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Purine nucleobase transport in the intraerythrocytic malaria parasite
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International Journal for Parasitology 38 (2008) 203–209 www.elsevier.com/locate/ijpara
Purine nucleobase transport in the intraerythrocytic malaria parasite Megan J. Downie a, Kevin J. Saliba a
a,b
, Stefan Bro¨er a, Susan M. Howitt a, Kiaran Kirk
a,*
School of Biochemistry and Molecular Biology, Faculty of Science, The Australian National University, Canberra, ACT 0200, Australia b Medical School, The Australian National University, Canberra, ACT 0200, Australia Received 29 May 2007; received in revised form 13 July 2007; accepted 17 July 2007
Abstract Hypoxanthine, a nucleobase, serves as the major source of the essential purine group for the intraerythrocytic malaria parasite. In this study we have measured the uptake of hypoxanthine, and that of the related purine nucleobase adenine, by mature blood-stage Plasmodium falciparum parasites isolated from their host cells by saponin-permeabilisation of the erythrocyte and parasitophorous vacuole membranes. The uptake of both [3H]hypoxanthine and [3H]adenine was comprised of at least two components; in each case there was a rapid equilibration of the radiolabel between the intra- and extracellular solutions via a low-affinity transport mechanism, and an accumulation of radiolabel (such that the estimated intracellular concentration exceeded the extracellular concentration) via a higher-affinity process. The uptake of [3H]adenine was studied in more detail. The rapid, low-affinity equilibration of [3H]adenine between the intra-and extracellular solution was independent of the energy status of the parasite whereas the higher-affinity accumulation of the radiolabel was ATP-dependent. A kinetic analysis of adenine uptake revealed that the low-affinity (equilibrative) process had a Km of 1.2 mM, similar to the value of 0.82 mM estimated here (using the Xenopus laevis oocyte expression system) for the Km for the transport of adenine by PfENT1, a parasite-encoded member of the ‘equilibrative nucleoside/nucleobase transporter’ family. The results indicate that nucleobases enter the intraerythrocytic parasite via a rapid, equilibrative process that has kinetic characteristics similar to those of PfENT1. 2007 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. Keywords: Plasmodium; Nucleobase; Transport; Xenopus oocytes; Malaria; Nutrient; Biochemistry
1. Introduction Like all protozoan parasites, the malaria parasite Plasmodium falciparum is unable to manufacture the purine ring de novo and, hence, is entirely dependent on the salvage of purine compounds from the environment. Several lines of evidence suggest that, for the intraerythrocytic form of the parasite, the nucleobase hypoxanthine serves as the main purine source: hypoxanthine is the major purine available in human serum (Murray, 1971); depletion of red blood cell hypoxanthine pools by xanthine oxidase inhibits parasite growth by up to 90% in vitro (Berman et al., 1991); and a recent study on parasites lacking the plasma membrane nucleoside/nucleobase transporter PfENT1 (‘P. falciparum Equilibrative Nucleoside/nucleo-
*
Corresponding author. Tel.: +61 2 6125 2284; fax: +61 2 6125 0313. E-mail address: [email protected] (K. Kirk).
base Transporter 1’ (Parker et al., 2000), also known as PfNT1 (Carter et al., 2000; Rager et al., 2001)) has provided evidence for the conversion of exogenous purines into hypoxanthine by host erythrocyte enzymes, followed by the uptake of hypoxanthine by the intraerythrocytic parasite (El Bissati et al., 2006). Hypoxanthine is the standard purine source supplied during the in vitro culture of P. falciparum, and [3H]hypoxanthine incorporation serves as a standard measure of parasite proliferation when conducting P. falciparum growth assays (Desjardins et al., 1979; Asahi et al., 1996). Yet despite the reliance of both the parasite and the parasitologist on this compound, the mechanism(s) by which hypoxanthine, or any other nucleobase, enters the parasite cytosol has not been characterised in any detail. In this study we have investigated the uptake of nucleobases across the plasma membrane of blood-stage P. falciparum parasites, functionally isolated from their host cells by saponin-permeabilisation of the erythrocyte and parasi-
0020-7519/$30.00 2007 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijpara.2007.07.005
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tophorous vacuole membranes. The characteristics of nucleobase transport in the parasite are compared with those of PfENT1 expressed in Xenopus laevis oocytes. Characterising the transport of rapidly metabolised substrates such as hypoxanthine is fraught with difficulty; distinguishing the different contributions of transport and metabolism to the measured uptake/accumulation of radiolabelled solute is not straightforward (Wohlhueter and Plagemann, 1989). In the case of hypoxanthine, analysis is also complicated by its high hydrophobicity and hence low water-solubility, making it difficult to measure uptake kinetics over a wide concentration range. For these reasons, the majority of the experiments in this study were performed with the related purine adenine which is both more soluble and less readily metabolised by the parasite (Queen et al., 1989), and which therefore serves as a useful model substrate for the characterisation of purine nucleobase transport. 2. Materials and methods 2.1. Materials [3H]Adenine and [3H]hypoxanthine were purchased from Amersham Biosciences. Oligonucleotides were custom-synthesised by Proligo. Restriction endonucleases were purchased from Invitrogen. Immucillin-H was a kind gift from Vern L. Schramm; it was synthesised by Peter C. Tyler and Gary B. Evans at Industrial Research Ltd. and dissolved in H2O. 2.2. Parasite culture Experiments were carried out using the 3D7 strain of P. falciparum at the mature trophozoite-stage (36–40 h postinvasion). The parasites were cultured in Group O, Rh+ erythrocytes and synchronised as described elsewhere (Allen and Kirk, 2004). 2.3. Parasite preparation Parasites were ‘isolated’ from their host erythrocytes by treating parasitised erythrocytes with saponin as described elsewhere (Saliba et al., 1998). Saponin renders the erythrocyte and parasitophorous vacuole membranes permeable to macromolecules (Ansorge et al., 1996) but leaves the parasite plasma membrane intact and able to maintain transmembrane ion gradients (Saliba and Kirk, 1999; Alleva and Kirk, 2001) as well as a substantial membrane potential (Allen and Kirk, 2004). Following saponin-treatment the isolated parasites were washed (>3·) and resuspended in a HEPES-buffered saline (125 mM NaCl, 5 mM KCl, 1 mM MgCl2, 20 mM glucose and 25 mM HEPES, pH 7.1). In experiments that required parasites to be depleted of ATP, parasites were resuspended in a glucose-free HEPES-buffered saline (135 mM NaCl, 5 mM KCl, 1 mM MgCl2 and 25 mM HEPES, pH 7.1) and incubated at 37 C for 15 min (Saliba and Kirk, 1999).
