H+-coupled Pantothenate Transport in the Intracellular Malaria Parasite*

Kevin J. Saliba and Kiaran KirkDagger

From the School of Biochemistry and Molecular Biology, Australian National University, Canberra, Australian Capital Territory 0200, Australia

Received for publication, December 5, 2000, and in revised form, February 1, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Pantothenate, the precursor of coenzyme A, is an essential nutrient for the intraerythrocytic stage of the malaria parasite Plasmodium falciparum. Pantothenate enters the malaria-infected erythrocyte via new permeation pathways induced by the parasite in the host cell membrane (Saliba, K. J., Horner, H. A., and Kirk, K. (1998) J. Biol. Chem. 273, 10190-10195). We show here that pantothenate is taken up by the intracellular parasite via a novel H+-coupled transporter, quite different from the Na+-coupled transporters that mediate pantothenate uptake into mammalian cells. The plasmodial H+:pantothenate transporter has a low affinity for pantothenate (Km ~23 mM) and a stoichiometry of 1 H+:1 pantothenate. It is inhibited by low concentrations of the bioflavonoid phloretin and the thiol-modifying agent p-chloromercuribenzene sulfonate. On entering the parasite, pantothenate is phosphorylated (and thereby trapped) by an unusually high affinity pantothenate kinase (Km ~300 nM). The combination of H+-coupled transporter and kinase provides the parasite with an efficient, high affinity pantothenate uptake system, which is distinct from that of the host and is therefore an attractive target for antimalarial chemotherapy.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

As it grows and matures within its host erythrocyte, the intracellular malaria parasite takes up nutrients from the external medium, doing so in competition with the cells of the host. The mechanisms by which it does so remain poorly understood. One important nutrient for which the human parasite P. falciparum has an absolute extracellular requirement is the vitamin pantothenic acid (PA1, vitamin B5) (1), a precursor of the enzyme cofactor coenzyme A (CoA) (2). PA is impermeant to normal, uninfected human erythrocytes but enters P. falciparum-infected erythrocytes via "new permeation pathways" induced by the intracellular parasite in the host cell membrane (3). Once inside the parasitized cell, PA is taken up by the parasite where it is phosphorylated by pantothenate kinase (3), the first step in its conversion to CoA.

The aim of this study was to investigate the mechanism by which PA is taken up across the plasma membrane of the intracellular malaria parasite. The results demonstrate the presence in the parasite membrane of a low affinity H+:pantothenate symporter, which acts in combination with a high affinity kinase within the parasite to form a highly efficient uptake system. The characteristics of the H+-dependent pantothenate transporter are quite different from those of its Na+-dependent counterparts in mammalian cells, raising the possibility that it may be a suitable antimalarial drug target.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Parasite Culture and Isolation

P. falciparum parasites (FAF6, cloned from ITG2, (4)) were maintained in synchronous in vitro cultures as described previously (5), with modifications (6). All experiments were carried out on late-stage trophozoites (36-40 h post-invasion) isolated from their host cells using saponin (0.05% w/v) as described previously (3, 7). Saponin permeabilizes both the host cell membrane and the parasitophorous vacuole membrane (8), thereby allowing solutes present in the extracellular medium unimpeded access to the parasite plasma membrane.

[14C]PA Uptake Measurements

Uptake into Isolated Parasites-- Measurements of the uptake of [14C]PA into parasites within saponin-permeabilized erythrocytes were carried out as described previously (3). In experiments designed to allow investigation of the uptake of [14C]PA under conditions in which the phosphorylation of the radiolabeled compound within the cell was minimized, isolated parasites were depleted of ATP by suspending them in a glucose-free, HEPES-buffered saline (135 mM NaCl, 5 mM KCl, 25 mM HEPES, 1 mM MgCl2, pH 7.4) for 30-60 min at 37 °C (7, 9). The ATP-depleted parasites were then harvested by centrifugation (2060 × g, 8 min) and resuspended in a weakly buffered solution (135 mM NaCl, 5 mM KCl, 0.1 mM HEPES, 1 mM MgCl2, pH 7.4) at 0.6-1.7 × 109 cells/ml, before combining 1 volume of the ATP-depleted parasite suspension with 12 volumes of a heavily buffered solution (135 mM NaCl, 5 mM KCl, 25 mM HEPES, 20 mM MES, 1 mM MgCl2), pH-adjusted to either 5.5 or 7.4 and containing [14C]PA (± inhibitors), to give a final PA concentration of 2 µM and a [14C]PA activity of 0.1 µCi/ml. At predetermined intervals 0.2-ml aliquots of the suspension were removed and centrifuged through an oil layer to terminate the flux as described elsewhere (3, 7).

