Acidification of the Malaria Parasite's Digestive Vacuole by a H+-ATPase and a H+-pyrophosphatase*

Kevin J. SalibaDagger §, Richard J. W. AllenDagger §||, Stephanie ZissisDagger , Patrick G. Bray**DaggerDagger, Stephen A. Ward**DaggerDagger, and Kiaran KirkDagger

From the Dagger  School of Biochemistry and Molecular Biology, Faculty of Science, Australian National University, Canberra, Australian Capital Territory 0200, Australia and the ** Molecular and Biochemical Parasitology Group, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L5 3QA, United Kingdom

Received for publication, August 22, 2002, and in revised form, November 8, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

As it grows within the human erythrocyte, the malaria parasite, Plasmodium falciparum, ingests the erythrocyte cytosol, depositing it via an endocytotic feeding mechanism in the "digestive vacuole," a specialized acidic organelle. The digestive vacuole is the site of hemoglobin degradation, the storage site for hemozoin (an inert biocrystal of toxic heme), the site of action of many antimalarial drugs, and the site of proteins known to be involved in antimalarial drug resistance. The acidic pH of this organelle is thought to play a critical role in its various functions; however, the mechanisms by which the pH within the vacuole is maintained are not well understood. In this study, we have used a combination of techniques to demonstrate the presence on the P. falciparum digestive vacuole membrane of two discrete H+ pumping mechanisms, both capable of acidifying the vacuole interior. One is a V-type H+-ATPase, sensitive to concanamycin A and bafilomycin A1. The other is a H+-pyrophosphatase, which was inhibited by NaF and showed a partial dependence on K+. The operation of the H+-pyrophosphatase was dependent on the presence of a Mg2+-pyrophosphate complex, and kinetic experiments gave results consistent with free pyrophosphate acting as an inhibitor of the protein. The presence of the combination of a H+-ATPase and a H+-pyrophosphatase on the P. falciparum digestive vacuole is similar to the situation in the acidic tonoplasts (vacuoles) of plant cells.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The malaria parasite is a unicellular eukaryote (genus Plasmodium) that during the course of its life cycle invades the erythrocytes of its vertebrate host. As it grows within its host erythrocyte, the parasite ingests the host cell cytosol via an endocytotic feeding mechanism. The host cell proteins (predominantly hemoglobin) are deposited in an internal acidic compartment, the digestive vacuole, where they are broken down by acid hydrolases to their component amino acids (1), some of which are used for protein synthesis (2). As well as being the major site for hemoglobin digestion, the digestive vacuole serves as a site for the storage of hemozoin (inert crystals of the potentially toxic heme that is liberated in the course of hemoglobin degradation). It is believed to be the primary site of action of many of the antimalarial drugs, and the two proteins known to play a role in antimalarial drug resistance in the most virulent of the human malaria parasites, Plasmodium falciparum, Pgh1 (3) and PfCRT (4), have been localized to the digestive vacuole membrane.

The acidic pH of the digestive vacuole is thought to play a key role in the various physiological functions of this organelle and may also play a role in the phenomenon of antimalarial drug resistance (5, 6). Despite the likely significance of vacuolar pH, the mechanism(s) underlying the acidification of the vacuole interior are not well understood. There is immunological evidence for the presence on the digestive vacuole membrane of a V-type H+-ATPase1 (7-9). Krogstad et al. (10) did show a Mg2+ATP-stimulated acidification of the digestive vacuole, but this was not characterized in any detail. In a later study (11), it was shown that the ATPase activity of isolated digestive vacuoles can be inhibited by the nonspecific H+ pump inhibitors N-ethylmaleimide and 7-chloro-4-nitrobenz-2-oxa-1,3-diazole, but the nature of the pump(s) involved was not investigated.

The aim of this study was to identify and characterize the mechanism(s) involved in the acidification of the digestive vacuole of P. falciparum. Using a combination of techniques, we have obtained direct physiological evidence for the presence on the digestive vacuole membrane of a V-type H+-ATPase, as well as a second H+ pump, a H+-pyrophosphatase, which couples the hydrolysis of the phosphoanhydride bond of pyrophosphate (PPi) to the influx of H+ into the vacuole.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Parasite Culture-- P. falciparum parasites (strain D10) were maintained in synchronous in vitro cultures as described previously (12) with modifications (13). All experiments were carried out on late stage trophozoites, 36-40 h postinvasion.

Loading Fluorescent pH Indicators into the Parasite's Digestive Vacuole-- A membrane-impermeant, dextran-linked form of the fluorescent pH indicator fluorescein (pKa ~6.4) was loaded into the parasite's digestive vacuole as described previously (10, 14), with only minor modifications. Packed erythrocytes (1 ml) were lysed by the addition of 2.25 ml of lysis buffer (5 mM HEPES, 11 mM glucose, 2 mM MgCl2, and 2 mM ATP, pH 7.4) containing 110 µM fluorescein-dextran (Mr 10,000; Molecular Probes, Inc., Eugene, OR). The lysate was incubated for 10 min at 30 °C, and then the cells were resealed by the addition of 2.25 ml of a resealing buffer (280 mM NaCl, 40 mM KCl, 11 mM glucose, 1 mM HEPES, pH 7.4). The dye-loaded erythrocytes were washed three times in RPMI (1,200 × g, 5 min) and then resuspended in culture medium and inoculated with ~1 ml of a trophozoite-stage P. falciparum-infected erythrocyte suspension (3-4% hematocrit, 10-20% parasitemia). The parasites were maintained in 50 ml of medium on an orbital shaker until the parasitemia was higher than 10-15% (rarely more than two complete cycles), at which point the cells were used for experiments.

Selective Permeabilization of the Erythrocyte and Parasite Plasma Membranes-- Fig. 1 illustrates the different parasite/vacuole preparations used throughout this study.