2.4. Transport measurements in isolated parasites The uptake of [3H]adenine and [3H]hypoxanthine was measured as described previously for nucleosides (Downie et al., 2006). Briefly, uptake of radiolabelled nucleobase was initiated by addition of a 200 lL volume of isolated parasites to an equal volume of radiolabelled substrate solution (at twice the intended final concentration) layered over a 200 lL dibutyl phthalate/dioctyl phthalate (5:4, v/v) oil mix. Due to the rapid nature of nucleoside and nucleobase transport by isolated parasites, all experiments were carried out at 4 C (in a cold room). Uptake was stopped at predetermined times by sedimenting the parasites below the oil using centrifugation (15,800g, 2 min) in a rapid-acceleration microcentrifuge (Beckman Microfuge E). Time points less than 15 s were sampled to the beat of a digital metronome (model MT40, Wittner GmbH and Co.). The cell pellets were processed for scintillation counting as described previously (Saliba et al., 1998). Estimates of the volume of extracellular fluid trapped in the cell pellets were made as described previously (Downie et al., 2006). Time courses are expressed in terms of ‘distribution ratios’ (i.e. the calculated intracellular concentration relative to the extracellular concentration of a particular solute). The use of distribution ratios permits an assessment of the accumulation of the radiolabel within the cell. Accumulation of radiolabel to concentrations higher than those in the extracellular medium (i.e. to a distribution ratio greater than 1) indicates the contribution of processes other than equilibrative transport to the measured uptake (i.e. active transport, binding or metabolism). The concentration of radiolabel inside the cell was calculated as described previously (Saliba et al., 1998; Downie et al., 2006). Distribution ratios were calculated by dividing this intracellular concentration of radiolabel by the concentration of radiolabel in the extracellular solution. 2.5. Expression and functional characterisation of PfENT1 in Xenopus oocytes The cRNA encoding PfENT1 was prepared as described previously (Downie et al., 2006). The sequence of the PfENT1 gene used here is identical to that deposited in the NCBI database by Parker et al. (2000). Xenopus laevis oocytes were prepared and injected with cRNA, dissolved in Ambion MEGAclear elution buffer, as described previously (Bro¨er, 2003; Downie et al., 2006). Nucleobase transport assays in oocytes were performed 4–5 days post-injection, as described for PfENT1induced nucleoside transport (Downie et al., 2006). 3. Results 3.1. Hypoxanthine uptake by isolated parasites Fig. 1 shows time courses for the uptake of hypoxanthine by saponin-isolated parasites. When unlabelled hypo-
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Fig. 1. Time courses for the uptake of [3H]hypoxanthine by Plasmodium falciparum trophozoites isolated from their host cells by saponin-permeabilisation of the erythrocyte and parasitophorous vacuole membranes. Unlabelled hypoxanthine was present at 10 lM (closed circles) or 200 lM (open circles). The experiments were performed at 4 C and data are presented as a distribution ratio, which denotes the intracellular concentration of hypoxanthine relative to that in the extracellular solution. The 10 lM data are averaged from three independent experiments (shown ± SEM), each carried out in duplicate, while the 200 lM data are averaged from two independent experiments (shown ± range/2), each carried out in duplicate. Where not shown, error bars fall within the symbols.
xanthine was present at an extracellular concentration of 10 lM, [3H]hypoxanthine was accumulated rapidly by the cells, reaching an apparent intracellular concentration some four times higher than that outside the cell within the 2 s required to acquire the first time point. There was then a subsequent, slower accumulation to a distribution ratio of 13 over the next 10 min, reminiscent of the behaviour seen previously for the purine-nucleosides adenosine and inosine (Downie et al., 2006). When the extracellular levels of unlabelled hypoxanthine were increased to 200 lM, the distribution ratio of [3H]hypoxanthine reached values close to 1 (i.e. the concentration was approximately the same inside and out) within 2 s, and increased only slightly (to 3) over the next 10 min. The hydrophobic nature, and consequent low solubility, of hypoxanthine, made it infeasible to look at the effect of higher concentrations of unlabelled hypoxanthine on [3H]hypoxanthine uptake by the parasite. 3.2. Adenine uptake by isolated parasites Adenine is another purine nucleobase which, in contrast to hypoxanthine, is not readily metabolised by the parasite (Queen et al., 1989) and which therefore serves as a useful model substrate with which to investigate purine nucleobase transport. As shown in Fig. 2, in the presence of 10 lM unlabelled extracellular adenine, [3H]adenine showed a rapid association with the isolated parasites, reaching a distribution ratio of 1 within 2 s. This distribution ratio continued to rise rapidly to 1.5 at 6 s. There
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Fig. 2. Time courses for the uptake (at 4 C) of [3H]adenine by isolated Plasmodium falciparum parasites. Unlabelled adenine was present at extracellular concentrations of 10 lM (closed circles), 200 lM (open circles) or 10 mM (closed triangles). The grey bar represents the uptake, over 10 s, of [3H]thymidine in the presence of 10 lM thymidine, which acted as a control in each experiment. Under these conditions [3H]thymidine equilibrates between the intra- and extracellular solutions (reaching a distribution ratio of 1) but is not accumulated (Downie et al., 2006). The data, expressed as a distribution ratio, are averaged from four independent experiments (shown ± SEM), each performed at 4 C and carried out in duplicate. Where not shown, error bars fall within the symbols.
was then a slower accumulation, with [3H]adenine reaching a distribution ratio of just over 2 at 60 s, increasing only slightly (to 2.5) by 10 min. When extracellular unlabelled adenine was increased to 200 lM, the association of [3H]adenine with the parasites was slowed substantially. A distribution ratio of 1 was reached after 30 s, and this increased only slightly over the next 10 min, to 1.5. A further increase in unlabelled extracellular adenine to 10 mM led to a further reduction in the rate of adenine association with the cell. The distribution ratio increased only slowly, reaching a final level of close to 1 by 10 min. Distribution ratios as low as those seen here for [3H]adenine (i.e below 3) are difficult to measure accurately. Another substrate shown previously to equilibrate across the parasite plasma membrane, giving a distribution ratio close to 1, is the non-metabolised (by the parasite) pyrimidine nucleoside, thymidine (Downie et al., 2006). In order to confirm that the relatively low distribution ratios obtained with [3H]adenine were accurate, we included in each [3H]adenine uptake experiment a single measurement of the uptake of [3H]thymidine (measured over 10 s in the presence of 10 lM extracellular unlabelled thymidine) which is transported by an equilibrative process (reaching a distribution ratio of 1) but not metabolised (Downie et al., 2006). Under the conditions in Fig. 2, [3H]thymidine reached a distribution ratio of 1 (represented by a grey bar in Fig. 2), consistent with previous results (Downie et al., 2006) and indicating that the distribution ratios estimated here for adenine were accurate. The mechanism(s) underlying the accumulation of adenine to distribution ratios >1 observed at the lower adenine
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concentrations tested was investigated by measuring time courses for the uptake of adenine (in the presence of 10 lM extracellular unlabelled adenine) in parasites either depleted of their ATP content or treated with ImmucillinH, an inhibitor of the parasite’s purine nucleoside phosphorylase (PfPNP) (Kicska et al., 2002a,b). Nucleoside phosphorylases are involved in the interconversion of nucleobases and nucleosides. The results are shown in Fig. 3. In control cells (ATP replete, no Immucillin-H; solid circles) the [3H]adenine distribution ratio increased rapidly to a value of 3 after 1 min, then remained approximately constant for the remainder of the time course (10 min). Cells treated with 500 nM Immucillin-H (open circles) showed a rapid accumulation of radiolabel to levels just below that of the control cells, indicating that adenine accumulation is unlikely to be a consequence of its metabolism by PfPNP. This contrasts to the situation with the nucleoside, adenosine, the accumulation of which is inhibited by Immucillin-H (under the same conditions as those used here; Downie et al., 2006). In parasites that were depleted of ATP (by incubation in glucose-free saline; closed triangles) the [3H]adenine distribution ratio reached a value close to 1 within 2 s and remained at this level for the duration of the time course. The distribution ratio estimates were confirmed with a thymidine uptake control (grey bar), similar to that shown for Fig. 2.