Rat Hepatoma Cells-- In one series of experiments the ion dependence of [14C]PA uptake into isolated parasites was compared with that of [14C]PA uptake into mammalian cells (the rat hepatoma cell line, HTC). The HTC cells were grown as adherent cultures under standard conditions and were released into suspension (to allow the use of identical methodology to that used with the isolated parasites) by trypsinization. Uptake of [14C]PA into HTC cells in suspension was carried out using the same solutions and under identical conditions to those used for the isolated parasites.

Intracellular Water Volume Measurements

The water volume of the cell pellets (both for isolated parasites and HTC cells) was estimated using a combination of [3H]water and [14C]sucrose as described elsewhere (7). The water volumes of the isolated parasites and HTC cells used in this study were estimated as 20 ± 2 fl (mean ± S.E.; n = 13) and 67 ± 1 fl (mean ± range/2; n = 2), respectively.

Phosphorylation of [14C]PA by Parasite Lysates

Phosphorylation of [14C]PA by cell lysates prepared from isolated parasites was monitored as described previously (3). Cell lysates were either used immediately or frozen and stored at -70 °C for subsequent analysis. It was confirmed in control experiments that no loss of activity occurred during cryostorage.

Cytosolic pH Measurements and Estimation of the Cytosolic Buffering Capacity

The cytosolic pH (pHi) of the isolated parasites was monitored using the intracellular fluorescent pH indicator BCECF, as described previously (7). Measurements were carried out at 37 °C (except where specified otherwise) using a PerkinElmer Life Sciences LS-50B spectrofluorometer, with gentle stirring of the cell suspension.

The buffering capacity of the parasite cytosol (beta i) was measured both at 37 °C and 5 °C by the additions of varying concentrations (10-40 mM) of either NH<UP><SUB>4</SUB><SUP>+</SUP></UP> or butyrate to isolated parasites suspended in either the presence or absence of glucose. The removal of glucose results in a decrease in pHi of about 0.2 pH units (7) and served the purpose of increasing the pHi range over which beta i could be estimated. The discrepancy between the change in pHi predicted to occur upon addition of different concentrations of either NH<UP><SUB>4</SUB><SUP>+</SUP></UP> or butyrate, and that actually observed, allows the calculation of beta i (10).

For parasites at 37 °C, beta i decreased approximately linearly with pHi, from ~30 mmol of H+/(l cell H2O.pH unit) at pHi 7.5, to ~50 mmol of H+/(l cell H2O.pH unit) at pHi 7.1. At pHi values below 7.1, beta i increased more steeply with decreasing pHi, reaching a value of ~150 mmol of H+/(l cell H2O.pH unit) at a pHi of 6.9. Linear regression analysis of the pHi dependence of beta i in the pHi range 7.5-7.1 yielded an estimated beta i value of 42.2 mmol of H+/(l cell H2O.pH unit) at the normal resting pHi of 7.3. This value was used in the evaluation of the H+ transport rates in all experiments carried out at 37 °C.

For parasites at 5 °C, beta i remained at ~30 mmol of H+/(l·pH unit) over the pHi range 7.5-7.1, and increased only marginally to ~50 mmol of H+/(l cell H2O.pH unit) when the pHi was decreased further to 7.0. The beta i value at the resting pHi at 5 °C (7.22 ± 0.02; mean ± S.E.; n = 4) was estimated to be 32.3 mmol of H+/(l cell H2O.pH unit).

Because the beta i values at the resting pHi values at 37 °C and 5 °C were similar, an average beta i value of 37.3 mmol of H+/(l cell H2O.pH unit) was used for the estimation of H+ influx rates in the analysis of the experiment (giving rise to Fig. 6) in which H+ influx was measured as a function of temperature in the range 16-37 °C.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

PA Uptake by P. falciparum Trophozoites Is Na+-independent and pH-dependent-- Uptake of [14C]PA into isolated P. falciparum trophozoites was independent of the presence of Na+ in the extracellular solution. The initial rate of accumulation of [14C]PA in parasites suspended in a HEPES-buffered saline solution, estimated from the initial slope of the uptake time course (Fig. 1A), was 2.8 ± 0.4 µmol/(1012 parasites.h) (mean ± S.E.; n = 4). This was not significantly different from that in parasites suspended in Na+-free solution, containing choline in place of Na+ (2.7 ± 0.4 µmol/(1012 parasites.h); n = 4; p = 0.2, paired t test).