Parasitized erythrocytes (Fig. 1A) were permeabilized (as described previously (15)) by brief exposure to saponin (0.005%, w/v sapogenin), a plant-derived detergent that permeabilizes both the host cell membrane and the parasitophorous vacuole membrane (16), thereby allowing solutes present in the extracellular solution unimpeded access to the parasite plasma membrane (Fig. 1B). Parasites within saponin-permeabilized erythrocytes are referred to throughout this paper as "isolated parasites." For all experiments other than those giving rise to Figs. 2, 8, and 10C, the plasma membrane of the parasite (within the saponin-permeabilized erythrocyte membrane) was itself permeabilized using digitonin (10), thereby permitting solutes added to the extracellular medium immediate access to the digestive vacuole membrane (Fig. 1C). Isolated parasites were suspended in 2 ml of ice-cold intracellular saline A (110 mM KCl, 30 mM NaCl, 2 mM MgCl2, 5 mM HEPES, pH 7.3). The cell suspension (typically 5 × 107 cells/ml) was kept on ice for 5-10 min before adding digitonin (0.01%, w/v) from a 200 mg/ml stock (in Me2SO). The cells were mixed gently and returned to ice for a further 2 min, after which 1 ml of ice-cold intracellular saline A containing 1 mg/ml bovine serum albumin was added. The permeabilized parasites were centrifuged immediately (15,800 × g) for 1 min and then washed twice (1 min at 15,800 × g) with 1 ml of the same bovine serum albumin-containing solution and once in intracellular saline A without bovine serum albumin. The digitonin-permeabilized parasites were suspended in intracellular saline A at an average cell density of ~8 × 106/ml and kept at 37 °C until being used in experiments (usually within 2 h, but up to 5 h in the experiment giving rise to Fig. 4). Within digitonin-permeabilized parasites, the digestive vacuole remained intact, as indicated by the retention of the dextran-linked fluorescent dye within the digestive vacuole of parasites isolated from dye-loaded erythrocytes (see above).

Isolation of Digestive Vacuoles-- Digestive vacuoles (Fig. 1D) were isolated as described previously (17), with some modifications. Approximately 2 × 109 isolated parasites (see above) were lysed by suspension in ~1 ml of an acidic, ice-cold, hypotonic lysis buffer (20 mM MES, pH 4.5). The lysate was transferred to a watchglass on ice, triturated forcefully twice through a 27-gauge needle, and then centrifuged (15,800 × g, 0.5 min). The supernatant solution was removed, and the pellet was resuspended in ~1 ml of intracellular saline B (120 mM KCl, 10 mM NaCl, 25 mM HEPES, 2 mM MgCl2, 5 mM Na2HPO4, pH 7.3), and triturated (as described above) six more times. The suspension was centrifuged (15,800 × g, 0.5 min), and the supernatant solution was removed. The pellet was again resuspended in ~1 ml of intracellular saline B, to which was added DNase I (300-400 units/ml), and the suspension was incubated at 37 °C for 5 min. The sample was centrifuged once more (15,800 × g, 0.5 min) and then resuspended in 100 µl of intracellular saline B and mixed with 1.3 ml of a Percoll solution (80% v/v Percoll, 1.5 mM MgCl2, 200 mM sucrose, pH 7.3). The suspension was again triturated (2 ×) onto the watchglass, returned to microcentrifuge tubes, and centrifuged at 5,200 × g for 8 min at 4 °C. Isolated vacuoles were retrieved from the bottom ~50 µl of the Percoll solution, avoiding the hemozoin crystal pellet at the very bottom of the tube, and resuspended in ice-cold intracellular saline B until being used in experiments within 20 min of their isolation. Microscopic examination of the preparation revealed a homogeneous population of approximately spherical, hemozoin-filled organelles that appeared to be free of contaminants.

Fluorescence Measurements-- Throughout this study, the pH within the parasite's digestive vacuole (pHDV) was monitored using the fluorescence arising from either the membrane-impermeant pH indicator fluorescein-dextran, loaded into the digestive vacuole as described above, or (in one series of experiments with isolated digestive vacuoles) from the membrane-permeant compound quinacrine. In preliminary experiments with a range of dextran-linked pH indicators, we attempted to calibrate the measured fluorescence with pHDV but found that the estimated pHDV varied with the fluorescent indicator used. The origin of this variation is presently under investigation. For the purpose of the present study, the fluorescence was simply used as a semiquantitative indicator of pHDV, and there was no attempt made to calibrate the fluorescence measurements. For both fluorescein-dextran and quinacrine, an upward deflection of the trace is indicative of an alkalinization, and a downward deflection of the trace is indicative of an acidification.

Experiments with Cell Populations-- In experiments with intact isolated parasites (Fig. 1B) in which the digestive vacuole was preloaded with fluorescein-dextran, the cells were suspended at a density of ~5 × 106 cells/ml in a solution comprised of 125 mM NaCl, 5 mM KCl, 1 mM MgCl2, 20 mM glucose, 25 mM HEPES, pH 7.1. With the exceptions of the experiments giving rise to Figs. 5B and 6B, digitonin-permeabilized parasites (Fig. 1C) were suspended (at ~5 × 106 cells/ml) in intracellular saline A. For the experiment giving rise to Fig. 5B, permeabilized parasites were suspended in intracellular saline A without MgCl2. For the experiment giving rise to Fig. 6B, they were suspended in a K+-free solution containing 140 mM NaCl, 2 mM MgCl2, 5 mM HEPES, pH 7.3.

Changes in pHDV were monitored at 37 °C on a Perkin-Elmer Life Sciences LS-50B spectrofluorometer using a dual excitation Fast Filter accessory. The sample was excited at 490 and 450 nm successively, and the fluorescence was measured at 520 nm. The ratio of the fluorescence intensity measured using the two excitation wavelengths (490 nm/450 nm) provides a measure of pHDV.

