02.008

Research Focus

Pyrimidine transport activities in trypanosomes Vivian Bellofatto Department of Microbiology and Molecular Genetics, University of Medicine and Dentistry of New Jersey, Newark, NJ 07101, USA

Parasites of the Trypanosomatidae family are unable to synthesize purines. Instead, they rely on their hosts to supply these necessary compounds. The article by Gudin et al. identifies three transport mechanisms of the equilibrative nucleoside transporter family by which nucleosides and nucleobases are transported in this medically important family of organisms. The work by Gudin et al. characterizes the dynamics of these transporters and points to further areas for future genetic and therapeutic experiments.

Purine and pyrimidine biosynthesis Parasites derive the nutrients they need from their hosts [1]. The Trypanosomatidae family of protozoan parasites depends upon purine acquisition because they cannot synthesize this class of heterocyclic nitrogenous compounds [2]. Purine synthesis requires more energy and is metabolically more complex than pyrimidine synthesis. Trypanosomes can synthesize pyrimidines de novo but scavenge a range of purine molecules. Many trypanosomatids, including Trypanosoma and Leishmania, transition from mammalian host to arthropod vector during their complex life cycle [3]. In humans, African trypanosomes spend weeks or months in the bloodstream, depending on the specific subspecies of Trypanosome brucei, and eventually lodge in the brain [4]. In either locale, trypanosomes avail themselves of various purines. Although it is extremely difficult to measure serum purine and pyrimidine levels in the bloodstream accurately, hypoxanthine and xanthine [both nucleobases (Table 1)] are present in serum and help to satisfy the purine needs of the trypanosomes. During parasite invasion, immunostimulaCorresponding author: Bellofatto, V. ([email protected]). Available online 19 March 2007. www.sciencedirect.com

tion causes an increase in blood levels of extracellular adenosine [5]. In the final stages of human African trypanosomiasis (HAT), parasites migrate and proliferate in the host’s central nervous system (CNS) and eventually cause a coma [6]. The CNS is replete with extracellular purine nucleosides, specifically adenosine, that function as neuromodulators [7]. Arthropod vectors produce saliva that is rich in nucleotide-metabolizing enzymes that function as an anticoagulatory and anti-inflammatory defense mechanism [8]. The resultant purine nucleosides and bases satisfy the nutritional needs of the metacylic parasites that are primed to enter the mammalian host from the tsetse vector. Membrane transporters Membrane transporters that salvage nucleosides and nucleobases have been intensely studied [9,10]. The class of transporters that is designated as equilibrative nucleoside transporters (ENTs) is common in eukarya. ENTs have 11 transmembrane helices and are situated in the cellular membrane [11]. The ENT family includes proteins that bind to and deliver both purine and pyrimidine nucleosides from the extracellular milieu across the cell membrane (Table 1). ENT transporters are equilibrative or facilitative permeases in metazoa but several have been shown to be proton-dependent concentrative transporters in protozoa [12]. Fungal systems lack ENT-type permeases. Yeast and pathogenic fungi salvage nucleobases and nucleosides through a set of transmembrane proteins in the structurally distinct family of Fur permeases, which includes the proton symporters Fui1p and Fur4p [13]. Fur orthologs have not been found in trypanosome database searches. In trypanosomatids, all purine and pyrimidine transporters that have been studied to date are members of the ENT family. Although these ENT-type transporters take up a range of nucleosides and nucleobases, some are purine

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Table 1. Nitrogenous base precursorsa Common purines

Common pyrimidines

Nucleobases Adenine Guanine Hypoxanthine Xanthine Cytosine Thymine Uracil

Nucleosides Adenosine Guanosine Inosine Xanthosine Cytidine Thymidine Uridine

a These bases are precursors to DNA, RNA, coenzymes and carriers of high-energy phosphate bonds.