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Fig. 1.   Effect of extracellular Na+ and pH on the accumulation of [14C]PA by isolated P. falciparum trophozoites. A, time courses for the accumulation of [14C]PA (2 µM) by isolated parasites suspended in either HEPES-buffered saline (containing 125 mM NaCl; closed circles) or a Na+-free solution containing choline in place of Na+ (open circles). The data are averaged from four separate experiments, each carried out in duplicate, and are shown ± S.E. B, time courses for the accumulation of [14C]PA by rat hepatoma (HTC) cells suspended in the Na+-containing (closed circles) or Na+-free (open circles) media used in A. The data are from a single experiment representative of those obtained from two separate experiments. C, time courses for the uptake of [14C]PA (2 µM) by isolated parasites suspended in HEPES-buffered RPMI 1640 at pH 6.5 (closed diamonds), 7.4 (open squares), or 8.5 (open triangles). Data are averaged from two separate experiments, each carried out in duplicate and are shown ± range/2.

This contrasts with the situation in mammalian cells. As illustrated in Fig. 1B, the uptake of [14C]PA into rat hepatoma (HTC) cells was wholly dependent upon the presence of Na+ in the extracellular medium, decreasing to a negligible level on replacement of Na+ with choline.

Although independent of the presence of extracellular Na+, the rate of [14C]PA uptake did show a marked dependence on the extracellular pH (Fig. 1C). For isolated parasites suspended in RPMI at pH 8.5 the initial rate of uptake was 0.5 ± 0.2 µmol/(1012 parasites.h). Decreasing the pH of the medium (pHo) from 8.5 to 7.4 caused the initial rate of uptake to increase significantly (p = 0.042) to 1.1 ± 0.2 µmol/(1012 parasites.h). Decreasing the pHo further to 6.5 caused the initial rate of uptake to increase even further (p = 0.009), to 1.7 ± 0.2 µmol/(1012 parasites.h).

Isolated trophozoites accumulated [14C]PA to levels many-fold higher than that in the extracellular medium (Fig. 1, A and C) and the data of Fig. 1C are consistent with a role for H+ in the PA uptake process. As has been shown previously (3), however, PA entering the parasite is phosphorylated, the first step in its conversion to coenzyme A. The accumulation of radiolabel under the conditions of Fig. 1 therefore arises from a combination of the transport of the compound into the parasites and its subsequent "metabolic trapping" within the parasites (7).

PA Transport into ATP-depleted P. falciparum Trophozoites Is H+-dependent and Inhibited by Phloretin and by Sulfhydryl Reagents-- To investigate the possible role of H+ ions in the transport of PA into the parasite, in the absence of interference from metabolism, uptake of [14C]PA was measured into parasites depleted of ATP (by preincubation in a glucose-free solution at pH 7.4; see "Experimental Procedures") and therefore unable to carry out the phosphorylation of PA. The ATP-depleted parasites lose the ability to regulate their cytosolic pH (pHi), which, over a period of several minutes, therefore approaches the pH of the preincubation medium (7). Following ATP depletion, the cells were resuspended (at t = 0) in media of either pH 5.5 or 7.4 (i.e. in either the presence or absence, respectively, of a substantial initial inward [H+] gradient).

In the absence of a significant inward [H+] gradient (Fig. 2A, open symbols) there was a gradual uptake of [14C]PA, with the intracellular radiolabel reaching a concentration of around 0.8 times that in the extracellular solution by 5 min. By contrast, in ATP-depleted parasites suspended in the presence of a substantial inward [H+] gradient (Fig. 2A, closed symbols), there was a rapid uptake of radiolabel into the parasites. The ratio of the intracellular concentration of radiolabel to the extracellular concentration of radiolabel (the so-called "distribution ratio") reached a maximum value of 6-7 at 1-2 min, before declining.


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Fig. 2.   H+-dependent uptake of [14C]PA into ATP-depleted P. falciparum trophozoites. A, time courses for the uptake of [14C]PA (2 µM) into isolated parasites depleted of ATP by preincubation in glucose-free media (pH 7.4) then resuspended (at t = 0) in a HEPES- and MES-buffered solution at a pHo of 7.4 (open circles), or 5.5 (closed circles). Data are averaged from three separate experiments, each carried out in duplicate and are shown ± S.E. Analysis of the cell extracts using Somogyi reagent (3) revealed that, throughout the time course at pHo of 5.5, >80% of the intracellular label remained non-phosphorylated. B, time courses for the dissipation of the inward H+ gradient (as indicated by the "BCECF fluorescence ratio" (i.e. 495 nm/440 nm)) in cells depleted of ATP by preincubation in glucose-free medium at pH 7.4 then resuspended in medium of pH 5.5 at t = 0, in the absence of PA and in the presence or absence of the pharmacological agents used in C. The ordinate shows the BCECF fluorescence ratio rather than pHi as the dissipation of the H+ gradient caused pHi to decrease to values outside the effective range of the dye. C, effect of pharmacological agents on the H+-induced [14C]PA (2 µM) influx into ATP-depleted, saponin-permeabilized P. falciparum-infected erythrocytes. Uptake was measured over 1 min in ATP-depleted cells suspended (at t = 0, at concentrations of ~20 × 106 cells/ml) in medium at a pH of either 5.5 or 7.4 (as indicated). The inhibitors were added to the parasite suspension in combination with the [14C]PA (i.e. there was no preincubation of the cells with inhibitors). The data are averaged from four separate experiments, each performed in duplicate, and are shown ± S.E. NEM, N-ethylmaleimide.