3.3. Concentration dependence of adenine uptake by isolated parasites Uptake of [3H]adenine by (ATP-replete) parasites suspended in media containing a range of adenine concentrations was measured using a 4 s incubation period. The results are shown in Fig. 4. The data conformed to a Michaelis–Menten curve (Fig. 4a), with a Km of 1.2 ± 0.4 mM and a Vmax of 0.19 ± 0.04 nmol/lL cell water per second (equivalent to 5.3 ± 1.1 lmol/1012 parasites per second; n = 3, ± SEM). When the data were converted to a Hanes plot (Hanes, 1932; Roberts, 1977), two lines of differing slope were observed, indicating the presence of both low- and high-affinity components in the uptake process (Fig. 4b). While there are not enough data points in either slope to give accurate Km estimates, the steeper slope (closed symbols) indicates a Km of 0.17 mM, while the slope fitted to the open symbols indicates a Km of 1.5 mM. 3.4. Nucleobase transport by PfENT1 in Xenopus oocytes The parasite plasma membrane nucleoside/nucleobase transporter, PfENT1, has previously been expressed in Xenopus oocytes, but with somewhat varying results, particularly with regard to nucleobase transport. Parker et al. (2000) reported that PfENT1 was able to transport nucleobases, including hypoxanthine and adenine. By contrast, Carter et al. (2000) reported that PfENT1-induced adenosine transport was not inhibited to any significant extent by either nucleobase. In our hands, oocytes expressing PfENT1 show an increased ability to take up both hypoxanthine and adenine from the extracellular solution, compared to oocytes injected with elution buffer alone (Fig. 5a). Fig. 5b shows the concentration dependence of adenine transport by PfENT1 expressed in Xenopus oocytes. The transport was saturable, with an estimated Km of 0.82 ± 0.17 mM and a Vmax of 200 ± 32 pmol/oocyte per 10 min (n = 2, ± range/2). 4. Discussion
Fig. 3. The effect of ATP depletion and of the PfPNP inhibitor Immucillin-H on the uptake of [3H]adenine by isolated Plasmodium falciparum parasites suspended in medium containing 10 lM adenine. [3H]Adenine uptake was measured over 10 min in ATP-replete parasites in the presence (open circles) or absence (closed circles) of Immucillin-H, and in ATP-depleted parasites in the absence of Immucillin-H (closed triangles). Parasites were depleted of ATP by incubation at 37 C in a glucose-free saline for 15 min before being cooled to 4 C for the experiment. For experiments involving Immucillin-H, parasites were incubated (at 4 C) in the presence of 500 nM Immucillin-H for 15 min prior to the start of the time course, which was carried out with the same concentration of Immucillin-H present throughout. The grey bar represents the uptake, over 10 s, of [3H]thymidine (in the presence of 10 lM thymidine) by ATP replete cells (as for Fig. 2). The data are expressed as a distribution ratio and are averaged from four independent experiments (shown ± SEM), each carried out in duplicate.
The results obtained here for the uptake of the nucleobase, hypoxanthine, by isolated parasites are similar to those obtained previously for the nucleosides adenosine and inosine, both of which are converted to hypoxanthine prior to being metabolised further (Hyde, 2007). Adenosine and inosine equilibrate rapidly between the intra- and extracellular solutions (i.e. they reach a distribution ratio of 1) via a low-affinity transport process (postulated to involve PfENT1; Downie et al., 2006) before being accumulated to higher levels via one or more high-affinity mechanisms (postulated to involve metabolism and/or accumulation in an intracellular compartment; Downie et al., 2006). The data obtained here (Fig. 1) are consistent with the same being true for hypoxanthine. The data obtained at 10 lM represent the combined effects of equilibrative transport and a subsequent high-affinity accumulation, attributable to metabolism,
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Fig. 4. Concentration dependence of the influx of adenine into isolated Plasmodium falciparum trophozoites. Uptake was measured over a 4 s time period in parasites exposed to varying concentrations of adenine. The data are averaged from three independent experiments (shown ± SEM), each carried out in duplicate at 4 C. In (a) the data are fitted to a Michaelis–Menten curve (Km = 1.2 ± 0.4 mM; Vmax = 0.19 ± 0.04 nmol/lL cell water per second) whereas in (b), the same data are expressed as a Hanes plot. The Hanes plot may be resolved into two lines of differing slope, indicating the presence of two distinct components to the uptake of [3H]adenine by isolated parasites. To aid clarity, the data points indicating the higher-affinity component in the Hanes plot are shown as closed symbols whereas the data points indicating the lower-affinity component are shown as open symbols. Where not shown, error bars fall within the symbols.
Fig. 5. (a) Uptake of [3H]hypoxanthine and [3H]adenine by Xenopus oocytes injected with either the cRNA encoding PfENT1 (black bars) or with an equivalent volume of the elution buffer (white bars). The data shown are from a single experiment but are representative of those obtained in two separate experiments (adenine) or >10 experiments (hypoxanthine). Unlabelled nucleobase was present at 10 lM in each experiment. Hypoxanthine uptake was measured over 20 min, 5 days post-injection. Adenine uptake was measured over 10 min, 4 days post-injection. Data shown are the mean uptake of 8–10 oocytes. Error bars represent SD. (b) Concentration dependence of the adenine uptake by PfENT1 expressed in Xenopus oocytes. Transport via PfENT1 was calculated by subtracting the mean uptake in oocytes injected with elution buffer alone from the mean uptake of oocytes injected with the cRNA encoding PfENT1. The data are shown fitted to a Michaelis–Menten curve (Km = 0.82 ± 0.17 mM; Vmax of 200 ± 32 pmol/oocyte per 10 min). Experiments were performed 4 days post-injection. The data are averaged from two independent experiments (shown ± range/2). Where not shown, error bars fall within the symbol.
binding or accumulative transport. At 200 lM the low-affinity equilibrative transport process (by which the distribution ratio reached a value close to 1 within a few seconds) continued to operate whereas the high-affinity accumulation processes were largely saturated. Adenine, another purine nucleobase, is more soluble than hypoxanthine and undergoes limited metabolism within the parasite (Queen et al., 1988, 1989). The uptake of adenine by isolated parasites was comprised of at least
two components. There was an ATP-independent, lowaffinity component responsible for the distribution ratio of 1 seen in ATP depleted cells (Fig. 3, closed triangles) or, within 30 s, in ATP replete cells when extracellular adenine was present at 200 lM (Fig. 2, open circles). There was also a second ATP-dependent high-affinity component, responsible for the radiolabel reaching distribution ratios above 1, as seen in ATP replete cells when adenine was present at 10 lM (Fig. 2, closed circles).
