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J. Biol. Chem., Vol. 282, Issue 35, 25395-25405, August 31, 2007

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Feedback Inhibition of Pantothenate Kinase Regulates Pantothenol Uptake by the Malaria Parasite^*

Adele M. Lehane, Rosa V. Marchetti, Christina Spry, Donelly A. van Schalkwyk, Rongwei Teng, Kiaran Kirk, and Kevin J. Saliba¹

From the School of Biochemistry and Molecular Biology, Medical School, The Australian National University, Canberra, ACT 0200, Australia

Received for publication, June 5, 2007

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
To survive, the human malaria parasite Plasmodium falciparum must acquire pantothenate (vitamin B₅) from the external medium. Pantothenol (provitamin B₅) inhibits parasite growth by competing with pantothenate for pantothenate kinase, the first enzyme in the coenzyme A biosynthesis pathway. In this study we investigated pantothenol uptake by P. falciparum and in doing so gained insights into the regulation of the parasite's coenzyme A biosynthesis pathway. Pantothenol was shown to enter P. falciparum-infected erythrocytes via two routes, the furosemide-inhibited "new permeation pathways" induced by the parasite in the infected erythrocyte membrane (the sole access route for pantothenate) and a second, furosemide-insensitive pathway. Having entered the erythrocyte, pantothenol is taken up by the intracellular parasite via a mechanism showing functional characteristics distinct from those of the parasite's pantothenate uptake mechanism. On reaching the parasite cytosol, pantothenol is phosphorylated and thereby trapped by pantothenate kinase, shown here to be under feedback inhibition control by coenzyme A. Furosemide reduced this inherent feedback inhibition by competing with coenzyme A for binding to pantothenate kinase, thereby increasing pantothenol uptake.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Pantothenate (vitamin B₅) is a precursor of coenzyme A (CoA), an obligate enzyme cofactor required in numerous metabolic processes. The five-step conversion of pantothenate to CoA is common to prokaryotes and eukaryotes, although the amino acid sequences of the enzymes involved vary considerably (1). The first step in this pathway is the conversion of pantothenate to 4'-phosphopantothenate by pantothenate kinase (PanK).² Plants, fungi, and various bacteria synthesize pantothenate de novo, whereas animals and certain microbes lack this ability and must acquire exogenous pantothenate (1).

The human malaria parasite Plasmodium falciparum cannot synthesize pantothenate and is completely dependent on its uptake from the external medium (2, 3). The membranes of uninfected human erythrocytes are largely impermeant to pantothenate (4), but the vitamin enters infected erythrocytes via the new permeation pathways (NPP) induced in the erythrocyte membrane after P. falciparum infection (4). Upon entering the host erythrocyte, pantothenate is taken up by the parasite via a low affinity H⁺-coupled transporter then, once in the parasite cytosol, is phosphorylated by the parasite's pantothenate kinase (PfPanK) as the first step in its conversion to CoA (4, 5).

There have been several reports of antibacterial (6–8) and antiplasmodial (9, 10) pantothenate analogs. Recent recognition of the sequence dissimilarity between prokaryotic and eukaryotic CoA biosynthesis enzymes (11) has renewed interest in the CoA biosynthesis pathway as a target for the development of novel antibacterial agents (12–14). Similarly, recent evidence indicates that malaria parasite proteins required for pantothenate transport and metabolism hold promise as much-needed chemotherapeutic targets (15). The P. falciparum pantothenate transporter and PfPanK (neither of which have been characterized at a molecular level) differ from studied mammalian counterparts in their biochemical characteristics (e.g. substrate affinity) (5). The value of this pathway as an antimalarial target was validated by the discovery that the widely used provitamin pantothenol (which differs from pantothenate only in the replacement of the terminal carboxyl group with a hydroxyl group) as well as a range of other pantothenate analogs (16, 17) inhibit the growth of P. falciparum in vitro via a mechanism that involves competitive inhibition of pantothenate phosphorylation by PfPanK (3).