Experiments with Single Cells-- Digitonin-permeabilized parasites (Fig. 1C) preloaded with fluorescein-dextran were immobilized on poly-L-lysine-coated coverslips and then mounted in a Bioptechs FCS2 perfusion chamber and immersed in intracellular saline A at 37 °C. The cells were imaged using a Zeiss LSM510 confocal microscope through a Plan-Apochromat 63 × 1.4 NA oil objective. The fluorescein-dextran was excited using the 488-nm wavelength line of an argon ion laser through a 488-nm HFT filter. Emitted fluorescence was captured off a 545-nm dichroic mirror through a 505-550-nm bandpass filter.

Experiments with Suspensions of Isolated Vacuoles-- pHDV in isolated P. falciparum digestive vacuole suspensions was monitored using a method based on one used previously by Hirata et al. (18) to measure the pH in vacuoles isolated from yeast. Vacuoles were isolated from unloaded parasites and suspended (at 4-8 × 107 vacuoles/ml) in intracellular saline B to which had been added 25 nM quinacrine. Fluorescence intensity was measured using excitation and emission wavelengths of 425 and 495 nm, respectively. The experiments with this preparation were carried out at 20 °C, as in Ref. 18.

31P NMR Measurements-- Hydrolysis of PPi by suspensions of digitonin-permeabilized parasites (Fig. 1C) and isolated P. falciparum digestive vacuoles (Fig. 1D) was monitored using 31P NMR spectroscopy.


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Fig. 1.   Schematic representation of the different parasite preparations used in this study. Mature P. falciparum trophozoites (A) were "isolated" from their host (human) erythrocytes by permeabilization of the erythrocyte and parasitophorous vacuole membranes by brief exposure to saponin (B). The plasma membrane of isolated parasites was permeabilized (releasing the contents of the parasite cytosol and giving solutes in the external solution direct access to the digestive vacuole) by brief exposure of isolated parasites to digitonin (C). Digestive vacuoles were isolated (D) by hypotonic lysis of isolated trophozoite-stage parasites and purified using Percoll density gradient centrifugation (see "Experimental Procedures").

In experiments with digitonin-permeabilized parasites, the cells were suspended at a cell density of ~6 × 107 cells/ml in intracellular saline A containing 4 mM PPi and incubated in the presence or absence of 10 mM NaF at 37 °C. At predetermined time intervals, 250-µl aliquots were removed and centrifuged (0.5 min at 15,800 × g). A 200-µl aliquot of the supernatant solution was transferred to 100 µl of ice-cold 30% perchloric acid in order to halt any enzyme activity. The pH was subsequently adjusted to 7.4 using NaOH.

In experiments with isolated vacuoles, the organelles were suspended in intracellular saline A at a density of ~1 × 107 vacuoles/ml and incubated in the presence of 0.5 mM PPi for 2 h at 37 °C. Samples were then processed as described above.

The volume of each neutralized extract sample was made up to 400 µl, and the solution was then transferred to a 5-mm (outside diameter) NMR tube. Methylene diphosphonic acid was added to a final concentration of either 5 mM (in the permeabilized parasite experiments) or 0.5 mM (in the isolated vacuole experiments) as an internal 31P NMR reference standard. EDTA was added to a concentration of 24 mM to preclude signal broadening by trace concentrations of multivalent metal ions, and 2H2O (10% v/v) was added to provide a field frequency lock. 31P NMR spectra were acquired at 25 °C and at 121.4 MHz, using a Varian VXR-300 spectrometer with a 45° pulse and a 1-s relaxation delay between successive pulses.

Calculation of the Concentrations of Free PPi and Mg·PPi Complexes-- The concentrations of free PPi (the sum of PP<UP><SUB>i</SUB><SUP>4−</SUP></UP>, HPP<UP><SUB>i</SUB><SUP>3−</SUP></UP>, H2PP<UP><SUB>i</SUB><SUP>2−</SUP></UP>, H3PP<UP><SUB>i</SUB><SUP>−</SUP></UP>, and H4PPi) and PPi complexed with Mg2+ (the sum of MgPP<UP><SUB>i</SUB><SUP>2−</SUP></UP>, Mg2PPi, and MgHPP<UP><SUB>i</SUB><SUP>−</SUP></UP>) in the experiments giving rise to Figs. 5A and 10B were calculated using the program GEOCHEM-PC (version 2.0) (19) and the following stability constants (at zero ionic strength), expressed as log K (where K is in M): MgPP<UP><SUB>i</SUB><SUP>2−</SUP></UP>, 6.6; Mg2PPi, 9.5; MgHPP<UP><SUB>i</SUB><SUP>−</SUP></UP>, 13.1; HPP<UP><SUB>i</SUB><SUP>3−</SUP></UP>, 9.4; H2PP<UP><SUB>i</SUB><SUP>2−</SUP></UP>, 16.2; H3PP<UP><SUB>i</SUB><SUP>−−</SUP></UP>, 18.8; H4PPi, 20.8; KPP<UP><SUB>i</SUB><SUP>3−</SUP></UP>, 2.3; NaPP<UP><SUB>i</SUB><SUP>3−</SUP></UP>, 2.3; MgP<UP><SUB>i</SUB><SUP>−</SUP></UP>, 15.3; HP<UP><SUB>i</SUB><SUP>2−</SUP></UP>, 12.35; H2P<UP><SUB>i</SUB><SUP>−</SUP></UP>, 19.55; H3Pi, 21.7. The calculations simulated the presence of different concentrations of PPi and (in the case of Fig. 10B) Pi in the presence of 2 mM Mg2+, 110 mM K+, and 30 mM Na+, at pH 7.3.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Regulation of pHDV in Isolated Parasites-- When isolated parasites in which the digestive vacuole was preloaded with fluorescein-dextran were suspended in a glucose-containing medium, they gave rise to a steady fluorescent signal. The addition of the V-type H+-ATPase inhibitors concanamycin A (Fig. 2A) or bafilomycin A1 (not shown) resulted in a progressive increase in the (490 nm/450 nm) fluorescence ratio, indicative of the dye-loaded digestive vacuole undergoing an alkalinization. This is consistent with a V-type H+-ATPase playing a role in maintaining the acidic pH of this organelle.