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The Michaelis–Menten curve fitted to the concentrationdependence data obtained in parasites over an adenine concentration range of 0.01–4 mM (Fig. 4a) is dominated by the low-affinity component (estimated from the fitted curve to have a Km of 1.2 ± 0.4 mM). However, the different kinetics of the two components are revealed in the Hanes plot of Fig. 4b which shows that as well as the low-affinity component (estimated from the slope to have a Km of 1.5 mM, similar to the value of 1.2 mM estimated from the Michaelis–Menten curve) there is a higher-affinity component with a Km of below 0.2 mM. As with hypoxanthine and the various nucleosides studied previously (Downie et al., 2006), the rapid, low-affinity, ATP-independent component of adenine uptake is likely to be mediated by equilibrative transport of this compound across the parasite plasma membrane. It has been noted previously (Downie et al., 2006) that the characteristics of nucleoside transport in isolated parasites are similar to those of the parasite plasma membrane transporter PfENT1 expressed in Xenopus oocytes. However, there has been some inconsistency regarding the question of whether PfENT1 transports nucleobases (Carter et al., 2000; Parker et al., 2000). In our hands PfENT1 did transport both hypoxanthine and adenine (Fig. 5a), confirming the findings of Parker et al. (2000). The Km of 0.82 ± 0.17 mM obtained here for PfENT1-mediated adenine transport into Xenopus oocytes is similar to the value of 1.2 ± 0.4 mM obtained here for the transport of adenine in isolated parasites. It is somewhat higher than the value of 0.32 ± 0.10 mM estimated by Parker et al. (2000) for PfENT1-mediated adenine transport in Xenopus oocytes, measured over a more limited concentration range than that used here. Nevertheless, both are indicative of a lowaffinity transport process. The ATP-dependent high-affinity component of the association of adenine with the parasite, responsible for the distribution ratios above 1 may be attributed to the rapid metabolic trapping of the compound, and/or to an energy-dependent transport or binding of the radiolabelled substrate within a subcellular compartment. The finding that 500 nM Immucillin-H had little effect on adenine accumulation, indicates that adenine accumulation is unlikely to be due to the conversion of adenine to adenosine by PfPNP, consistent with earlier findings (Kicska et al., 2002a; Ting et al., 2005). The levels of adenine accumulation were significantly lower than those seen for the purine-nucleosides inosine and adenosine and the purine nucleobase hypoxanthine, consistent with earlier reports that hypoxanthine, adenosine and inosine are metabolised much more rapidly than adenine (Queen et al., 1988; Queen et al., 1989). On entering the parasite adenosine and inosine are first converted to hypoxanthine, which is rapidly phosphoribosylated by the enzyme hypoxanthine–guanine–xanthine phosphoribosyltransferase (HGXPRTase). Phosphoribosyltransferases catalyse the transfer of a phosphoribosyl group from phosphoribosylpyrophosphate (the production of which is dependent upon ATP; Roth et al.,
1986) to a purine base. Parasite adenine phosphoribosyltransferase (APRTase) activity is 1,500 times lower than HGXPRTase activity, and it has been reported that the two activities are catalysed by two separate enzymes with different molecular weights (Queen et al., 1988, 1989). More recently it has been suggested that the Plasmodium genome does not encode a specific APRTase enzyme (Chaudhary et al., 2004), and that, rather than being phosphoribosylated directly, adenine may be deaminated to hypoxanthine by an adenine deaminase activity (Hyde, 2007). Thus, while the data in Figs. 2 and 3 are consistent with the presence of an APRTase activity, it is not clear whether this activity might be due to a separate adeninespecific phosphoribosyltransferase enzyme (as suggested by Queen et al., 1989), to a residual adenine PRTase activity associated with the parasite’s HGXPRTase, or to deamination of adenine to hypoxanthine (followed by its subsequent phosphoribosylation). In conclusion, in this study we have shown that hypoxanthine, the primary purine source for the intraerythrocytic malaria parasite, enters the parasite via a rapid, equilibrative, low-affinity mechanism. The same is true of adenine. Both compounds undergo accumulation within the parasite, hypoxanthine more so than adenine, via a mechanism that is likely to involve the metabolism and/ or accumulative transport of the radiolabelled compounds within the parasite. The low-affinity of the equilibrative transport process is consistent with the involvement of PfENT1, shown here (as in a previous study; Parker et al., 2000) to transport both hypoxanthine and adenine, and to have a Km for adenine similar to that estimated for adenine transport in the parasite. The hypothesis that PfENT1 serves as a major route for the uptake of purine nucleobases is consistent with the recent finding that transgenic parasite lacking a functional PfENT1 show reduced uptake of [3H]hypoxanthine (El Bissati et al., 2006). Acknowledgements We are grateful to the Canberra Branch of the Australian Red Cross Blood Service for the provision of blood, to Dr. Vern Schramm for the kind gift of the ImmucillinH, and to the Australian Research Council and Australian National Health and Medical Research Council for grant support. References Allen, R.J., Kirk, K., 2004. The membrane potential of the intraerythrocytic malaria parasite Plasmodium falciparum. J. Biol. Chem. 279, 11264–11272. Alleva, L.M., Kirk, K., 2001. Calcium regulation in the intraerythrocytic malaria parasite Plasmodium falciparum. Mol. Biochem. Parasitol. 117, 121–128. Ansorge, I., Benting, J., Bhakdi, S., Lingelbach, K., 1996. Protein sorting in Plasmodium falciparum-infected red blood cells permeabilized with the pore-forming protein streptolysin O. Biochem. J. 315, 307–314.