specific, whereas others recognize both purines and pyrimidines. Two of the best-studied nucleoside transporters (NTs) in trypanosomatids (NT1 and NT2 of Leishmania donovani) are clear examples of these phenomena [14]. Radiolabelled-ligand uptake studies and genetic analyses demonstrate that LdNT2 is specific for purines because it has a high affinity and selectivity for inosine, guanosine and xanthosine. LdNT1 transports adenosine in addition to pyrimidine nucleosides with reasonable efficiency [15]. Except for the Fur-related family, pyrimidine-specific transporters have not been identified in trypanosomatids or other eukaryotic cell types at the molecular level [16]. Pyrimidine transport in trypanosomes Gudin et al. [16] discuss pyrimidine transport in Trypanosoma brucei brucei procyclic parasites, which divide in the tsetse midgut. These organisms cause a wasting disease (nagana) in cattle and are akin to T. brucei subspecies that cause human disease. Gudin et al. outline careful radiolabelled-substrate uptake and inhibitor studies in intact parasites. They characterized the TbNT family members that function in the trypanosome plasma membrane with classic transport methodologies. They discovered two new transporter activities: the C1 transporter, the first trypanosomatid membrane transporter that is a high-affinity cytosine permease and the U2 transporter, the first trypanosome transporter that has a high affinity for uridine (Km of 4.1 mM). A third transporter has an affinity for uridine (Km of 34 mM) that is approximately an order of magnitude less than that observed for U2 and is probably the U1 transporter [17]. The first hint that T. brucei procyclic cells possess two distinct uridine transporters (U1 and U2) came from the observation that uridine uptake was inhibited in a biphasic manner by excess unlabelled uridine or uracil [16]. One transporter activity (U1) was resistant to inhibition by thymidine and cytidine, whereas the other was sensitive to these pyrimidine nucleosides. Comparison of kinetic data with previous findings revealed that the thymidine-resistant permease is the previously identified U1 transporter [17]. The other activity was because of a new permease (U2), which has a high affinity for uridine and is inhibited by thymidine and cytidine. An important next experiment is to determine the ligand-specificity range of C1 and U2 and note if these permeases function in purine transport. Exclusive pyrimidine transporters would be expected to be nonessential proteins and possibly regulated as a function of growth and environmental conditions. Although pyrimidine uptake is not essential in trypanosomes, kinetic analyses [16] indicate that the T. brucei www.sciencedirect.com

C1 transporter has an unusually high affinity for cytosine, consistent with a robust scavenging activity that is advantageous for a parasite [9], but a low capacity, consistent with low C1 levels. The authors suggest that the apparent low capacity of the newly identified cytosine (C1) permease might reflect regulated C1 gene expression. It is possible that the C1 mRNA and protein levels are downregulated under the experimental cell-culture conditions. The parasites used here, as in many studies of procyclic and bloodstream stage trypanosomes, are monomorphic organisms that are well adapted to axenic culture media. It would be interesting to assess C1 levels as T. brucei makes its way through its five-stage life cycle: (i) procyclics, (ii) epimastigotes, (iii) metacyclics, (iv) long slender bloodstream forms and (v) short stumpy bloodstream forms. Nucleobase and nucleoside transporters are included in the group of differentially expressed genes in trypanosomatids (see Ref. [2]). Because the parasite resides in different environments (ranging from the tsetse salivary gland to human intracranial spaces), it presumably modulates transporters in tune with the purine profile of the host or vector. Purine transport is essential for parasite survival. There are several interesting examples of stage-specific purine and pyrimidine transporters in the literature. To date, studies of mRNA steady-state levels and/or protein-activity levels of various purine and pyrimidine transporters in T. brucei (e.g. TbAT1, TbNT2, TbNT5 and TbH2) have shown that transporter expression is dynamic [18]. A recent report identified a novel purinenucleoside transporter, TbNT10, whose expression is greatly increased in parasites that have transformed into the short stumpy form and are primed to leave the human host and take up residence in the tsetse fly [19]. The extent (and, thus, the overall uptake rate) and specificity, of purine transport into parasites is probably under the control of molecular mechanisms that affect mRNA stability and translation. Because pyrimidines are not essential for trypanosomes, the evolutionary pressures on pyrimidine transporters and their regulation might differ markedly from wellstudied purine permeases [20]. Have purine transporters evolved to recognize a subset of pyrimidines in the continual quest of the trypanosome to parasitize its host or are there pyrimidine-specific transporters in trypanosomes? It will be exciting to tease apart the different membrane transporters in both the procyclic and bloodstream forms of T. brucei. To perform such experiments, individual transporter genes must be cloned and expressed in a suitable background. Further analysis of transporter mutants The next challenge for the field of trypanosome nucleoside and nucleobase transport is the fusion of biochemical and genetic analysis of transporter mutants. Genetic analysis requires molecular cloning of candidate transporters and their expression in either heterologous backgrounds or in trypanosomes that have been engineered to rely on a subset of transporters. The recent trypanosome genomes (http://www.genedb.org) gives valuable access to hypothetical nucleoside and nucleobase transporters and helps to