Cell extracts corresponding to each of the time points shown by the closed symbols in Fig. 2A were analyzed for the presence of phosphorylated [14C]PA using Somogyi reagent (3). Throughout the time course the majority (>80%) of the PA taken up by the parasites remained unphosphorylated. The accumulation of [14C]PA, within the ATP-depleted parasites, to concentrations much higher than that in the extracellular medium, as seen in parasites suspended in the presence (but not in the absence) of an inward [H+] gradient, may therefore be attributed primarily to active accumulation of (non-phosphorylated) [14C]PA.

The inability of the ATP-depleted parasites to regulate their pHi meant that those suspended in the acidic medium underwent a progressive acidification (Fig. 2B). This accounts for the observed decline in the PA distribution ratio following its reaching a maximum at 1-2 min (Fig. 2A, closed symbols).

Fig. 2C shows the effect of a number of reagents on the uptake of [14C]PA by ATP-depleted parasites suspended in the presence of an inward [H+] gradient, whereas Fig. 2B illustrates the effect of the different reagents tested on the time-dependent dissipation of the [H+] gradient. In this set of experiments, the distribution ratio in the absence of inhibitors reached a value of ~4.5 in the 1-min incubation period. The H+ ionophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) (10 µM) caused a rapid dissipation of the imposed [H+] gradient (Fig. 2C) and caused the distribution ratio attained in 1 min to decrease to just below 1, a similar value to that seen in cells suspended in the absence of a transmembrane [H+] gradient at pHo 7.4 (Fig. 2C). Phloretin (200 µM), and the two sulfhydryl reagents pCMBS (10 µM) and N-ethylmaleimide (2 mM), were all found to reduce the uptake of [14C]PA by >=  70% (p <=  0.008; Fig. 2B). Unlike CCCP, however, these reagents did not increase the rate of dissipation of the imposed [H+] gradient (Fig. 2B) and their inhibitory effect on [14C]PA uptake may therefore be attributed to an inhibition of the actual transport mechanism rather than a dissipation of the driving force for PA influx.

Pantothenate Influx into P. falciparum Trophozoites Is Accompanied by H+ Influx-- The data of Fig. 2, demonstrating H+-dependent PA transport, are consistent with the hypothesis that pantothenate is transported across the parasite plasma membrane via a H+-coupled process. This was explored further by investigating the effect of pantothenate on the pHi of the isolated parasites. As shown in Fig. 3A, the initial resting pHi of the parasites at 37 °C was in the range 7.30-7.35. Addition of pantothenate (2-40 mM) to the suspending medium resulted in a transient, concentration-dependent acidification of the parasite cytosol, consistent with H+ entering the parasite. The time courses for the initial acidification were fitted to a first order exponential equation (Fig. 3B) and the initial slope (i.e. the initial rate of acidification of the cytosol) was calculated for each of the fitted curves. This was multiplied by the intracellular buffering capacity (42.2 mmol of H+/(l cell H2O.pH unit)) to give an initial rate of H+ influx at each of the pantothenate concentrations tested. Fig. 3C shows the initial H+ influx rate as a function of the extracellular pantothenate concentration. The data, showing a non-linear dependence of H+ influx rate on pantothenate concentration, were fitted to the Michaelis-Menten equation, giving an estimated Km of 22.6 ± 2.3 mM and Vmax of 56.3 ± 2.2 mmol of H+/(l cell H2O.min) (mean ± S.E.; n = 4). The water volume of a single isolated P. falciparum trophozoite estimated in this study was 20 fl ("Experimental Procedures"); the estimated Vmax is therefore equivalent to a PA-induced H+ influx of 1.13 ± 0.04 mmol of H+/(1012 cells.min).