M.J. Downie et al. / International Journal for Parasitology 38 (2008) 203–209 Asahi, H., Kanazawa, T., Kajihara, Y., Takahashi, K., Takahashi, T., 1996. Hypoxanthine: a low molecular weight factor essential for growth of erythrocytic Plasmodium falciparum in a serum-free medium. Parasitology 113, 19–23. Berman, P.A., Human, L., Freese, J.A., 1991. Xanthine oxidase inhibits growth of Plasmodium falciparum in human erythrocytes in vitro. J. Clin. Invest. 88, 1848–1855. Bro¨er, S., 2003. Xenopus laevis oocytes. Methods Mol. Biol. 227, 245–258. Carter, N.S., Ben Mamoun, C., Liu, W., Silva, E.O., Landfear, S.M., Goldberg, D.E., Ullman, B., 2000. Isolation and functional characterization of the PfNT1 nucleoside transporter gene from Plasmodium falciparum. J. Biol. Chem. 275, 10683–10691. Chaudhary, K., Darling, J.A., Fohl, L.M., Sullivan Jr., W.J., Donald, R.G., Pfefferkorn, E.R., Ullman, B., Roos, D.S., 2004. Purine salvage pathways in the apicomplexan parasite Toxoplasma gondii. J. Biol. Chem. 279, 31221–31227. Desjardins, R.E., Canfield, C.J., Haynes, J.D., Chulay, J.D., 1979. Quantitative assessment of antimalarial activity in vitro by a semiautomated microdilution technique. Antimicrob. Agents Chemother. 16, 710–718. Downie, M.J., Saliba, K.J., Howitt, S.M., Bro¨er, S., Kirk, K., 2006. Transport of nucleosides across the Plasmodium falciparum parasite plasma membrane has characteristics of PfENT1. Mol. Microbiol. 60, 738–748. El Bissati, K., Zufferey, R., Witola, W.H., Carter, N.S., Ullman, B., Ben Mamoun, C., 2006. The plasma membrane permease PfNT1 is essential for purine salvage in the human malaria parasite Plasmodium falciparum. Proc. Natl. Acad. Sci. USA 103, 9286–9291. Hanes, C.S., 1932. Studies on plant amylases: the effect of starch concentration upon the velocity of hydrolysis by the amylase of germinated barley. Biochem. J. 26, 1406–1421. Hyde, J.E., 2007. Targeting purine and pyrimidine metabolism in human apicomplexan parasites. Curr. Drug Targets 8, 31–47. Kicska, G.A., Tyler, P.C., Evans, G.B., Furneaux, R.H., Kim, K., Schramm, V.L., 2002a. Transition state analogue inhibitors of purine nucleoside phosphorylase from Plasmodium falciparum. J. Biol. Chem. 277, 3219–3225. Kicska, G.A., Tyler, P.C., Evans, G.B., Furneaux, R.H., Schramm, V.L., Kim, K., 2002b. Purine-less death in Plasmodium falciparum induced
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by immucillin-H, a transition state analogue of purine nucleoside phosphorylase. J. Biol. Chem. 277, 3226–3231. Murray, A.W., 1971. The biological significance of purine salvage. Annu. Rev. Biochem. 40, 811–826. Parker, M.D., Hyde, R.J., Yao, S.Y., McRobert, L., Cass, C.E., Young, J.D., McConkey, G.A., Baldwin, S.A., 2000. Identification of a nucleoside/nucleobase transporter from Plasmodium falciparum, a novel target for anti-malarial chemotherapy. Biochem. J. 349, 67–75. Queen, S.A., Vander Jagt, D., Reyes, P., 1988. Properties and substrate specificity of a purine phosphoribosyltransferase from the human malaria parasite, Plasmodium falciparum. Mol. Biochem. Parasitol. 30, 123–133. Queen, S.A., Vander Jagt, D.L., Reyes, P., 1989. Characterization of adenine phosphoribosyltransferase from the human malaria parasite, Plasmodium falciparum. Biochim. Biophys. Acta 996, 160–165. Rager, N., Ben Mamoun, C., Carter, N.S., Goldberg, D.E., Ullman, B., 2001. Localization of the Plasmodium falciparum PfNT1 nucleoside transporter to the parasite plasma membrane. J. Biol. Chem. 276, 41095–41099. Roberts, D.V., 1977. Enzyme Kinetics. Cambridge University Press, Cambridge. Roth Jr., E.F., Ruprecht, R.M., Schulman, S., Vanderberg, J., Olson, J.A., 1986. Ribose metabolism and nucleic acid synthesis in normal and glucose-6-phosphate dehydrogenase-deficient human erythrocytes infected with Plasmodium falciparum. J. Clin. Invest. 77, 1129–1135. Saliba, K.J., Horner, H.A., Kirk, K., 1998. Transport and metabolism of the essential vitamin pantothenic acid in human erythrocytes infected with the malaria parasite Plasmodium falciparum. J. Biol. Chem. 273, 10190–10195. Saliba, K.J., Kirk, K., 1999. pH regulation in the intracellular malaria parasite, Plasmodium falciparum: H+ extrusion via a V-type H+ATPase. J. Biol. Chem. 274, 33213–33219. Ting, L.M., Shi, W., Lewandowicz, A., Singh, V., Mwakingwe, A., Birck, M.R., Ringia, E.A., Bench, G., Madrid, D.C., Tyler, P.C., Evans, G.B., Furneaux, R.H., Schramm, V.L., Kim, K., 2005. Targeting a novel Plasmodium falciparum purine recycling pathway with specific immucillins. J. Biol. Chem. 280, 9547–9554. Wohlhueter, R.M., Plagemann, P.G., 1989. Measurement of transport versus metabolism in cultured cells. Methods Enzymol. 173, 714–732.
International Journal for Parasitology 38 (2008) 203–209 www.elsevier.com/locate/ijpara
Purine nucleobase transport in the intraerythrocytic malaria parasite Megan J. Downie a, Kevin J. Saliba a
a,b
, Stefan Bro¨er a, Susan M. Howitt a, Kiaran Kirk
a,*
School of Biochemistry and Molecular Biology, Faculty of Science, The Australian National University, Canberra, ACT 0200, Australia b Medical School, The Australian National University, Canberra, ACT 0200, Australia Received 29 May 2007; received in revised form 13 July 2007; accepted 17 July 2007
Abstract Hypoxanthine, a nucleobase, serves as the major source of the essential purine group for the intraerythrocytic malaria parasite. In this study we have measured the uptake of hypoxanthine, and that of the related purine nucleobase adenine, by mature blood-stage Plasmodium falciparum parasites isolated from their host cells by saponin-permeabilisation of the erythrocyte and parasitophorous vacuole membranes. The uptake of both [3H]hypoxanthine and [3H]adenine was comprised of at least two components; in each case there was a rapid equilibration of the radiolabel between the intra- and extracellular solutions via a low-affinity transport mechanism, and an accumulation of radiolabel (such that the estimated intracellular concentration exceeded the extracellular concentration) via a higher-affinity process. The uptake of [3H]adenine was studied in more detail. The rapid, low-affinity equilibration of [3H]adenine between the intra-and extracellular solution was independent of the energy status of the parasite whereas the higher-affinity accumulation of the radiolabel was ATP-dependent. A kinetic analysis of adenine uptake revealed that the low-affinity (equilibrative) process had a Km of 1.2 mM, similar to the value of 0.82 mM estimated here (using the Xenopus laevis oocyte expression system) for the Km for the transport of adenine by PfENT1, a parasite-encoded member of the ‘equilibrative nucleoside/nucleobase transporter’ family. The results indicate that nucleobases enter the intraerythrocytic parasite via a rapid, equilibrative process that has kinetic characteristics similar to those of PfENT1. 2007 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. Keywords: Plasmodium; Nucleobase; Transport; Xenopus oocytes; Malaria; Nutrient; Biochemistry
1. Introduction Like all protozoan parasites, the malaria parasite Plasmodium falciparum is unable to manufacture the purine ring de novo and, hence, is entirely dependent on the salvage of purine compounds from the environment. Several lines of evidence suggest that, for the intraerythrocytic form of the parasite, the nucleobase hypoxanthine serves as the main purine source: hypoxanthine is the major purine available in human serum (Murray, 1971); depletion of red blood cell hypoxanthine pools by xanthine oxidase inhibits parasite growth by up to 90% in vitro (Berman et al., 1991); and a recent study on parasites lacking the plasma membrane nucleoside/nucleobase transporter PfENT1 (‘P. falciparum Equilibrative Nucleoside/nucleo-
*
Corresponding author. Tel.: +61 2 6125 2284; fax: +61 2 6125 0313. E-mail address: [email protected] (K. Kirk).