In this study we have investigated the mechanism of pantothenol uptake by P. falciparum-infected human erythrocytes and by parasites functionally isolated from their host cells by a saponin permeabilization technique. The results demonstrate that, like pantothenate, pantothenol gains access into infected erythrocytes via the NPP, although an additional pathway accounts for a minor component of its uptake. Once inside the erythrocyte, pantothenol is taken up across the parasite plasma membrane via a mechanism(s) that is functionally distinct from that which mediates the uptake of pantothenate. On entering the parasite, pantothenol is, like pantothenate, phosphorylated by PfPanK, which we show here to be inhibited by CoA. Furosemide was found to alleviate this CoA-mediated negative feedback inhibition of PfPanK and thereby increases the uptake of pantothenol by the parasite despite reducing its initial rate of uptake into P. falciparum-infected erythrocytes by inhibiting the NPP.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Reagents—[¹⁴C]Pantothenol (50 mCi/mmol) and [¹⁴C]pantothenate (55 mCi/mmol) were purchased from American Radiolabeled Chemicals. [¹⁴C]Choline (55 mCi/mmol) and [³H]hypoxanthine (14.7 Ci/mmol) were purchased from Amersham Biosciences.

Parasite Culture and Isolation—Experiments were performed using the 3D7 or FAF6 strains of P. falciparum. The parasites were cultured in Group O, Rh⁺ erythrocytes and synchronized with sorbitol as described elsewhere (18). Parasites were "isolated" from their host erythrocytes by saponin treatment (4), which permeabilizes the cholesterol-containing erythrocyte plasma membrane and parasitophorous vacuole membrane (19) while leaving the parasite plasma membrane intact (20).

[¹⁴C]Pantothenol and [¹⁴C]Pantothenate Uptake across the Erythrocyte Membrane—The uptake of [¹⁴C]pantothenol or [¹⁴C]pantothenate into intact trophozoite-infected and uninfected erythrocytes was measured essentially as described previously for [¹⁴C]pantothenate (4). Uptake was commenced by adding [¹⁴C]pantothenol (2 µM, 0.10 µCi/ml in final reaction) or [¹⁴C]pantothenate (2 µM, 0.11 µCi/ml) to the cell suspension and terminated by centrifuging (15,800 x g, 2 min) 200-µl aliquots of the cell suspension (7 x 10⁷ cells/ml for infected erythrocytes and between 7 x 10⁷ and 3 x 10⁹ cells/ml for uninfected erythrocytes) through 300 µl of dibutyl phthalate, thus separating the cells from the supernatant solution containing the radiolabeled compound. Trophozoite-infected erythrocytes were separated from uninfected cells using the Miltenyi Biotec VarioMACS magnet (21, 22), giving a parasitemia of 95–97%. A cell volume of 75 fl was assumed for both infected and uninfected erythrocytes (4).

[¹⁴C]Pantothenol, [¹⁴C]Pantothenate, and [¹⁴C]Choline Uptake into Isolated Parasites— The uptake of radiolabeled compounds by isolated trophozoites suspended in HEPES-buffered saline (125 mM NaCl, 5 mM KCl, 1 mM MgCl₂,20mM glucose, 25 mM HEPES, pH 7.1 unless stated otherwise) at 37 °C was measured essentially as described previously (23). In experiments with ATP-depleted parasites, glucose was omitted from the saline (and the NaCl concentration was increased to 135 mM so as to maintain the osmolarity of the saline), and parasites were preincubated in this solution for 15–30 min at 37 °C. In the experiments giving rise to Fig. 5C, an inwardly negative membrane potential was imposed on ATP-depleted parasites as described previously (23). In all experiments the concentration of the extracellular radiolabeled compound was 2 µM, and the cells were suspended at a density of 10⁸ cells/ml. An isolated parasite volume of 28 fl was assumed (4). Furosemide, phloretin, N-ethylmaleimide (NEM), carbonyl cyanide m-chlorophenylhydrazone, and valinomycin were added to the parasites (at the same time as the addition of radiolabel) as stock solutions in Me₂SO, and an equal volume of Me₂SO was added to the relevant controls (the maximum Me₂SO concentration in the reaction solution was 0.4%, v/v). p-Chloromercuribenzene sulfonate (pCMBS) was dissolved in water.