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lend molecular genetic identity to the biochemical activities of transporters that have varied affinity for different pyrimidines and, thus, might be pyrimidine specific [16]. ENTs have gained importance as the delivery site for a range of pharmacological agents that can enter and destroy cells that are abnormal, cells that are infected with parasites or cells that are growing uncontrollably in cancer. The initial work of Carter and Fairlamb [21], complemented by others [22,23], revealed that the targets of the arsenical derivatives (including melarsorpol), in addition to the diamidines (including pentamidine, in use since the early 1900s to combat HAT), include the adenine–adenosine transporter encoded by the TbAT1 gene (the P2 transporter). Recent work indicates that this P2 transporter is responsible for the uptake of DB75, a drug under study as the first orally administered HAT drug [24]. However, because the loss of the P2 transport activity is already associated with reduced sensitivity to melaminophenyl arsenicals and diamidines, other transporters must be characterized to develop effective trypanocidals that are not crossresistant to currently available drugs [18,25]. In addition, genetic changes engineered to alter substrate specificity are key to the design of drugs that mimic the natural substrates but work as pharmacological poisons. Characterizing transporters Characterizing transporters requires the identification of the key amino acids that determine transport function and substrate specificity. Work by the Landfear and Ullman laboratories recently demonstrated that a K153R mutant alters the ability of the LdNT1.1 transporter to bind inosine. Wild type NT1 does not transport inosine, whereas the mutant binds tightly with a Km of 27 mM [26]. An exciting use of either directed or forward genetic-based mutagenesis of transporter genes is the analysis of ligand discrimination among purines and the less bulky, and nonessential, pyrimidines. Missing from the literature is the molecular identification, in protozoa or higher eukaryotes, of nucleoside or nucleobase transporters that are specific for pyrimidines. Although Gudin et al. [16] identify three new pyrimidine transporters in T. brucei, including one with a high affinity for cytosine, it would be interesting to test if purines are permeates for either U2 and/or C1. Such a study would, for the first time, clearly define a class of pyrimidine-specific carriers in protozoa [16,17]. Discoveries in these parasites have been known to foreshadow what furtively exists in other eukaryotes. Acknowledgements The author is supported by NIAID grants AI29478 and AI53835 and was previously a Burroughs-Wellcome New Investigator in molecular parasitology.

References 1 Zimmer, C. (2000) Parasite Rex, Simon and Schuster Inc.

