Transport of purines and purine salvage pathway inhibitors by the Plasmodium falciparum equilibrative nucleoside transporter PfENT1

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Abstract

Plasmodium falciparum is a purine auxotroph. The transport of purine nucleosides and nucleobases from the host erythrocyte to the parasite cytoplasm is essential to support parasite growth. P. falciparum equilibrative nucleoside transporter 1 (PfENT1) is a major route for purine transport across the parasite plasma membrane. Malarial parasites are sensitive to inhibitors of purine salvage pathway enzymes. The immucillin class of purine nucleoside phosphorylase inhibitors and the adenosine analog, tubercidin, block growth of P. falciparum under in vitro culture conditions. We sought to determine whether these inhibitors utilize PfENT1 to gain access to the parasite cytosol. There is considerable controversy in the literature regarding the Km and/or Ki for purine transport by PfENT1 in the Xenopus oocyte expression system. We show that oocytes metabolize adenosine but not hypoxanthine. For adenosine, metabolism is the rate limiting step in oocyte uptake assays, making hypoxanthine the preferred substrate for PfENT1 transport studies in oocytes. We demonstrate that the Ki for PfENT1 transport of hypoxanthine and adenosine is in the 300–700 μM range. Effects of substrate metabolism on uptake studies may explain conflicting results in the literature regarding the PfENT1 adenosine transport Km. PfENT1 transports the tubercidin class of compounds. None of the immucillin compounds tested inhibited PfENT1 transport of [3H]hypoxanthine or [3H]adenosine. Although nucleobases are transported, modifications of the ribose ring in corresponding nucleoside analogs affect substrate recognition by PfENT1. These results provide new insights into PfENT1 and the mechanism by which purine salvage pathway inhibitors are transported into the parasite cytoplasm.

Introduction

Malaria, caused by infection with parasites of the Plasmodium genus, remains a devastating global health problem. It accounts for 300–500 million clinical cases and 1–2 million deaths each year. Due to the increasing emergence of resistance to current antimalarial drugs, efforts to establish new drug targets within the parasite have become increasingly important. The essential role of DNA synthesis during malaria's 48 h intra-erythrocytic growth phase suggests that purine metabolic pathways may represent promising targets for the development of new antimalarial drugs. Like many protozoan parasites, Plasmodium parasites are purine auxotrophs incapable of synthesizing purines de novo [1], [2]. While the parasite's dependence upon an external purine source has been known for nearly two decades [3], recent studies have begun to elucidate the molecular details involved in purine transport and metabolism.

The intra-erythrocytic malarial parasite transports purine nucleosides and nucleobases from the erythrocyte cytoplasm into the parasite cytosol via the PfENT1 equilibrative nucleoside transporter [4], [5], [6]. In the parasite cytoplasm, purine nucleosides and nucleobases are metabolized to generate nucleotides needed for nucleic acid synthesis, ATP generation, and intracellular signaling. However, the set of purine metabolic enzymes within the malarial parasite are more limited than those found in most mammalian cells. Plasmodium parasites do not contain a gene for adenosine kinase (AK) and thus cannot directly convert adenosine to AMP [7], [8]. For this reason, adenosine that is transported into the parasite cytosol is converted to hypoxanthine via the successive action of adenosine deaminase (PfADA) and purine nucleoside phosphorylase (PfPNP). Hypoxanthine is then utilized by hypoxanthine–guanine–xanthine phosphoribosyltransferase (PfHGXPRT) to generate inosine 5′-mono-phosphate (IMP) [9]. IMP is the branch-point for the synthesis of all other parasite purine nucleotides. The majority of purines salvaged by Plasmodium falciparum are metabolized through this pathway.

During malaria infection in humans, plasma purines provide a source of purines that the parasites can use. The concentrations of various purines in human plasma are in the range of 0.4–6 μM [10]. During growth under in vitro culture conditions P. falciparum can proliferate in media containing a single purine source (hypoxanthine, adenine, guanine, xanthine, inosine, adenosine or guanosine) at a concentration greater than ∼2–5 μM [11]. Parasite growth, however, with just guanine, guanosine or xanthine as the sole purine source is less robust than with the other purines and they are toxic at concentrations >50 μM [11]. Thus, during malaria infection the total plasma purine concentration available to the intra-erythrocytic parasites is ∼10–30 μM [10]. Pfnt1 knockout parasites can survive in culture but only in media supplemented with supraphysiological purine concentrations [6], [11].