[¹⁴C]Pantothenol and [¹⁴C]Pantothenate Phosphorylation— The amount of [¹⁴C]pantothenol and [¹⁴C]pantothenate phosphorylated by PfPanK in parasite lysate (prepared from 7 x 10⁸ cells/ml) was measured under different conditions at predetermined time points by precipitating phosphorylated compounds from solution using the Somogyi reagent (ZnSO₄ and Ba(OH)₂), as described previously (4). The results of key experiments were confirmed with an alternate assay that relies on the separation of the phosphorylated from the unphosphorylated [¹⁴C]substrate by binding the phosphorylated [¹⁴C]substrate to DE81 filter disks followed by scintillation counting, as described previously (14, 24) with the following two modifications; the [¹⁴C]substrate concentration was 0.1 Ci/ml (2 µM), and the filter disks were washed 3 times, each time with 3 ml of an acetic acid:ethanol:water (1:95:4, v/v/v) mixture. In those experiments in which CoA and furosemide were added to the parasite lysate, they were added at the same time as the radiolabeled compound, and the relevant solvent (water and Me₂SO) controls were included in each case.

Biosynthesis, Purification, and Analysis of 4'-Phosphopantothenol—A mixture of pantothenol, ATP, MgCl₂, and furosemide in 50 mM KH₂PO₄ buffer, pH 7.4, was at time 0 combined with lysate prepared aseptically from 3 x 10⁸ P. falciparum parasites, yielding a final volume of 1.8 ml and final concentrations as follows: pantothenol (1 mM), ATP (15 mM), MgCl₂ (15 mM), and furosemide (100 µM). Furosemide was included in the reaction to alleviate feedback inhibition of PanK by CoA or CoA thioesters that may have been present in the parasite lysate (see "Results"). The mixture was incubated for 48 h at 37 °C, and the reaction was terminated by transferring the mixture to 95 °C for 5 min. Precipitated protein was pelleted by centrifugation at 16,000 x g for 5 min. A reaction mixture identical to the one described above but also containing 0.5 µCi/ml [¹⁴C]pantothenol was incubated at 37 °C for 48 h and processed in the same manner. The supernatant from this reaction was spotted on a plastic-backed 0.2-mm-thick silica gel 60 plate (Merck), and the plate was developed to 14 cm from the base line in a solvent of ethanol, 28% NH₄OH (6:4, v/v). Horizontal strips of the thin-layer chromatography (TLC) plate (0.5 cm in width) were transferred to scintillation vials, to which 100 µl of water followed by 3 ml of scintillation fluid was added. The vials were vortexed, and the radioactivity on each section of the plate was determined by scintillation counting. Scintillation counting revealed the production of a ¹⁴C-labeled pantothenol metabolite with an R_f (0.61) distinct from that of pantothenol (R_f = 0.88). The product of the unlabeled reaction could thereby be purified from unreacted pantothenol and other components in the supernatant by TLC. The reaction supernatant was applied in a band to a TLC plate, and the plate was developed to 14 cm from the base line in ethanol, 28% NH₄OH (6:4, v/v). The region bracketing the product (R_f = 0.61) was scraped from the plate, and the product was extracted from the silica gel by suspending the silica in water (5 times) and each time centrifuging at 14,000 x g for 2 min. The water-soluble extracts were combined, concentrated in vacuo, and subjected to ¹H NMR and ESI-MS analysis. NMR spectra were recorded on a Bruker Avance 800 NMR spectrometer operating at 800.13 MHz. The chemical shifts () are reported as the shift in ppm from trimethylsilyl-2,2,3,3-tetradeuteropropionic acid (0.00 ppm). ESI-MS analysis was performed by the Research School of Chemistry Mass Spectrometry Facility, The Australian National University. Low resolution mass spectra were recorded on a Micromass-Waters LC-ZMD single quadrupole liquid chromatograph-mass spectrometer, and high resolution mass spectra were recorded on a Bruker Apex III 4.7T Fourier transform ion cyclotron resonance mass spectrometer using negative ion detection.