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Fig. 3.   Pantothenate-induced H+ influx in isolated P. falciparum trophozoites suspended in a solution of pH 7.1. A, effect of the addition (at the points indicated by the filled arrowheads) of increasing concentrations of pantothenate (pH 7.1) on the pHi of isolated parasites. B, time-dependent change in pHi in the initial period following the addition of increasing concentrations of pantothenate (pH 7.1). Delta pHi is the difference between the resting pHi prior to the addition of pantothenate (an average of the pHi values obtained over the 20 s immediately prior to the addition of pantothenate) and the pHi at a given time (t) following pantothenate addition. The data were fitted to the first order equation Delta pH = Delta pH<UP><SUB><IT>i</IT></SUB><SUP>max</SUP></UP> × (1 - e-kt), where Delta pH<UP><SUB><IT>i</IT></SUB><SUP>max</SUP></UP> is the maximum decrease in pH following the addition of pantothenate and k is the first order rate constant for the acidification. C, concentration dependence of the initial rate of pantothenate-induced H+ influx. The initial rate of pantothenate-induced H+ influx at each concentration was estimated in each case by the expression H+ influx = beta i × Delta pH<UP><SUB><IT>i</IT></SUB><SUP>max</SUP></UP> × k, where beta i is the intracellular buffering capacity (42.2 mmol of H+/(l cell H2O.pH unit)) and Delta pH<UP><SUB><IT>i</IT></SUB><SUP>max</SUP></UP> and k are from the fitted curves shown in B. The data were fitted to the Michaelis-Menten equation (H+ influx = Vmax × [pantothenate]o/(Km + [pantothenate]o)). Km was estimated as 22.6 ± 2.3 mM and Vmax as 56.3 ± 2.2 mmol of H+/(l cell H2O.min) (mean ± S.E.; n = 4). The data in A are from a single experiment representative of those obtained in four separate experiments. The data in B and C are averaged from four separate experiments; error bars in B have been omitted for clarity; error bars in C represent S.E.

Pantothenate-induced H+ Influx Is Inhibited by Phloretin and pCMBS-- Phloretin and pCMBS, shown in Fig. 2C to inhibit H+-dependent [14C]PA uptake, were both found to be effective inhibitors of the pantothenate-induced acidification. The addition of 200 µM phloretin to ATP-replete parasites caused a slight apparent alkalinization of the cytosol (by an undetermined mechanism) and an almost complete inhibition of the acidification caused by the addition of 40 mM PA (not shown). To eliminate the possibility that the inhibition of H+ influx was related to the apparent phloretin-induced alkalinization, the experiment was repeated in parasites depleted of ATP, which caused pHi to decrease from 7.3 to 7.1 (Fig. 4A and Ref. 7) and which abolished the apparent phloretin-induced alkalinization. As shown in Fig. 4A, 200 µM phloretin inhibited the pantothenate-induced acidification in ATP-depleted parasites almost completely. Fig. 4B shows the concentration dependence of the inhibition by phloretin of the pantothenate-induced acidification; the IC50 (i.e. the concentration of phloretin at which inhibition was half-maximal) was 16.4 ± 2.6 µM (mean ± S.E.; n = 3; Fig. 4B).


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Fig. 4.   Effect of the transport inhibitors phloretin and pCMBS on PA-induced H+ influx into isolated P. falciparum trophozoites suspended in a solution of pH 7.1. A, the effect of Me2SO (upper trace) and phloretin (200 µM added in Me2SO; lower trace) on the cytosolic acidification induced by the addition of 40 mM pantothenate (pH 7.1) to ATP-depleted isolated parasites. The point of addition of the Me2SO/inhibitor is indicated by the open arrowheads, and the point of addition of the pantothenate is indicated by the closed arrowheads. The data are from a single experiment, representative of those obtained in three separate experiments. B, dose-response curve for the effect of phloretin on the initial rate of pantothenate-induced H+ influx. Data averaged from three separate experiments (shown ± S.E.) were fitted by non-linear least squares regression to a sigmoidal curve. C, the effect of water (upper trace) and pCMBS (6.25 µM added in water; lower trace) on the cytosolic acidification induced by the addition of 40 mM pantothenate (pH 7.1). The point of addition of the water/inhibitor is indicated by the open arrowheads, and the point of addition of the pantothenate is indicated by the closed arrowheads. The data are from a single experiment, representative of those obtained in three separate experiments. D, dose-response curve for the effect of pCMBS on the initial rate of PA-induced H+ influx. Data are from a single experiment, representative of those obtained in three separate experiments.

The sulfhydryl reagent, pCMBS, was an even more potent inhibitor of the pantothenate-induced acidification, exerting an unusual "all-or-none" effect. In initial experiments it was found that pCMBS concentrations as low as 0.3 µM caused complete inhibition of the pantothenate-induced acidification (Fig. 4C). However, it was also found that inhibition of the pantothenate-induced acidification by pCMBS was dependent upon the concentration of cells present in the suspension; 0.391 µM pCMBS abolished pantothenate-induced acidification in parasites suspended at a concentration of 4.3 × 106 cells/ml while having no effect on pantothenate-induced acidification in parasites suspended at 8.6 × 106 cells/ml (not shown). Fig. 4D, showing the concentration dependence of the effect of pCMBS on pantothenate-induced acidification in a single representative experiment, illustrates the all-or-none effect. Under the conditions of the experiment, 0.781 µM pCMBS caused a complete inhibition of pantothenate-induced H+ influx whereas 0.391 µM pCMBS was without significant effect.