base Transporter 1’ (Parker et al., 2000), also known as PfNT1 (Carter et al., 2000; Rager et al., 2001)) has provided evidence for the conversion of exogenous purines into hypoxanthine by host erythrocyte enzymes, followed by the uptake of hypoxanthine by the intraerythrocytic parasite (El Bissati et al., 2006). Hypoxanthine is the standard purine source supplied during the in vitro culture of P. falciparum, and [3H]hypoxanthine incorporation serves as a standard measure of parasite proliferation when conducting P. falciparum growth assays (Desjardins et al., 1979; Asahi et al., 1996). Yet despite the reliance of both the parasite and the parasitologist on this compound, the mechanism(s) by which hypoxanthine, or any other nucleobase, enters the parasite cytosol has not been characterised in any detail. In this study we have investigated the uptake of nucleobases across the plasma membrane of blood-stage P. falciparum parasites, functionally isolated from their host cells by saponin-permeabilisation of the erythrocyte and parasi-
0020-7519/$30.00 2007 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijpara.2007.07.005
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tophorous vacuole membranes. The characteristics of nucleobase transport in the parasite are compared with those of PfENT1 expressed in Xenopus laevis oocytes. Characterising the transport of rapidly metabolised substrates such as hypoxanthine is fraught with difficulty; distinguishing the different contributions of transport and metabolism to the measured uptake/accumulation of radiolabelled solute is not straightforward (Wohlhueter and Plagemann, 1989). In the case of hypoxanthine, analysis is also complicated by its high hydrophobicity and hence low water-solubility, making it difficult to measure uptake kinetics over a wide concentration range. For these reasons, the majority of the experiments in this study were performed with the related purine adenine which is both more soluble and less readily metabolised by the parasite (Queen et al., 1989), and which therefore serves as a useful model substrate for the characterisation of purine nucleobase transport. 2. Materials and methods 2.1. Materials [3H]Adenine and [3H]hypoxanthine were purchased from Amersham Biosciences. Oligonucleotides were custom-synthesised by Proligo. Restriction endonucleases were purchased from Invitrogen. Immucillin-H was a kind gift from Vern L. Schramm; it was synthesised by Peter C. Tyler and Gary B. Evans at Industrial Research Ltd. and dissolved in H2O. 2.2. Parasite culture Experiments were carried out using the 3D7 strain of P. falciparum at the mature trophozoite-stage (36–40 h postinvasion). The parasites were cultured in Group O, Rh+ erythrocytes and synchronised as described elsewhere (Allen and Kirk, 2004). 2.3. Parasite preparation Parasites were ‘isolated’ from their host erythrocytes by treating parasitised erythrocytes with saponin as described elsewhere (Saliba et al., 1998). Saponin renders the erythrocyte and parasitophorous vacuole membranes permeable to macromolecules (Ansorge et al., 1996) but leaves the parasite plasma membrane intact and able to maintain transmembrane ion gradients (Saliba and Kirk, 1999; Alleva and Kirk, 2001) as well as a substantial membrane potential (Allen and Kirk, 2004). Following saponin-treatment the isolated parasites were washed (>3·) and resuspended in a HEPES-buffered saline (125 mM NaCl, 5 mM KCl, 1 mM MgCl2, 20 mM glucose and 25 mM HEPES, pH 7.1). In experiments that required parasites to be depleted of ATP, parasites were resuspended in a glucose-free HEPES-buffered saline (135 mM NaCl, 5 mM KCl, 1 mM MgCl2 and 25 mM HEPES, pH 7.1) and incubated at 37 C for 15 min (Saliba and Kirk, 1999).
2.4. Transport measurements in isolated parasites The uptake of [3H]adenine and [3H]hypoxanthine was measured as described previously for nucleosides (Downie et al., 2006). Briefly, uptake of radiolabelled nucleobase was initiated by addition of a 200 lL volume of isolated parasites to an equal volume of radiolabelled substrate solution (at twice the intended final concentration) layered over a 200 lL dibutyl phthalate/dioctyl phthalate (5:4, v/v) oil mix. Due to the rapid nature of nucleoside and nucleobase transport by isolated parasites, all experiments were carried out at 4 C (in a cold room). Uptake was stopped at predetermined times by sedimenting the parasites below the oil using centrifugation (15,800g, 2 min) in a rapid-acceleration microcentrifuge (Beckman Microfuge E). Time points less than 15 s were sampled to the beat of a digital metronome (model MT40, Wittner GmbH and Co.). The cell pellets were processed for scintillation counting as described previously (Saliba et al., 1998). Estimates of the volume of extracellular fluid trapped in the cell pellets were made as described previously (Downie et al., 2006). Time courses are expressed in terms of ‘distribution ratios’ (i.e. the calculated intracellular concentration relative to the extracellular concentration of a particular solute). The use of distribution ratios permits an assessment of the accumulation of the radiolabel within the cell. Accumulation of radiolabel to concentrations higher than those in the extracellular medium (i.e. to a distribution ratio greater than 1) indicates the contribution of processes other than equilibrative transport to the measured uptake (i.e. active transport, binding or metabolism). The concentration of radiolabel inside the cell was calculated as described previously (Saliba et al., 1998; Downie et al., 2006). Distribution ratios were calculated by dividing this intracellular concentration of radiolabel by the concentration of radiolabel in the extracellular solution. 2.5. Expression and functional characterisation of PfENT1 in Xenopus oocytes The cRNA encoding PfENT1 was prepared as described previously (Downie et al., 2006). The sequence of the PfENT1 gene used here is identical to that deposited in the NCBI database by Parker et al. (2000). Xenopus laevis oocytes were prepared and injected with cRNA, dissolved in Ambion MEGAclear elution buffer, as described previously (Bro¨er, 2003; Downie et al., 2006). Nucleobase transport assays in oocytes were performed 4–5 days post-injection, as described for PfENT1induced nucleoside transport (Downie et al., 2006). 3. Results 3.1. Hypoxanthine uptake by isolated parasites Fig. 1 shows time courses for the uptake of hypoxanthine by saponin-isolated parasites. When unlabelled hypo-
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Fig. 1. Time courses for the uptake of [3H]hypoxanthine by Plasmodium falciparum trophozoites isolated from their host cells by saponin-permeabilisation of the erythrocyte and parasitophorous vacuole membranes. Unlabelled hypoxanthine was present at 10 lM (closed circles) or 200 lM (open circles). The experiments were performed at 4 C and data are presented as a distribution ratio, which denotes the intracellular concentration of hypoxanthine relative to that in the extracellular solution. The 10 lM data are averaged from three independent experiments (shown ± SEM), each carried out in duplicate, while the 200 lM data are averaged from two independent experiments (shown ± range/2), each carried out in duplicate. Where not shown, error bars fall within the symbols.
xanthine was present at an extracellular concentration of 10 lM, [3H]hypoxanthine was accumulated rapidly by the cells, reaching an apparent intracellular concentration some four times higher than that outside the cell within the 2 s required to acquire the first time point. There was then a subsequent, slower accumulation to a distribution ratio of 13 over the next 10 min, reminiscent of the behaviour seen previously for the purine-nucleosides adenosine and inosine (Downie et al., 2006). When the extracellular levels of unlabelled hypoxanthine were increased to 200 lM, the distribution ratio of [3H]hypoxanthine reached values close to 1 (i.e. the concentration was approximately the same inside and out) within 2 s, and increased only slightly (to 3) over the next 10 min. The hydrophobic nature, and consequent low solubility, of hypoxanthine, made it infeasible to look at the effect of higher concentrations of unlabelled hypoxanthine on [3H]hypoxanthine uptake by the parasite. 3.2. Adenine uptake by isolated parasites Adenine is another purine nucleobase which, in contrast to hypoxanthine, is not readily metabolised by the parasite (Queen et al., 1989) and which therefore serves as a useful model substrate with which to investigate purine nucleobase transport. As shown in Fig. 2, in the presence of 10 lM unlabelled extracellular adenine, [3H]adenine showed a rapid association with the isolated parasites, reaching a distribution ratio of 1 within 2 s. This distribution ratio continued to rise rapidly to 1.5 at 6 s. There
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Fig. 2. Time courses for the uptake (at 4 C) of [3H]adenine by isolated Plasmodium falciparum parasites. Unlabelled adenine was present at extracellular concentrations of 10 lM (closed circles), 200 lM (open circles) or 10 mM (closed triangles). The grey bar represents the uptake, over 10 s, of [3H]thymidine in the presence of 10 lM thymidine, which acted as a control in each experiment. Under these conditions [3H]thymidine equilibrates between the intra- and extracellular solutions (reaching a distribution ratio of 1) but is not accumulated (Downie et al., 2006). The data, expressed as a distribution ratio, are averaged from four independent experiments (shown ± SEM), each performed at 4 C and carried out in duplicate. Where not shown, error bars fall within the symbols.