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2 Carter, N.S. et al. (2002) . In Molecular Medical Parasitology (Marr, J.J. et al., eds), pp. 197–224, Academic Press 3 Hoare, C.A. (1966) The classification of mammalian trypanosomes. Ergeb. Mikrobiol. Immunitatsforsch. Exp. Ther. 39, 43–57 4 Fevre, E.M. et al. (2006) Human African trypanosomiasis: epidemiology and control. Adv. Parasitol. 61, 167–221 5 Hasko, G. and Cronstein, B.N. (2004) Adenosine: an endogenous regulator of innate immunity. Trends Immunol. 25, 33–39 6 Kennedy, P.G. (2006) Diagnostic and neuropathogenesis issues in human African trypanosomiasis. Int. J. Parasitol. 36, 505–512 7 Hasko, G. et al. (2005) Adenosine receptor signaling in the brain immune system. Trends Pharmacol. Sci. 26, 511–516 8 Gounaris, K. and Selkirk, M.E. (2005) Parasite nucleotidemetabolizing enzymes and host purinergic signalling. Trends Parasitol. 21, 17–21 9 Cohn, C.S. and Gottlieb, M. (1997) The acquisition of purines by trypanosomatids. Parasitol. Today 13, 231–235 10 de Koning, H.P. et al. (2005) Purine and pyrimidine transport in pathogenic protozoa: from biology to therapy. FEMS Microbiol. Rev. 29, 987–1020 11 Hyde, R.J. et al. (2001) The ENT family of eukaryote nucleoside and nucleobase transporters: recent advances in the investigation of structure/function relationships and the identification of novel isoforms. Mol. Membr. Biol. 18, 53–63 12 Landfear, S.M. (2001) Molecular genetics of nucleoside transporters in Leishmania and African trypanosomes. Biochem. Pharmacol. 62, 149– 155 13 Zhang, J. et al. (2006) Characterization of the transport mechanism and permeant binding profile of the uridine permease fui1p of Saccharomyces cerevisiae. J. Biol. Chem. 281, 28210–28221 14 Liu, W. et al. (2006) Functional characterization of nucleoside transporter gene replacements in Leishmania donovani. Mol. Biochem. Parasitol. 150, 300–307 15 Aronow, B. et al. (1987) Two high affinity nucleoside transporters in Leishmania donovani. Mol. Biochem. Parasitol. 22, 29–37 16 Gudin, S. et al. (2006) Trypanosoma brucei: a survey of pyrimidine transport activities. Exp. Parasitol. 114, 118–125 17 de Koning, H.P. and Jarvis, S.M. (1998) A highly selective, high-affinity transporter for uracil in Trypanosoma brucei brucei: evidence for proton-dependent transport. Biochem. Cell Biol. 76, 853–858 18 Geiser, F. et al. (2005) Molecular pharmacology of adenosine transport in Trypanosoma brucei: P1/P2 revisited. Mol. Pharmacol. 68, 589– 595 19 Sanchez, M.A. et al. (2004) A novel purine nucleoside transporter whose expression is up-regulated in the short stumpy form of the Trypanosoma brucei life cycle. Mol. Biochem. Parasitol. 136, 265–272 20 Landfear, S.M. et al. (2004) Nucleoside and nucleobase transporters in parasitic protozoa. Eukaryot. Cell 3, 245–254 21 Carter, N.S. and Fairlamb, A.H. (1993) Arsenical-resistant trypanosomes lack an unusual adenosine transporter. Nature 361, 173–176 22 Barrett, M.P. et al. (1995) A diamidine-resistant Trypanosoma equiperdum clone contains a P2 purine transporter with reduced substrate affinity. Mol. Biochem. Parasitol. 73, 223–229 23 Maser, P. et al. (1999) A nucleoside transporter from Trypanosoma brucei involved in drug resistance. Science 285, 242–244 24 Lanteri, C.A. et al. (2006) Roles for the Trypanosoma brucei P2 transporter in DB75 uptake and resistance. Mol. Pharmacol. 70, 1585–1592 25 Matovu, E. et al. (2003) Mechanisms of arsenical and diamidine uptake and resistance in Trypanosoma brucei. Eukaryot. Cell 2, 1003–1008 26 Valdes, R. et al. (2006) Comprehensive examination of charged intramembrane residues in a nucleoside transporter. J. Biol. Chem. 281, 22647–22655 1471-4922/$ – see front matter ß 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.pt.2007.03.003