Pantothenate:H+ Transport Operates with a Stoichiometry of 1:1 and Shows a Strong Temperature Dependence-- The data in Figs. 2 and 3, together, constitute good evidence for PA entering the parasite via a H+:pantothenate symport process. The stoichiometry of the process was investigated by comparing the maximum decrease in pHi (Delta pH<UP><SUB><IT>i</IT></SUB><SUP>max</SUP></UP>) observed following the addition of increasing concentrations of pantothenate (2.5-40 mM) with that observed following the addition of the same concentrations of the more lipophilic monocarboxylate, butyrate. Butyrate crosses membranes rapidly in the undissociated form; i.e. with an effective stoichiometry of transport of 1 H+:1 butyrate (11). For the purpose of these experiments the isolated parasites were depleted of ATP to minimize H+ pump-mediated pH recovery within the time taken for the pantothenate- and butyrate-induced acidification.

The addition of butyrate to the isolated parasites caused an extremely rapid acidification (Fig. 5A); Delta pH<UP><SUB><IT>i</IT></SUB><SUP>max</SUP></UP> was attained within 4 s and was followed by a slow, progressive alkalinization, reflecting the leakage of H+ out of the cell, down the outward [H+] gradient. The pantothenate-induced acidification was substantially slower than the butyrate-induced acidification, taking ~150 s for Delta pH<UP><SUB><IT>i</IT></SUB><SUP>max</SUP></UP> to be reached in the ATP-depleted parasites (Fig. 5A). Fig. 5B compares the Delta pH<UP><SUB><IT>i</IT></SUB><SUP>max</SUP></UP> following the addition of increasing concentrations of pantothenate or butyrate. In the case of pantothenate, the influx was sufficiently slow for there to have been significant leakage of H+ out of the parasite, down the [H+] gradient, during the ~150 s required for Delta pH<UP><SUB><IT>i</IT></SUB><SUP>max</SUP></UP> to be attained. To make a quantitative comparison of the relative magnitude of the H+ uptake induced by the influx of butyrate and pantothenate, it was therefore necessary to correct the Delta pH<UP><SUB><IT>i</IT></SUB><SUP>max</SUP></UP> measured following addition of pantothenate, for the significant H+ leakage that occurred during the period of pantothenate influx (as described in the legend to Fig. 5). The corrected pantothenate-induced Delta pH<UP><SUB><IT>i</IT></SUB><SUP>max</SUP></UP> values (Fig. 5B, open circles) were very similar to the butyrate-induced Delta pH<UP><SUB><IT>i</IT></SUB><SUP>max</SUP></UP> values (Fig. 5B, closed circles), consistent with pantothenate and H+ entering the parasite with a stoichiometry of 1:1. The transport process is therefore electroneutral.


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Fig. 5.   Comparison of the effect of butyrate and pantothenate on pHi of ATP-depleted, P. falciparum trophozoites suspended in a solution of pH 7.1. A, traces showing the time-dependent decrease in pHi following the addition of butyrate or pantothenate at the point indicated by the closed arrowheads, each at a concentration of 40 mM. B, maximum decrease in pHi (Delta pH<UP><SUB><IT>i</IT></SUB><SUP>max</SUP></UP>) following the addition of increasing concentrations of butyrate (closed circles) or pantothenate (closed triangles). The open circles show the Delta pH<UP><SUB><IT>i</IT></SUB><SUP>max</SUP></UP> for pantothenate, corrected for the alkalinization that occurred as a result of H+ leakage out of the parasites during the extended time period (~150 s) taken for the pantothenate-induced Delta pHi to reach its maximum value. In making the correction, the extent of alkalinization to have occurred at each time point was estimated from the (pH-dependent) alkalinization rates observed following the fast acidification induced by the addition of different concentrations of butyrate. This was then used to estimate the Delta pH<UP><SUB><IT>i</IT></SUB><SUP>max</SUP></UP> that would have been observed following the addition of pantothenate, had H+ leakage not occurred. It is important to recognize that Delta pH<UP><SUB><IT>i</IT></SUB><SUP>max</SUP></UP> is not a transport rate. The non-linearity of the graph of Delta pH<UP><SUB><IT>i</IT></SUB><SUP>max</SUP></UP> versus [monocarboxylate] is not due to a saturation of the transport process but is due, instead, to the fact that the buffering power of the parasite cytosol (beta i) increases in a non-linear manner with decreasing pH (see "Experimental Procedures"). The data are averaged from three separate experiments and are shown as mean ± S.E.