View larger version (17K): [in this window] [in a new window]   FIGURE 1. [14C]Pantothenol uptake by P. falciparum-infected and uninfected erythrocytes in the presence or absence of furosemide. A, time courses for the uptake of [14C]pantothenol (2 µM) into infected erythrocytes in the absence (open circles) and presence (filled circles) of 100 µM furosemide. Furosemide was added to the cell suspension in combination with the [14C]pantothenol (i.e. there was no preincubation of the cells with furosemide). Data are averaged from three separate experiments, each performed in duplicate, and are shown ± S.E. B, time courses for the uptake into uninfected erythrocytes of [14C]pantothenate (2 µM; open squares) or of[14C]pantothenol (2 µM) in the absence (open circles) and presence (filled circles) of 100 µMfurosemide (added to the cells at the same time as the [14C]pantothenol). Data are shown relative to the pantothenol distribution ratio at the 20-min time point and are averaged from 4 separate experiments for [14C]pantothenol uptake in the absence of furosemide (shown ± S.E.), from 3 experiments for [14C]pantothenate uptake (shown ± S.E.), and from 2 experiments for [14C]pantothenol uptake in the presence of furosemide (shown ± range/2). All experiments were carried out in duplicate. For clarity, only positive error bars are shown for pantothenol uptake, and only negative error bars are shown for pantothenol uptake in the presence of furosemide. Where not shown, error bars fall within the symbol. Inset, Arrhenius plot for [14C]pantothenol (2 µM) transport into uninfected erythrocytes in the temperature range 5–37 °C. Data are averaged from three separate experiments, each performed in duplicate, and are shown ± S. E.

Measurement of Cytosolic pH— The pH of the parasite cytosol was monitored using 2',7'-bis-(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF) as described elsewhere (20).

Statistics—Statistical comparisons were made using Student's two-tailed t tests for paired or unpaired samples, as appropriate.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Pantothenol Is Taken Up by Infected and Uninfected Erythrocytes—[¹⁴C]Pantothenol entry into human erythrocytes infected with P. falciparum trophozoites was initially rapid, reaching a distribution ratio ([pantothenol]_i/[pantothenol]_o) of 1 within 1 min, but slowing thereafter, reaching a distribution ratio of 2.2 ± 0.2 (mean ± S.E.) by 20 min (Fig. 1A, open circles). Furosemide (100 µM), an effective inhibitor of the NPP, reduced the initial rate of [¹⁴C]pantothenol uptake from a value 19 ± 3 µmol/(10¹² cells·h) to 2.7 ± 0.3 µmol/(10¹² cells·h) (p = 0.027), consistent with the NPP being the major but not the sole route for the uptake of pantothenol. However, furosemide significantly increased (approximately doubled; p = 0.036) the total accumulation seen over the 20-min time course (Fig. 1A, filled circles).