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Fig. 2.   Alkalinization and reacidification of the digestive vacuole in intact isolated parasites. The digestive vacuole was loaded with fluorescein-dextran, and the host erythrocyte membrane was permeabilized with saponin as described under "Experimental Procedures." Fluorescence was measured at 520 nm and is expressed as a ratio of that measured on excitation at 490/450 nm. An increase in the ratio (i.e. an upward deflection) indicates an alkalinization. The addition of the H+-ATPase inhibitor concanamycin A (75 nM) to parasites suspended in a glucose-containing medium (A, black arrowhead) caused an alkalinization of the digestive vacuole, as did the removal of glucose from the medium (B-E, black arrowheads). The readdition of 20 mM glucose to glucose-depleted cells (B, white arrowhead) caused a reacidification of the digestive vacuole, as did the addition to the extracellular medium of 2 mM ATP (C, gray arrowhead). The ATP/ADP translocase inhibitor, atractyloside (100 µM, white arrowhead) prevented the ATP-induced reacidification (gray arrowhead) but did not prevent the glucose-induced reacidification (D, white arrowhead). The addition of 2 mM PPi to glucose-depleted cells (E, white arrowhead) slowed the rate of alkalinization slightly but failed to induce a reacidification. The traces are from a single experiment and are representative of those obtained in at least three separate experiments.

The intraerythrocytic parasite derives its ATP from glycolysis (20). As shown in Fig. 2B, removal of glucose from the extracellular medium (a maneuver shown previously to result in a rapid depletion of parasite ATP (21)) resulted in an alkalinization of the vacuole. Upon restoration of glucose (20 mM) to the medium, the vacuoles reacidified. A similar reacidification was seen upon the addition to glucose-depleted parasites of 2 mM ATP (Fig. 2C), which is known to cross the parasite plasma membrane via an ATP/ADP translocase (22, 23). Atractyloside (100 µM), an inhibitor of the ATP/ADP translocase, prevented the ATP-induced reacidification while having no effect on the reacidification caused by the subsequent addition of glucose (Fig. 2D). Unlike ATP, PPi (added to the extracellular solution at a concentration of 2 mM) failed to induce an acidification of the vacuole in glucose-depleted parasites, although it did cause a modest decrease in the rate of alkalinization (Fig. 2E).

ATP- and PPi-induced Acidification of the Parasite's Digestive Vacuole in Suspensions of Digitonin-permeabilized Parasites-- In isolated parasites (as used in the experiments giving rise to Fig. 2), the parasite plasma membrane remains intact, and compounds added to the extracellular medium have to traverse this membrane in order to gain access to the digestive vacuole (Fig. 1B). The parasite plasma membrane can be permeabilized, thereby exposing the surface of the digestive vacuole directly to the extracellular solution (as well as releasing the contents of the cytosol, including those compounds required to energize the acidification of the vacuole), by treating the isolated parasites with digitonin (Fig. 1C).

Fig. 3 illustrates the effects of PPi and ATP on pHDV in digitonin-permeabilized parasites. As shown in Fig. 3A, the addition of 0.2 mM PPi to digitonin-permeabilized parasites suspended in a high K+ solution resulted in a rapid decrease in the fluorescence ratio, indicative of the vacuole undergoing an acidification. The subsequent addition of the H+-ATPase inhibitor concanamycin A (75 nM) had no significant effect. The data are consistent with the presence in the vacuole membrane of a H+ pumping PPase, distinct from the V-type H+-ATPase.


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Fig. 3.   Effect of ATP and PPi on the pH of the digestive vacuole in isolated P. falciparum trophozoites in which the plasma membrane was permeabilized by digitonin. The digestive vacuole was loaded with fluorescein-dextran, and the erythrocyte membrane, parasitophorous vacuole membrane, and parasite plasma membrane were permeabilized as described under "Experimental Procedures." The addition of either PPi (A, 0.2 mM, white arrowhead) or ATP (B, 1 mM, gray arrowhead) caused an acidification. Concanamycin A (75 nM), added at the points indicated by the black arrowheads, had no effect on the pH of PPi-acidified vacuoles (A) but reversed the ATP-induced acidification (B). The addition of 0.2 mM PPi to ATP- and concanamycin A-treated cells (B, white arrowhead) resulted in the complete reacidification of the digestive vacuoles (B). The traces are from a single experiment and are representative of those obtained in at least three separate experiments.

The addition of 1 mM ATP to digitonin-permeabilized parasites also caused a marked acidification of the digestive vacuole (Fig. 3B). The acidification was reversed upon the addition of concanamycin A, consistent with the ATP-induced acidification being mediated by a V-type H+-ATPase. The subsequent addition of 0.2 mM PPi resulted in the complete reacidification of the vacuole (Fig. 3B).

The rate (and magnitude) of the vacuolar acidification induced by 2 mM ATP decreased with the time after permeabilization of the parasite plasma membrane by digitonin (Fig. 4, closed symbols). By ~1 h after the addition of digitonin, the ATP-induced acidification was reduced to an almost negligible level. By contrast, the rate of acidification measured in response to the addition of 0.2 mM PPi was maintained at an approximately constant level for >4 h following exposure of the parasites to digitonin (Fig. 4, open symbols).