was then a slower accumulation, with [3H]adenine reaching a distribution ratio of just over 2 at 60 s, increasing only slightly (to 2.5) by 10 min. When extracellular unlabelled adenine was increased to 200 lM, the association of [3H]adenine with the parasites was slowed substantially. A distribution ratio of 1 was reached after 30 s, and this increased only slightly over the next 10 min, to 1.5. A further increase in unlabelled extracellular adenine to 10 mM led to a further reduction in the rate of adenine association with the cell. The distribution ratio increased only slowly, reaching a final level of close to 1 by 10 min. Distribution ratios as low as those seen here for [3H]adenine (i.e below 3) are difficult to measure accurately. Another substrate shown previously to equilibrate across the parasite plasma membrane, giving a distribution ratio close to 1, is the non-metabolised (by the parasite) pyrimidine nucleoside, thymidine (Downie et al., 2006). In order to confirm that the relatively low distribution ratios obtained with [3H]adenine were accurate, we included in each [3H]adenine uptake experiment a single measurement of the uptake of [3H]thymidine (measured over 10 s in the presence of 10 lM extracellular unlabelled thymidine) which is transported by an equilibrative process (reaching a distribution ratio of 1) but not metabolised (Downie et al., 2006). Under the conditions in Fig. 2, [3H]thymidine reached a distribution ratio of 1 (represented by a grey bar in Fig. 2), consistent with previous results (Downie et al., 2006) and indicating that the distribution ratios estimated here for adenine were accurate. The mechanism(s) underlying the accumulation of adenine to distribution ratios >1 observed at the lower adenine
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concentrations tested was investigated by measuring time courses for the uptake of adenine (in the presence of 10 lM extracellular unlabelled adenine) in parasites either depleted of their ATP content or treated with ImmucillinH, an inhibitor of the parasite’s purine nucleoside phosphorylase (PfPNP) (Kicska et al., 2002a,b). Nucleoside phosphorylases are involved in the interconversion of nucleobases and nucleosides. The results are shown in Fig. 3. In control cells (ATP replete, no Immucillin-H; solid circles) the [3H]adenine distribution ratio increased rapidly to a value of 3 after 1 min, then remained approximately constant for the remainder of the time course (10 min). Cells treated with 500 nM Immucillin-H (open circles) showed a rapid accumulation of radiolabel to levels just below that of the control cells, indicating that adenine accumulation is unlikely to be a consequence of its metabolism by PfPNP. This contrasts to the situation with the nucleoside, adenosine, the accumulation of which is inhibited by Immucillin-H (under the same conditions as those used here; Downie et al., 2006). In parasites that were depleted of ATP (by incubation in glucose-free saline; closed triangles) the [3H]adenine distribution ratio reached a value close to 1 within 2 s and remained at this level for the duration of the time course. The distribution ratio estimates were confirmed with a thymidine uptake control (grey bar), similar to that shown for Fig. 2.
3.3. Concentration dependence of adenine uptake by isolated parasites Uptake of [3H]adenine by (ATP-replete) parasites suspended in media containing a range of adenine concentrations was measured using a 4 s incubation period. The results are shown in Fig. 4. The data conformed to a Michaelis–Menten curve (Fig. 4a), with a Km of 1.2 ± 0.4 mM and a Vmax of 0.19 ± 0.04 nmol/lL cell water per second (equivalent to 5.3 ± 1.1 lmol/1012 parasites per second; n = 3, ± SEM). When the data were converted to a Hanes plot (Hanes, 1932; Roberts, 1977), two lines of differing slope were observed, indicating the presence of both low- and high-affinity components in the uptake process (Fig. 4b). While there are not enough data points in either slope to give accurate Km estimates, the steeper slope (closed symbols) indicates a Km of 0.17 mM, while the slope fitted to the open symbols indicates a Km of 1.5 mM. 3.4. Nucleobase transport by PfENT1 in Xenopus oocytes The parasite plasma membrane nucleoside/nucleobase transporter, PfENT1, has previously been expressed in Xenopus oocytes, but with somewhat varying results, particularly with regard to nucleobase transport. Parker et al. (2000) reported that PfENT1 was able to transport nucleobases, including hypoxanthine and adenine. By contrast, Carter et al. (2000) reported that PfENT1-induced adenosine transport was not inhibited to any significant extent by either nucleobase. In our hands, oocytes expressing PfENT1 show an increased ability to take up both hypoxanthine and adenine from the extracellular solution, compared to oocytes injected with elution buffer alone (Fig. 5a). Fig. 5b shows the concentration dependence of adenine transport by PfENT1 expressed in Xenopus oocytes. The transport was saturable, with an estimated Km of 0.82 ± 0.17 mM and a Vmax of 200 ± 32 pmol/oocyte per 10 min (n = 2, ± range/2). 4. Discussion
Fig. 3. The effect of ATP depletion and of the PfPNP inhibitor Immucillin-H on the uptake of [3H]adenine by isolated Plasmodium falciparum parasites suspended in medium containing 10 lM adenine. [3H]Adenine uptake was measured over 10 min in ATP-replete parasites in the presence (open circles) or absence (closed circles) of Immucillin-H, and in ATP-depleted parasites in the absence of Immucillin-H (closed triangles). Parasites were depleted of ATP by incubation at 37 C in a glucose-free saline for 15 min before being cooled to 4 C for the experiment. For experiments involving Immucillin-H, parasites were incubated (at 4 C) in the presence of 500 nM Immucillin-H for 15 min prior to the start of the time course, which was carried out with the same concentration of Immucillin-H present throughout. The grey bar represents the uptake, over 10 s, of [3H]thymidine (in the presence of 10 lM thymidine) by ATP replete cells (as for Fig. 2). The data are expressed as a distribution ratio and are averaged from four independent experiments (shown ± SEM), each carried out in duplicate.
The results obtained here for the uptake of the nucleobase, hypoxanthine, by isolated parasites are similar to those obtained previously for the nucleosides adenosine and inosine, both of which are converted to hypoxanthine prior to being metabolised further (Hyde, 2007). Adenosine and inosine equilibrate rapidly between the intra- and extracellular solutions (i.e. they reach a distribution ratio of 1) via a low-affinity transport process (postulated to involve PfENT1; Downie et al., 2006) before being accumulated to higher levels via one or more high-affinity mechanisms (postulated to involve metabolism and/or accumulation in an intracellular compartment; Downie et al., 2006). The data obtained here (Fig. 1) are consistent with the same being true for hypoxanthine. The data obtained at 10 lM represent the combined effects of equilibrative transport and a subsequent high-affinity accumulation, attributable to metabolism,
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Fig. 4. Concentration dependence of the influx of adenine into isolated Plasmodium falciparum trophozoites. Uptake was measured over a 4 s time period in parasites exposed to varying concentrations of adenine. The data are averaged from three independent experiments (shown ± SEM), each carried out in duplicate at 4 C. In (a) the data are fitted to a Michaelis–Menten curve (Km = 1.2 ± 0.4 mM; Vmax = 0.19 ± 0.04 nmol/lL cell water per second) whereas in (b), the same data are expressed as a Hanes plot. The Hanes plot may be resolved into two lines of differing slope, indicating the presence of two distinct components to the uptake of [3H]adenine by isolated parasites. To aid clarity, the data points indicating the higher-affinity component in the Hanes plot are shown as closed symbols whereas the data points indicating the lower-affinity component are shown as open symbols. Where not shown, error bars fall within the symbols.