The pantothenate-induced H+ influx was highly sensitive to temperature. Upon reduction of the temperature from 37 °C to 5-10 °C, pantothenate-induced H+ influx was abolished (Fig. 6A). An Arrhenius plot was linear over the temperature range 16-37 °C (Fig. 6B) and yielded an estimated activation energy for pantothenate-induced H+ influx of 89 ± 6 kJ/mol (mean ± S.E.; n = 3).


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Fig. 6.   Temperature dependence of pantothenate-induced H+ influx into isolated P. falciparum trophozoites suspended in a solution of pH 7.1. A, traces, representative of those from four separate experiments, showing the abolition of the pantothenate-induced acidification on reduction of the temperature from 37 °C to 5 °C. B, Arrhenius plot for pantothenate-induced acidification in the temperature range 16-37 °C. The pantothenate concentration was 40 mM. The mean data (averaged from three separate experiments) are shown ± S.E. Linear regression of the data yielded an activation energy of 89 ± 6 kJ/mol.

Phosphorylation of PA within the Parasite Is by a High Affinity Kinase-- The kinetics of the phosphorylation of PA, within the parasite, was investigated in parasite lysates. The rate of phosphorylation was measured over the PA concentration range 0.1-0.75 µM, and the Michaelis-Menten equation was fitted to the data (Fig. 7), yielding a Km of 0.28 ± 0.03 µM and a Vmax of 10.8 ± 0.7 µmol/(1012 parasites.h).


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Fig. 7.   Concentration dependence of the rate of phosphorylation of [14C]PA by lysates of isolated P. falciparum trophozoites. The data (averaged from three separate experiments and shown as mean ± S.E.) were fitted to the Michaelis-Menten equation, yielding a Km of 0.28 ± 0.03 µM and a Vmax of 10.8 ± 0.7 µmol/(1012 cells.h).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Pantothenic acid, one of the relatively few nutrients for which the intracellular malaria parasite P. falciparum has an absolute extracellular requirement (1), enters infected erythrocytes primarily via the new permeation pathways induced by the parasite in the host cell membrane (7). From the host cell cytosol, PA is presumed to enter the parasitophorous vacuole in which the parasite is enclosed, via the large-diameter pores characterized previously in the vacuole membrane (12). From the parasitophorous vacuole, PA is taken up across the parasite plasma membrane. The results of this study shed light on the mechanism by which this is achieved.

In this study we have used parasites functionally isolated from their host cells by brief exposure of the parasitized cells to saponin. Saponin permeabilizes the erythrocyte membrane to both low and high molecular weight solutes (including host cell proteins), as well as making the parasitophorous vacuole membrane (already highly permeable to low molecular weight solutes (12)) permeable to proteins (8). By contrast the parasite plasma membrane remains intact, able to support transmembrane ion gradients (7). There is a possibility that the solution within the parasitophorous vacuole compartment acts as an unstirred layer, influencing the rate of passage of solutes into the isolated parasites as well as the concentration of solutes in the immediate vicinity of the parasite plasma membrane. However, the finding in this study that butyrate equilibrates between the intra- and extracellular solutions within 3-4 s, and the earlier observation that NH3 does so even faster (in < 2 s (7)) would indicate that our measurements of pantothenate transport (which is more than an order of magnitude slower than that of either butyrate or NH3) are not subject to significant unstirred layer effects (13).

PA is transported across the plasma membrane of mammalian cells via a well-characterized family of Na+:pantothenate symporters ((14-17) and see Fig. 1B). By contrast, the uptake of PA into the intracellular malaria parasite is independent of Na+, but is, instead, dependent on H+. The demonstration, in Fig. 2A, that an inward [H+] gradient can drive the active accumulation of [14C]PA (to a distribution ratio 1) and the demonstration in Fig. 3 that an inward [PA] gradient can drive the net influx of H+, together, constitute good evidence for the operation of a H+:pantothenate symport process, operating with a stoichiometry of 1:1 (Fig. 5). These data do not distinguish between simple diffusion of the undissociated acid, and a transporter-mediated process. However the findings that pantothenate-driven H+ influx is saturable (Km = 22.6 ± 2.3 mM; Fig. 3) and that both H+-driven PA influx (Fig. 2A) and pantothenate-driven H+ influx (Fig. 3A) are inhibited by phloretin and by sulfhydryl reagents are consistent with the involvement of a transporter. So too is the finding that the transport process has a relatively high activation energy (Ea = 89 ± 6 kJ/mol; Fig. 6), which is in the range of those reported previously for transporters in other systems and higher than that usually associated with the process of simple diffusion (18).