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Fig. 4.   Initial rate of acidification of the digestive vacuole of digitonin-permeabilized parasites following the addition of 2 mM ATP (closed symbols) or 0.2 mM PPi (open symbols), plotted as a function of the time postpermeabilization. Initial rates of acidification were estimated by fitting traces such as those illustrated in Fig. 3 to the first order equation Delta  fluorescence ratio = Delta  fluorescence ratiomax × (1 - e-kt), where: Delta  fluorescence ratio is the difference between the resting fluorescence ratio prior to the addition of ATP or PPi (averaged over the 20 s immediately prior to the addition) and the fluorescence ratio at a given time (t) following the addition of ATP or PPi; Delta  fluorescence ratiomax is the maximum decrease in fluorescence ratio attained following the addition of ATP or PPi; and k is the first order rate constant for the acidification. The time dependence of the ATP-induced acidification rate was fitted to a first-order exponential decay function. The PPi data were fitted to a straight line. The data are from a single experiment and are representative of those obtained in five separate experiments.

The time-dependent decrease of the ATP-induced acidification made an analysis of the ATP concentration dependence of the process difficult, and this was not attempted. It was, however, possible to analyze the concentration dependence of the PPi-induced acidification of the vacuole in digitonin-permeabilized parasites. The process showed biphasic kinetics, illustrated in Fig. 5A. The rate of acidification increased with [PPi] up to a concentration of ~0.5 mM and then decreased as [PPi] was increased further. When PPi was added at a concentration of 5 mM, the rate of acidification was less than 20% of its maximum value, whereas the addition of 10 mM PPi had a negligible effect on pHDV.


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Fig. 5.   [PPi] and Mg2+ dependence of the PPi-induced acidification of the digestive vacuole in digitonin-permeabilized parasites. A, initial rates of acidification were estimated as described in the legend to Fig. 4 and are expressed as a fraction of the maximum rate of acidification attained (0.08 ± 0.01 fluorescence ratio units/s (mean ± S.E.) at [PPi] = 0.3 mM). The data are averaged from three independent experiments and are fitted to the Weibull Distribution equation (SigmaPlot; Jandel). Error bars represent S.E. values. The dashed line represents the calculated concentration of free PPi; the dotted line represents the calculated concentration of PPi complexed with Mg2+ (i.e. the sum of the concentrations of MgPP<UP><SUB>i</SUB><SUP>2−</SUP></UP>, Mg2PPi, and MgHPP<UP><SUB>i</SUB><SUP>−</SUP></UP>). Both were obtained using the computer program GEOCHEM-PC (19). B, in digitonin-permeabilized parasites suspended in a MgCl2-free solution, pHDV was unaffected by the addition of 0.2 mM PPi (white arrowhead) but underwent an immediate decrease in response to the subsequent addition of 2 mM MgCl2 (black arrowhead). The trace is from a single experiment, representative of those obtained in three separate experiments.

A likely explanation for the biphasic kinetics is that the true substrate for the H+-PPase is a Mg·PPi complex (MgPP<UP><SUB>i</SUB><SUP>2−</SUP></UP>, Mg2PPi, and/or MgHPP<UP><SUB>i</SUB><SUP>−</SUP></UP>) and that, as has been shown in other systems (24, 25), free PPi (i.e. uncomplexed PPi or PPi complexed with H+) acts as a competitive inhibitor of the H+-PPase. The dotted line in Fig. 5A shows the calculated concentrations of the Mg·PPi complex (i.e. the sum of the concentrations of MgPP<UP><SUB>i</SUB><SUP>2−</SUP></UP>, Mg2PPi, and MgHPP<UP><SUB>i</SUB><SUP>−</SUP></UP>), and the dashed line shows the concentration of free PPi over the PPi concentration range tested. At low concentrations, the Mg·PPi complexes predominated. As the total concentration of PPi increased, however, the inhibitory free PPi ion became the predominant species present, and it is probably this that was responsible for the decrease in the PPi-induced acidification rate observed as the total [PPi] was increased above ~0.5 mM.

Further evidence in support of the view that Mg·PPi is the substrate for the H+-PPase is provided in Fig. 5B, which shows that in the absence of MgCl2 the addition of 0.2 mM PPi had no effect on pHDV, whereas the subsequent addition of 2 mM MgCl2 resulted in the immediate acidification of the digestive vacuole. The addition of MgCl2 in the absence of PPi had no effect on pHDV (not shown).

The rate and magnitude of the PPi-induced acidification of the digestive vacuole in digitonin-permeabilized parasites were reduced by replacement of K+ with Na+ in the medium (Fig. 6). The PPi-induced acidification was also susceptible to competitive inhibition by the H+-PPase inhibitor NaF (Fig. 7). When the digestive vacuole was acidified by the addition of 2 mM PPi to the digitonin-permeabilized parasite suspension, the subsequent addition of 10 mM NaF resulted in a modest alkalinization (Fig. 7A). Reduction of the PPi concentration increased the sensitivity to inhibition by NaF, indicative of competitive behavior. When the digestive vacuole was acidified by the addition to the permeabilized parasite suspension of 0.2 mM PPi, NaF at a concentration as low as 2.5 mM induced a progressive alkalinization of the vacuole (Fig. 7B).


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Fig. 6.   K+ dependence of the PPi-induced acidification of the digestive vacuole in digitonin-permeabilized parasites. The traces show the effect of 0.2 mM PPi, added as either the K+ salt (A) or the Na+ salt (B), at the point indicated by the white arrowhead, to digitonin-permeabilized parasites suspended in a solution containing either 110 mM K+ plus 30 mM Na+ (A) or 0 mM K+ plus 140 mM Na+ (B). Traces are from a single experiment, representative of those obtained in three separate experiments.