To determine whether the component of [¹⁴C]pantothenol uptake that was not inhibited by furosemide could be attributed to an endogenous erythrocyte pathway, we investigated [¹⁴C]pantothenol uptake by uninfected erythrocytes. Compared with [¹⁴C]pantothenate, which enters uninfected erythrocytes very slowly (Fig. 1B, open squares), [¹⁴C]pantothenol crossed the uninfected erythrocyte membrane rapidly (Fig. 1B, open circles), with an initial rate of 4.2 ± 0.4 µmol/(10¹² cells·h). Furosemide did not significantly affect this initial rate (4.2 ± 0.8 µmol/(10¹² cells·h)) or [¹⁴C]pantothenol accumulation after 20 min (p = 0.9 and 0.5, respectively; Fig. 1B, filled circles). Similarly, the addition of 10 mM pantothenol to the external medium had no significant effect on [¹⁴C]pantothenol uptake (not shown), consistent with pantothenol crossing the uninfected erythrocyte membrane either by simple diffusion through the membrane bilayer or through a low affinity transport system. An Arrhenius plot of the transport of pantothenol across the uninfected erythrocyte membrane (Fig. 1B, inset) was nonlinear and gave activation energies of 64 ± 11 kJ/mol at temperatures >25 °C and 106 ± 4 kJ/mol at temperatures <25 °C. Both the nonlinearity of the Arrhenius plot and the relatively high activation energies are consistent with a carrier-mediated process (25–28).

View larger version (8K): [in this window] [in a new window]   FIGURE 2. [14C]Pantothenol uptake by isolated parasites. Time course for the uptake of [14C]pantothenol (2 µM) by saponin-isolated parasites. Data are averaged from three separate experiments, each performed in duplicate, and shown ± S.E.

View larger version (13K): [in this window] [in a new window]   FIGURE 3. [14C]Pantothenol and [14C]pantothenate phosphorylation by PfPanK. Time courses for the phosphorylation of [14C]pantothenol (0.35 µM; open circles) and [14C]pantothenate (0.35 µM; open squares) by PfPanK in parasite lysate, as determined using the Somogyi reagent. The data are from a single experiment (which included duplicates, shown ± range/2) and are representative of those obtained in two separate experiments. Inset, the same experiment as in main figure, but the amount of [14C]pantothenol and [14C]pantothenate phosphorylation was determined by binding the phosphorylated substrate to DE81 filter disks followed by scintillation counting. The data are averaged from three separate experiments and shown ± S.E. In both experiments, where not shown, error bars fall within the symbol.

Pantothenol Is Taken up by Isolated Parasites and Phosphorylated—The uptake of [¹⁴C]pantothenol by the intracellular parasite was investigated using parasites isolated by saponin permeabilization of the erythrocyte (and parasitophorous vacuole) membranes. The accumulation of [¹⁴C]pantothenol by isolated parasites reached a distribution ratio of 7.9 ± 1.5 after 20 min (Fig. 2), with an initial rate (estimated from the first time point) 11 ± 2 mmol/(10¹² cells·h). The fact that the distribution ratio reached a value significantly >1 might be explained either by an active transport process or by the metabolism of pantothenol within the parasite. Because pantothenol has been shown to act as a competitive inhibitor of the phosphorylation of pantothenate (3), we investigated whether pantothenol is itself phosphorylated by PfPanK using lysate prepared from isolated parasites. ¹H NMR and ESI-MS analyses revealed that incubation of pantothenol with parasite lysate resulted in the generation of 4'-phosphopantothenol (see "Experimental Procedures" for details). We then compared the rate of pantothenol phosphorylation to that of pantothenate phosphorylation by parasite lysates. In paired experiments 62 ± 2% of [¹⁴C]pantothenol was phosphorylated after 40 min (Fig. 3, open circles) compared with 84 ± 1% of the same concentration of [¹⁴C]pantothenate (Fig. 3, open squares). The initial phosphorylation rates were 6.1 ± 0.1 mmol/ (10¹² cells·h) and 10 ± 2 mmol/(10¹² cells·h) for pantothenol and pantothenate, respectively. Similar results were obtained with an alternate assay for measuring pantothenate/pantothenol phosphorylation (Fig. 3, inset). Thus, pantothenol is phosphorylated by PfPanK but at a slower rate than pantothenate. Pantothenol uptake by the parasite, therefore, reflects a combination of transport and metabolism.