Several purine salvage pathway inhibitors have antimalarial activity under in vitro culture conditions. The immucillin family of nucleoside analogs inhibit PNP. Immucillins inhibit both the erythrocytic and malarial PNP enzymes [12], [13]. The inhibition constant for Immucillin-H inhibition of PfPNP is 29 nM [14]. Immucillins inhibit in vitro P. falciparum growth in cultures containing hypoxanthine at a concentration higher than that found in human plasma [15]. In the presence of 10 μM hypoxanthine, 10 μM Immucillin-H completely inhibited parasite growth [15]. Tubercidin, an adenosine analog, is a substrate for adenosine kinase (AK). It is phosphorylated by AK and can act as a competitive inhibitor of AK phosphorylation of adenosine. Tubercidin also blocks parasite growth [16], although P. falciparum lacks AK activity. It is unclear whether tubercidin compounds exert their effect solely upon erythrocyte AK or interact with an as yet unidentified target within the parasite. The transport pathway(s) by which these purine salvage pathway inhibitors enter into the parasite is unknown.

DNA sequence analysis suggests that the P. falciparum genome encodes four putative nucleoside transporters [17], however only PfENT1 has been characterized [4], [5], [18], [19]. PfENT1 is an equilibrative nucleoside transporter localized to the parasite plasma membrane [20] that transports both nucleosides and nucleobases. However, disparate values have been reported for the transport Km or Ki of PfENT1 for various physiologic substrates [4], [5], [18], [19], [21]. To date, nearly all functional studies of PfENT1 have been performed using radioactive substrate uptake in the Xenopus laevis oocyte heterologous expression system. Metabolism of transported substrates in the oocyte cytoplasm to non-transportable products can have significant effects on the interpretation of radiolabeled substrate uptake experiments, especially if the metabolic enzyme is the rate limiting step under the “uptake” assay conditions. Previous studies of PfENT1 mediated transport in Xenopus oocytes have not accounted for the potential effects of substrate metabolism in the oocyte cytoplasm in the interpretation of their experimental results. In order to study the transport of purine salvage pathway inhibitors that could alter the metabolism of transport substrates in the Xenopus oocyte expression system it was essential to characterize the effects of purine metabolism on the apparent transport properties of PfENT1 in the Xenopus oocyte system. We expressed PfENT1 in oocytes and investigated the metabolism of transported substrates, the transport of immucillin and tubercidin derivatives and the transport of purine nucleoside and nucleobase analogs to define the structural determinants of substrate specificity. The results help to resolve conflicting data in the literature, define the transport pathway for the tubercidins and demonstrate that immucillin uptake is mediated by a transport pathway other than PfENT1.

Section snippets

Materials

Oligonucleotides to clone PfENT1 were synthesized by Sigma Genosys Biotechnologies, Inc. (The Woodlands, TX). Restriction enzymes were purchased from New England Biolabs, Inc. (Beverly, MA). [3H]Adenosine was purchased from Amersham Biosciences and [3H]hypoxanthine was purchased from PerkinElmer NEN Radiochemicals (Waltham, MA). [3H]Tubercidin was purchased from Moravek Biochemicals and Radiochemicals (Brea, CA). All other reagents were purchased from Sigma–Aldrich (St. Louis, MO) unless

PfENT1 mediated transport of natural purine substrates

Substrate transport by PfENT1 was measured by tritiated substrate uptake into oocytes expressing PfENT1. Fig. 1 shows a time course for the uptake of either 1.5 μM adenosine or hypoxanthine into PfENT1 expressing oocytes. While the uptake of both adenosine and hypoxanthine proceeds linearly for the first 30 min (Fig. 1, inset), the rate of adenosine uptake is nearly four times faster than the rate of hypoxanthine uptake. Similar observations were reported elsewhere [19] but no explanation has

Discussion

We sought to examine PfENT1's ability to transport purine salvage pathway enzyme inhibitors in a heterologous Xenopus oocyte expression system. We found that oocyte mediated purine metabolism had a significant confounding effect upon PfENT1 transport studies. We showed that adenosine is phosphorylated to AMP, ADP, and ATP in the oocyte cytoplasm, presumably via adenosine kinase and adenylate kinase. This raises an important issue for the use of radiolabeled substrate uptake experiments for the

Acknowledgements

We thank I.J. Frame for expert technical assistance and Dr. Alan Finkelstein for helpful discussions. This work was supported in part by the National Institutes of Health [Grant AI064933 (to M.H.A.) and AI49512 (to V.S.)] and by a contract from the New Zealand Foundation for Science, Research and Technology.

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