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Fig. 7.   NaF-induced reversal of the PPi-induced acidification of the digestive vacuole in digitonin-permeabilized parasites. The addition of 10 mM NaF (black arrowhead) to parasites treated with 2 mM PPi caused a modest alkalinization of the digestive vacuole (A). The addition of 2.5-10 mM NaF (black arrowhead) to parasites treated with 0.2 mM PPi caused a much more pronounced alkalinization (B). The data are from a single experiment, representative of those obtained in at least three separate experiments.

ATP- and PPi-induced Acidification of the Parasite's Digestive Vacuole in Isolated Digestive Vacuoles and in Single Permeabilized Parasites-- In addition to the measurements carried out on suspensions of intact and digitonin-permeabilized parasites, two alternative approaches were taken to confirm the presence of discrete ATP- and PPi-dependent acidification mechanisms on the parasite's digestive vacuole. The first utilized preparations of isolated digestive vacuoles. The second entailed monitoring the fluorescence arising from within the (dye-loaded) digestive vacuoles of single digitonin-permeabilized parasites using confocal microscopy.

Fig. 8 shows representative fluorescence traces obtained from suspensions of isolated digestive vacuoles to which had been added quinacrine, a membrane-permeant fluorescent indicator that partitions into acidic compartments where it undergoes a decrease in fluorescence. The isolated vacuoles underwent a marked acidification in response to the addition of ATP to the medium, consistent with the presence of a functional H+-ATPase. The subsequent addition of the H+-ATPase inhibitor concanamycin A caused the vacuoles to realkalinize (Fig. 8A). The addition of PPi to isolated vacuoles resulted in an acidification, consistent with the operation of an H+-PPase (Fig. 8B). Similar results were obtained from suspensions of digestive vacuoles isolated from parasites grown in erythrocytes preloaded with fluorescein-dextran (not shown).


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Fig. 8.   ATP- and PPi-induced acidification of digestive vacuoles isolated from P. falciparum trophozoites. The traces show the effect of the addition of ATP (1 mM; gray arrowhead), followed by the addition of the H+-ATPase inhibitor concanamycin A (75 nM; black arrowhead) (A) and the addition of PPi (1 mM, white arrowhead) (B) on the fluorescence from a suspension of isolated digestive vacuoles containing 25 nM quinacrine. A downward deflection of the trace is indicative of vacuolar acidification. The traces are representative of those obtained in two or more similar experiments.

As a second means of confirming that the activities detected are both present on the digestive vacuole, we used confocal imaging to collect the fluorescence emanating from the digestive vacuole of individual digitonin-permeabilized parasites. The addition of ATP to the permeabilized parasite resulted in an acidification of the digestive vacuole, and the addition of the H+-ATPase inhibitor concanamycin A caused it to realkalinize (Fig. 9A). The addition of PPi also caused a vacuolar acidification (Fig. 9B).


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Fig. 9.   Single cell measurements of the ATP- and PPi-induced acidification of the digestive vacuole of digitonin-permeabilized parasites. Digitonin-permeabilized parasites (in which the digestive vacuole was preloaded with fluorescein-dextran) were attached to coverslips with polylysine and visualized using a confocal microscope. The fluorescence from the digestive vacuoles of individual parasites was recorded. The addition of 2 mM ATP (A, gray arrowhead) caused an acidification that was reversed by the addition of 70 nM concanamycin A (black arrowhead). The addition of 2 mM PPi (B, white arrowhead) induced a somewhat slower acidification. The data are averaged from measurements on 27 (A) and 40 (B) individual parasites and are expressed as a percentage of the initial resting fluorescence.

31P NMR Measurements of PPi Hydrolysis-- The fluorescence data presented so far are consistent with the presence in the digestive vacuole membrane of two discrete H+ pumping mechanisms: a concanamycin A-sensitive H+-ATPase and a NaF-sensitive H+-PPase. The presence of PPase activity (i.e. the hydrolysis of PPi to Pi) was confirmed in both digitonin-permeabilized parasites and in isolated digestive vacuoles using 31P NMR spectroscopy.

PPi added at a concentration of 4 mM to a suspension of digitonin-permeabilized parasites underwent progressive hydrolysis to Pi, with complete conversion by ~3 h (Fig. 10A). As is clear from the time courses of Fig. 10B, the rate of hydrolysis (indicated by the slope of the lines) increased as time progressed, and the concentration of PPi in the sample decreased. The dotted line in Fig. 10B indicates the changing ratio of the concentration of Mg·PPi complex to the concentration of free PPi as the experiment progressed. During the course of the experiment, the relative concentration of the (inhibitory) free PPi species decreased, and it is probably this that underlay the observed time-dependent increase in the rate of PPi hydrolysis. The inclusion of NaF (10 mM) in the medium had the effect of slowing the rate of hydrolysis substantially (Fig. 10B).


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Fig. 10.   Hydrolysis of PPi by digitonin-permeabilized parasites and by isolated digestive vacuoles. A, stacked 31P NMR spectra showing the time-dependent hydrolysis of PPi (added at a concentration of 4 mM) and the concomitant production of Pi by digitonin-permeabilized parasites. The spectra are derived from an average of 750 pulses, processed with a line-broadening factor of 3. B, time courses showing the hydrolysis of PPi (4 mM) in the absence (closed circles) or presence (open symbols) of digitonin-permeabilized parasites suspended in the presence (squares) or absence (triangles) of 10 mM NaF. The data were averaged from two separate experiments and are shown as mean ± range/2. There was no hydrolysis in the absence of parasites. The dotted line shows the calculated time-dependent change in the ratio of Mg·PPi to free PPi in the sample in which parasites were suspended in the absence of NaF (i.e. in the time course indicated by the open triangles). C, 31P NMR spectra showing the complete conversion of PPi (0.5 mM) to Pi after a 2-h incubation with isolated digestive vacuoles. The spectra are derived from an average of 4,000 pulses, processed with a line-broadening factor of 3.