View larger version (16K): [in this window] [in a new window]   FIGURE 4. [14C]Pantothenol uptake by isolated parasites in the presence of furosemide. A, time courses for the uptake of [14C]pantothenol (2 µM) by saponin-isolated parasites in the absence (open circles) and presence (filled circles) of 100 µM furosemide. Furosemide was added to the parasites at the same time as [14C]pantothenol. Inset,[14C]pantothenate (2 µM) uptake by isolated parasites after 5 min in the absence (open bar) and presence (filled bar) of 100 µM furosemide. B, time courses for the uptake of [14C]pantothenol (2 µM; filled bars) and [14C]pantothenate (2 µM; open bars) by isolated parasites in the presence of 500 µM unlabeled pantothenate. Time courses were performed in the presence and absence of furosemide and with ATP-replete and ATP-depleted parasites. In A (including inset) and B, data are averaged from three separate experiments, each performed in duplicate. Error bars represent S.E. Where not shown, error bars fall within the symbol.

The effect of furosemide on the transport of pantothenol and pantothenate across the parasite plasma membrane was investigated in parasites in which the contribution of the phosphorylation reaction to the measured uptake was inhibited by the inclusion of 500 µM unlabeled pantothenate in the solution. This concentration of pantothenate is sufficient to saturate PfPanK (which has a K_m of 0.3 µM) (5) but should have little effect on the pantothenate transport mechanism (which has a K_m of 23 mM) (5). Under these conditions both compounds equilibrated across the parasite plasma membrane, with pantothenol entering the parasite more rapidly than pantothenate (p = 0.03). The initial rates of pantothenate and pantothenol uptake were 4.2 ± 0.1 µmol/(10¹² cells·h) and 16 ± 2 µmol/(10¹² cells·h), respectively (Fig. 4B, open bars and filled bars, respectively). Furosemide did not significantly affect the transport rates of either compound (p > 0.06; Fig. 4B). Furthermore, ATP depletion did not affect the transport of pantothenol (p = 0.07; Fig. 4B) but caused a slight slowing of the initial rate of pantothenate transport to 2.5 ± 0.1 µmol/(10¹² cells·h) (p = 0.01; Fig. 4B).

Pantothenol Uptake by Isolated Parasites Is pH-dependent— The transport of pantothenate across the parasite plasma membrane is H⁺-dependent (5). The pH dependence of [¹⁴C]pantothenol uptake by isolated parasites was investigated over the extracellular pH (pH_o) range 6.1 to 8.1. The corresponding intracellular pH (pH_i) range is 7.07–7.72 (23). [¹⁴C]Pantothenol uptake showed a marked pH dependence, increasing with increasing pH_o (Fig. 5A). There was a significant difference (p = 0.01) between the initial rates at pH 6.1 (0.7 ± 0.3 µmol/(10¹² cells·h)) and 8.1 (5.0 ± 1.0 µmol/(10¹² cells·h)). This is the opposite pH dependence from that seen with pantothenate (5), consistent with a fundamentally different uptake mechanism being involved.

View larger version (14K): [in this window] [in a new window]   FIGURE 5. pH dependence of [14C]pantothenol uptake by isolated parasites. A, time courses for the uptake of [14C]pantothenol (2 µM) by saponin-isolated parasites at extracellular pH values (pHo) of 6.1, 7.1, and 8.1. Data are averaged from four separate experiments, each carried out in duplicate, and shown ± S.E. Where not shown, error bars fall within the symbol. B, effects of the additions of 40 mM pantothenol and 40 mM pantothenate on the intracellular pH (pHi) of BCECF-loaded isolated parasites. In the time denoted by the dashed lines, the cuvette chamber was open to add the substrate. C, the effect of imposing an inwardly negative membrane potential (by adding 1µM valinomycin to parasites suspended in a low-K+ medium) on [14C]pantothenol (2µM) uptake by ATP-depleted parasites after 2 min. [14C]Choline (2 µM) uptake was also measured as a positive control. The conditions were: low-K+, 1µM valinomycin; high-K+, 1µM valinomycin (negative control); low-K+, no valinomycin (negative control). The data are averaged from three separate experiments (each performed in duplicate) and shown + S.E.