The H+-coupling of pantothenate transport may allow the parasite to take advantage of the H+ gradient normally present across the parasite plasma membrane in accumulating PA from the extracellular medium. The normal pHi of the parasite is 7.3-7.4 (Refs. 7, 19-21 and this study), compared with an estimated pH of ~6.9 in a region of the erythrocyte cytosol immediately adjacent to the parasite (21). There is therefore a significant inward [H+] gradient, favoring the net accumulation of PA, although the intracellular concentration of PA will be determined not only by the stoichiometry of the transporter but by the relative rates of transport and metabolism of the vitamin under in vivo conditions.

The H+:pantothenate symporter of the intracellular malaria parasite differs from the Na+:pantothenate symporters of the host not only in its cation requirements, but in its substrate affinity. Although the relatively high Km for the process could not be measured with a high degree of accuracy (without exposing the parasites to excessively high concentrations of the pantothenate salt) it is evident that the transport of PA into the parasite occurs via a low affinity process. The estimated Km of ~23 mM contrasts with those of PA transporters elsewhere. The H+-coupled pantothenate transporter of the yeast Saccharomyces cerevisiae has a Km of 3.5 µM (22), whereas the prokaryotes Lactobacillus plantarum and Escherichia coli have Na+-coupled pantothenate transporters with Km values of 0.8 µM (23) and 0.4 µM (24), respectively. The Na+-coupled multivitamin transporters of mammalian cells have a Km for pantothenate of 1.5-4.9 µM (14-16), which falls within the normal range for the concentration of PA in the blood plasma of healthy individuals (0.6-17 µM (25)).

In contrast to the comparatively low affinity of the parasite's PA transporter, the parasite's pantothenate kinase has a significantly higher affinity for pantothenate than its mammalian counterparts. The kinase phosphorylates and thereby traps the PA within the parasite cytosol, doing so with a Km (~0.28 µM) that is some 40-fold lower than that of the highest affinity mammalian pantothenate kinase described to date (from rat liver; Km ~12 µM (26)), as well as being significantly lower than that of the high affinity transporters of the cells of the host. Thus, although the transporter in the parasite plasma membrane is a low affinity system, the combination of the transporter and the kinase serves as a high affinity uptake system that should enable the parasite to compete very effectively with the cells of the host for this key nutrient.

The H+:pantothenate symporter described here is, to our knowledge, the first example described of a H+-coupled nutrient transporter in P. falciparum. The only previous description of a H+:pantothenate symporter in a eukaryote cell is in the yeast S. cerevisiae (22). This is consistent with the view that the physiology of the malaria parasite resembles that of lower eukaryotes, such as plants and yeast (7), more closely than it does that of mammalian cells. The PA transporters of mammalian cells are Na+-dependent systems with characteristics quite different from those of the H+:pantothenate symporter described here. The finding here that the pantothenate kinase of the parasite has a substrate affinity substantially higher than that of any of the mammalian pantothenate kinases described to date may be indicative of there also being significant differences between the mammalian and parasite forms of this enzyme. Such differences, together with the previously demonstrated importance of PA for parasite growth (1), focus attention on the possibility that the mechanisms involved in the uptake of PA by the intracellular malaria parasite are suitable targets for new and much-needed antimalarial drugs.

    ACKNOWLEDGEMENTS

We are grateful to Pauline Junankar for the provision of HTC cells, to Jemma Elliott for the estimation of the buffering capacity of the isolated parasites, and to the staff of the Canberra branch of the Australian Red Cross Blood Service for the provision of blood for use in parasite culture.

    FOOTNOTES

* This work was supported by grants from the Australian National Health and Medical Research Council (971008 and 122814), the Australian Research Council (F97082), and the Ramaciotti Foundations.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Tel.: 61-2-6125-2284; Fax: 61-2-6125-0313; E-mail: kiaran.kirk@anu.edu.au.

Published, JBC Papers in Press, February 22, 2001, DOI 10.1074/jbc.M010942200

    ABBREVIATIONS

The abbreviations used are: PA pantothenic acid, beta i, intracellular buffering capacity; BCECF, 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein; pCMBS, p-chloromercuribenzene sulfonate; MES, 2-(N-morpholino)ethanesulfonic acid; pHi cytosolic pH, Delta pHi change in cytosolic pH; Delta pH<UP><SUB><IT>i</IT></SUB><SUP>max</SUP></UP>, maximum change in cytosolic pH; pHo, extracellular pH; CCCP, carbonyl cyanide m-chlorophenylhydrazone.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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