Fig. 5. (a) Uptake of [3H]hypoxanthine and [3H]adenine by Xenopus oocytes injected with either the cRNA encoding PfENT1 (black bars) or with an equivalent volume of the elution buffer (white bars). The data shown are from a single experiment but are representative of those obtained in two separate experiments (adenine) or >10 experiments (hypoxanthine). Unlabelled nucleobase was present at 10 lM in each experiment. Hypoxanthine uptake was measured over 20 min, 5 days post-injection. Adenine uptake was measured over 10 min, 4 days post-injection. Data shown are the mean uptake of 8–10 oocytes. Error bars represent SD. (b) Concentration dependence of the adenine uptake by PfENT1 expressed in Xenopus oocytes. Transport via PfENT1 was calculated by subtracting the mean uptake in oocytes injected with elution buffer alone from the mean uptake of oocytes injected with the cRNA encoding PfENT1. The data are shown fitted to a Michaelis–Menten curve (Km = 0.82 ± 0.17 mM; Vmax of 200 ± 32 pmol/oocyte per 10 min). Experiments were performed 4 days post-injection. The data are averaged from two independent experiments (shown ± range/2). Where not shown, error bars fall within the symbol.
binding or accumulative transport. At 200 lM the low-affinity equilibrative transport process (by which the distribution ratio reached a value close to 1 within a few seconds) continued to operate whereas the high-affinity accumulation processes were largely saturated. Adenine, another purine nucleobase, is more soluble than hypoxanthine and undergoes limited metabolism within the parasite (Queen et al., 1988, 1989). The uptake of adenine by isolated parasites was comprised of at least
two components. There was an ATP-independent, lowaffinity component responsible for the distribution ratio of 1 seen in ATP depleted cells (Fig. 3, closed triangles) or, within 30 s, in ATP replete cells when extracellular adenine was present at 200 lM (Fig. 2, open circles). There was also a second ATP-dependent high-affinity component, responsible for the radiolabel reaching distribution ratios above 1, as seen in ATP replete cells when adenine was present at 10 lM (Fig. 2, closed circles).
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The Michaelis–Menten curve fitted to the concentrationdependence data obtained in parasites over an adenine concentration range of 0.01–4 mM (Fig. 4a) is dominated by the low-affinity component (estimated from the fitted curve to have a Km of 1.2 ± 0.4 mM). However, the different kinetics of the two components are revealed in the Hanes plot of Fig. 4b which shows that as well as the low-affinity component (estimated from the slope to have a Km of 1.5 mM, similar to the value of 1.2 mM estimated from the Michaelis–Menten curve) there is a higher-affinity component with a Km of below 0.2 mM. As with hypoxanthine and the various nucleosides studied previously (Downie et al., 2006), the rapid, low-affinity, ATP-independent component of adenine uptake is likely to be mediated by equilibrative transport of this compound across the parasite plasma membrane. It has been noted previously (Downie et al., 2006) that the characteristics of nucleoside transport in isolated parasites are similar to those of the parasite plasma membrane transporter PfENT1 expressed in Xenopus oocytes. However, there has been some inconsistency regarding the question of whether PfENT1 transports nucleobases (Carter et al., 2000; Parker et al., 2000). In our hands PfENT1 did transport both hypoxanthine and adenine (Fig. 5a), confirming the findings of Parker et al. (2000). The Km of 0.82 ± 0.17 mM obtained here for PfENT1-mediated adenine transport into Xenopus oocytes is similar to the value of 1.2 ± 0.4 mM obtained here for the transport of adenine in isolated parasites. It is somewhat higher than the value of 0.32 ± 0.10 mM estimated by Parker et al. (2000) for PfENT1-mediated adenine transport in Xenopus oocytes, measured over a more limited concentration range than that used here. Nevertheless, both are indicative of a lowaffinity transport process. The ATP-dependent high-affinity component of the association of adenine with the parasite, responsible for the distribution ratios above 1 may be attributed to the rapid metabolic trapping of the compound, and/or to an energy-dependent transport or binding of the radiolabelled substrate within a subcellular compartment. The finding that 500 nM Immucillin-H had little effect on adenine accumulation, indicates that adenine accumulation is unlikely to be due to the conversion of adenine to adenosine by PfPNP, consistent with earlier findings (Kicska et al., 2002a; Ting et al., 2005). The levels of adenine accumulation were significantly lower than those seen for the purine-nucleosides inosine and adenosine and the purine nucleobase hypoxanthine, consistent with earlier reports that hypoxanthine, adenosine and inosine are metabolised much more rapidly than adenine (Queen et al., 1988; Queen et al., 1989). On entering the parasite adenosine and inosine are first converted to hypoxanthine, which is rapidly phosphoribosylated by the enzyme hypoxanthine–guanine–xanthine phosphoribosyltransferase (HGXPRTase). Phosphoribosyltransferases catalyse the transfer of a phosphoribosyl group from phosphoribosylpyrophosphate (the production of which is dependent upon ATP; Roth et al.,
1986) to a purine base. Parasite adenine phosphoribosyltransferase (APRTase) activity is 1,500 times lower than HGXPRTase activity, and it has been reported that the two activities are catalysed by two separate enzymes with different molecular weights (Queen et al., 1988, 1989). More recently it has been suggested that the Plasmodium genome does not encode a specific APRTase enzyme (Chaudhary et al., 2004), and that, rather than being phosphoribosylated directly, adenine may be deaminated to hypoxanthine by an adenine deaminase activity (Hyde, 2007). Thus, while the data in Figs. 2 and 3 are consistent with the presence of an APRTase activity, it is not clear whether this activity might be due to a separate adeninespecific phosphoribosyltransferase enzyme (as suggested by Queen et al., 1989), to a residual adenine PRTase activity associated with the parasite’s HGXPRTase, or to deamination of adenine to hypoxanthine (followed by its subsequent phosphoribosylation). In conclusion, in this study we have shown that hypoxanthine, the primary purine source for the intraerythrocytic malaria parasite, enters the parasite via a rapid, equilibrative, low-affinity mechanism. The same is true of adenine. Both compounds undergo accumulation within the parasite, hypoxanthine more so than adenine, via a mechanism that is likely to involve the metabolism and/ or accumulative transport of the radiolabelled compounds within the parasite. The low-affinity of the equilibrative transport process is consistent with the involvement of PfENT1, shown here (as in a previous study; Parker et al., 2000) to transport both hypoxanthine and adenine, and to have a Km for adenine similar to that estimated for adenine transport in the parasite. The hypothesis that PfENT1 serves as a major route for the uptake of purine nucleobases is consistent with the recent finding that transgenic parasite lacking a functional PfENT1 show reduced uptake of [3H]hypoxanthine (El Bissati et al., 2006). Acknowledgements We are grateful to the Canberra Branch of the Australian Red Cross Blood Service for the provision of blood, to Dr. Vern Schramm for the kind gift of the ImmucillinH, and to the Australian Research Council and Australian National Health and Medical Research Council for grant support. References Allen, R.J., Kirk, K., 2004. The membrane potential of the intraerythrocytic malaria parasite Plasmodium falciparum. J. Biol. Chem. 279, 11264–11272. Alleva, L.M., Kirk, K., 2001. Calcium regulation in the intraerythrocytic malaria parasite Plasmodium falciparum. Mol. Biochem. Parasitol. 117, 121–128. Ansorge, I., Benting, J., Bhakdi, S., Lingelbach, K., 1996. Protein sorting in Plasmodium falciparum-infected red blood cells permeabilized with the pore-forming protein streptolysin O. Biochem. J. 315, 307–314.
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