31P NMR spectroscopy was also used to monitor the conversion of PPi to Pi by isolated digestive vacuoles. The difficulty of preparing the large quantities of isolated digestive vacuoles required for the 31P NMR analysis limited the experiments that we were able to do using this approach. As illustrated in Fig. 10C, however, the isolated vacuoles mediated the complete conversion of 0.5 mM PPi to Pi within 2 h.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The digestive vacuole of the malaria parasite is an acidic organelle in which host erythrocyte proteins (predominantly hemoglobin) are degraded to small peptides and amino acids. In this paper, we present evidence for the presence on the P. falciparum digestive vacuole membrane of two discrete H+ pumps. One is a V-type H+-ATPase, inhibited by concanamycin A and bafilomycin A1. The other is a H+-PPase, inhibited by NaF and showing a partial dependence on K+. The presence of the two types of H+ pump resembles the situation in the acidic intracellular vacuole of plant cells (26) and perhaps in the acidocalcisome (an acidic organelle that serves as a calcium store) of a number of protozoan parasites (27, 28), in which a H+-ATPase and a H+-PPase act in concert to maintain a low internal pH.

The Characteristics and Physiological Role of the Digestive Vacuole H+-ATPase and H+-PPase-- The presence of a V-type H+-ATPase on the digestive vacuole of P. falciparum has been inferred previously from immunological data (7-9), from the previous demonstration of Mg2+-ATP-stimulated acidification of the digestive vacuole in permeabilized parasites (10), and from the demonstration that ATP hydrolysis by isolated digestive vacuoles can be inhibited by (nonspecific) H+ pump inhibitors such as N-ethylmaleimide and 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (11). In this study, we provide direct evidence (Fig. 2) that a V-type H+-ATPase plays a major role in maintaining the acidic pH of the digestive vacuole in intact parasites (within saponin-permeabilized erythrocytes). Under normal conditions, the pump was reliant on glucose in the medium to ensure that it was adequately supplied with ATP (Fig. 2B). However, the pump was able to function in the absence of glucose (for at least 10 min) under conditions in which the parasite was able to import ATP directly from the extracellular medium via the atractyloside-sensitive ATP/ADP exchanger (Fig. 2C).

The first reports of a H+-PPase in the intraerythrocytic malaria parasite were from Docampo and co-workers (29, 30), who presented evidence for PPi-dependent acidification of a subcellular compartment in total cell homogenates of mature Plasmodium parasites. The results of the present study, showing both PPi-induced acidification of the digestive vacuole and hydrolysis of PPi to Pi by isolated vacuoles, provide direct evidence for the presence of a functional H+-PPase on the digestive vacuole membrane.

As with other H+-PPases described to date (e.g. in mung bean (Vigna radiata) hypocotyls (31), Avena sativa L. roots (32), and Kalanchoë daigremontiana leaves (33)), the H+-PPase described in this study exhibited biphasic kinetics, with the PPi-induced acidification rate increasing as [PPi] was increased up to ~0.5 mM and then decreasing as [PPi] was increased further (Fig. 5A). These observations may be explained by inhibition of the H+-PPase by free PPi (24, 25), the concentration of which increased to high levels as the total concentration of PPi was increased under conditions of constant [Mg2+] (Fig. 5A). This explanation is also consistent with the results of the 31P NMR experiment (Fig. 10A) in which the rate of hydrolysis of PPi to Pi by digitonin-permeabilized parasites (indicated by the slope of the solid lines in Fig. 10B) increased with time. As the PPi was hydrolyzed (under conditions of constant [Mg2+]), the ratio of Mg·PPi to free PPi increased (Fig. 10B, dotted line), thereby alleviating inhibition of the H+-PPase and increasing the rate of hydrolysis of PPi.

The extent to which the H+-PPase contributes significantly to the maintenance of the acidic pH of the digestive vacuole under physiological conditions is not clear. The addition of the PPase inhibitor NaF to intact parasites did cause an alkalinization of the digestive vacuole (results not shown); however, the fact that NaF is also a potent inhibitor of glycolysis (and, thereby, of ATP production) makes the interpretation of this observation difficult. The observation that the specific H+-ATPase inhibitor concanamycin A caused a pronounced alkalinization shows that under the conditions of the experiment, the H+-PPase alone was not able to maintain pHDV.

The finding that PPi is present in the blood stage of at least one species of Plasmodium (P. berghei) (34) does suggest that the digestive vacuole H+-PPase may be active in situ, at least under some conditions. However, the fact that earlier 31P NMR studies in P. berghei failed to detect PPi (35), together with the observation that the PPi content of different stages of Trypanosoma cruzi has been shown to fluctuate with the growth stage and with the level of "stress" to which the parasites are subjected (36), raises the possibility that the concentration of PPi in the intraerythrocytic forms of P. falciparum may, at least under certain conditions, be too low (or perhaps even too high) (Fig. 5A) to allow the H+-PPase to contribute significantly to the maintenance of pHDV.

Following permeabilization of the parasite plasma membrane, the ability of ATP to induce a vacuolar acidification decreased in a time-dependent manner (Fig. 4). The fact that the PPi-induced acidification did not show a similar decrease over a longer period indicates that the decrease in H+ pumping by the ATPase is not simply a reflection of a general loss of digestive vacuole function or integrity. A loss of H+-ATPase activity has been reported to occur following fractionation of organelles in T. brucei (37). In yeast vacuoles, V-type H+-ATPase activity decreases in the prolonged absence of glucose, reportedly through disassembly of subunits from the multisubunit complex (38-42). Enzyme oxidation after isolation has also been implicated in loss of H+-ATPase activity (43).

Whatever the underlying mechanism(s), the observation that the H+-ATPase activity is lost under conditions in which the H+-PPase activity remains active suggests a possible role for the PPase as a "back-up system," which might operate to acidify the digestive vacuole under conditions in which the vacuolar H+-ATPase is impaired. However, the pronounced vacuolar alkalinization observed to occur upon inhibition of the H+-ATPase, either by concanamycin A (Fig. 2A) or by glucose deprivation (Fig. 2, B-E), suggests that the H+-PPase was unable to fulfill such a back-up role under the conditions of our experiments. The fact that in some systems V-type H+-ATPases have the ability to "run backward" and synthesize ATP when subjected to a sufficiently large H+ electrochemical gradient (e.g. see Refs. 18 and 44) raises the possibility that under conditions in which the parasite has a significant cytosolic [PPi] and low [ATP], the presence of a H+-PPase-induced H+ electrochemical gradient across the digestive vacuole membrane might cause the vacuolar H+-ATPase to run backward, thus enabling the digestive vacuole to act as an ATP-generating organelle. Again, however, this is unlikely to have occurred under the conditions of our experiment, since in the absence of parasite ATP the vacuolar pH increased to such an extent as to make it unlikely that there was a significant H+ gradient across the digestive vacuole membrane. Just as a H+-PPase-induced H+ gradient could, in principal, cause the H+-ATPase to run backward and synthesize ATP, so too could a H+-ATPase-induced H+ gradient cause the H+-PPase to run backward and synthesize PPi. Whether this occurred under the conditions of our experiments is unclear.

Whereas ATP added to intact parasites induced an acidification of the intracellular vacuole, the addition of PPi did not (cf. Fig. 2, C and E). This may be due to PPi not crossing the parasite plasma membrane at a sufficiently high rate to fuel the vacuolar H+-PPase and/or to the hydrolysis by cytosolic PPases (present in intact parasites but lost on permeabilization of the parasite plasma membrane) of the PPi entering the cell.

The Identity of the Digestive Vacuole H+-PPase-- The H+-PPases that have been characterized to date are composed of single polypeptides and fall into two distinct classes, distinguished on the basis of their K+ dependence; type I H+-PPases have an absolute requirement for K+ in order to function, whereas type II H+-PPases operate in the absence of K+ (45). Analysis of the P. falciparum genome has revealed the presence of two H+-PPase genes, PfVP1 and PfVP2, encoding homologues of the type I (K+-dependent) H+-PPase and type II (K+-independent) H+-PPase, respectively, of Arabidopsis (46). The mRNAs from both were shown to be expressed throughout the intraerythrocytic phase of the parasite life cycle, with the level of PfVP1 mRNA much higher than the level of PfVP2 mRNA (46). PfVP1 and PfVP2 have predicted molecular masses of 76.4 and 115.8 kDa, respectively. In two previous studies, Western blots of P. falciparum trophozoite lysates or membranes using polyclonal antibodies generated against a peptide sequence (TKAADVGADLVGKIE) from the A. thaliana H+-PPase showed a single band (albeit at two different estimated molecular masses, 77 kDa (29) and 67 kDa (46)). On the basis of immunofluorescence experiments, it was concluded that the protein is distributed over the parasite surface as well as intracellularly (29, 46). However, there was no fluorescence detected from the parasite's digestive vacuole (46). A similar localization pattern was seen in experiments in which parasites were transfected with green fluorescent protein-PfVP1 fusion protein (46).

In the course of this work, we used antibodies raised against the corresponding peptide from the mung bean H+-PPase (DVGADLVGKVE) (47) in an attempt to detect one or more proteins in Western blots of the isolated digestive vacuole preparation. The corresponding sequence of PfVP1 has the sequence DVGADLSGKNE, whereas that of PfVP2 has the sequence DIGADLVGKVE (46). Western blots of whole saponin-isolated parasites showed a single band at an estimated molecular mass of 65 kDa, close to the molecular mass predicted for PfVP1 and very similar to that observed by McIntosh et al. (46). However, the protein was not present at detectable levels in isolated digestive vacuole preparations (data not shown). This, together with the previous immunolocalization data, argues against PfVP1 being the digestive vacuole H+-PPase. It is possible that the vacuolar H+-PPase is the substantially larger PfVP2 and that this protein is not recognized by the antibodies used both by us and by others (despite the similarity of the target sequence) and/or that the level of expression of PfVP1 (or PfVP2) in the digestive vacuole is too low to have been detected on our Western blots. PfVP2, like its K+-independent Arabidopsis homologue, AVP2, possesses a conserved lysine (at position 817) (46) that has recently been shown to confer K+ independence to H+-PPases (48). PfVP2 might therefore also be expected to be K+-independent. The finding that the parasite digestive vacuole H+-PPase shows a pronounced stimulation by K+ (Fig. 6) might therefore argue against it being PfVP2, raising the possibility that the H+-PPase activity on the digestive vacuole is due to another, as yet unidentified, P. falciparum protein. The resolution of this issue awaits further research.

    ACKNOWLEDGEMENTS

We are grateful to the staff of the ACT Red Cross Blood Transfusion Service for the provision of blood, to Prof. M. Maeshima for the provision of H+-PPase antibody, and to Dr. P. Ryan for assistance with the use of GEOCHEM-PC.

    FOOTNOTES

* This work was supported by Australian National Health and Medical Research Council Grant 179804 and grants from the Australian National University Faculties Research Grants Scheme 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.

§ These two authors contributed equally to this work.

To whom correspondence should be addressed. Tel.: 61-2-6125-0640; Fax: 61-2-6125-0313; E-mail: kevin.saliba@anu.edu.au.

|| Supported by an Australian postgraduate student award.

Dagger Dagger Supported by the Wellcome Trust.

Published, JBC Papers in Press, November 8, 2002, DOI 10.1074/jbc.M208648200

    ABBREVIATIONS

The abbreviations used are: H+-ATPase, proton-translocating ATPase; H+-PPase, proton translocating PPase; pHDV, digestive vacuole pH; MES, 4-morpholineethanesulfonic acid.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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