A decrease in pH_o results in a depolarization of the parasite plasma membrane from its normal value of approximately –95 mV (18). An increase in the uptake of choline by the parasite at higher pH_o values has been attributed to the dependence of choline influx on the membrane potential (23). Whether the membrane potential plays a role in pantothenol influx was tested in ATP-depleted parasites in which an inwardly negative membrane potential was imposed by the addition of the K⁺ ionophore valinomycin (1 µM) to parasites suspended in a low K⁺ medium. The imposition of an inwardly negative membrane potential increased the accumulation of [¹⁴C]choline but not [¹⁴C]pantothenol by ATP-depleted parasites (Fig. 5C).

View larger version (14K): [in this window] [in a new window]   FIGURE 6. Effects of pantothenate transport inhibitors on pantothenol uptake by isolated parasites. The effects of phloretin (200 µM), N-ethylmaleimide (NEM,2mM), p-chloromercuribenzene sulfonate (pCMBS,10 µM), and carbonyl cyanide m-chlorophenylhydrazone (CCCP,10 µM) on the uptake of [14C]pantothenol (2 µM; filled bars) and [14C]pantothenate (2 µM; open bars) by isolated parasites. The pantothenate transport inhibitors were added to the parasite suspension at the same time as the radiolabel, and the uptake was terminated after 5 min. The data are averaged from three-four separate experiments for pantothenol (shown + S.E.) and two-three separate experiments for pantothenate (shown + range/2 or S.E.). All experiments were performed in duplicate.

PfPanK Is Inhibited by CoA—Because furosemide and pH do not affect pantothenol transport by the parasite (Figs. 4B and 5B), we hypothesized that they affect pantothenol uptake by influencing its metabolism. This was tested by examining their effects on pantothenol phosphorylation in parasite lysate. Experiments using lysate provide a useful tool for studying phosphorylation independently of transport but do not take into account the regulation of phosphorylation in situ. CoA and/or its esters have been shown to inhibit the PanKs of bacteria, plants, and animals (1), although recently the PanKs from Staphylococcus aureus and Helicobacter pylori have been shown to be refractory to this feedback inhibition (29, 30). We therefore investigated the effects of CoA on PfPanK activity. [¹⁴C]Pantothenate phosphorylation by PfPanK was inhibited by CoA (Fig. 7) in a concentration-dependent manner. The IC₅₀ value (the concentration at which inhibition was half-maximal) was 200 µM. Acetyl-CoA and malonyl-CoA were also effective inhibitors of pantothenate phosphorylation (data not shown).

View larger version (10K): [in this window] [in a new window]   FIGURE 7. Inhibition of PfPanK by CoA. Dose-response curve for the inhibition by CoA of the phosphorylation of [14C]pantothenate (0.68 µM, measured over 40 min) by PfPanK in parasite lysate. Data are averaged from two separate experiments, each performed in duplicate, and shown ± range/2. The linearity of the phosphorylation reaction over the time course of the experiment was verified in each experiment (to control for variability in activity from different batches of lysate; data not shown).

Furosemide Is a Competitive Inhibitor of CoA-mediated PfPanK Inhibition—The mechanism by which furosemide alleviates the CoA-mediated inhibition of PanK was investigated further. We first tested the effect of increasing concentrations of furosemide on PanK activity in the absence of CoA. As can be seen in Fig. 9A, relatively high concentrations of furosemide (in the absence of CoA) inhibited PfPanK activity. We next carried out a kinetic analysis to determine the interaction between furosemide and CoA. Dixon plots revealed that furosemide antagonized the inhibitory effect of CoA on pantothenate (Fig. 9B) and pantothenol (Fig. 9C) phosphorylation by PfPanK, doing so in a competitive manner, as illustrated by the increasing CoA K_i (x axis intercept) up to 17-fold as the furosemide concentration was increased up to 200 µM.

View larger version (15K): [in this window] [in a new window]   FIGURE 8. Effects of furosemide and pH on phosphorylation of pantothenol by PfPanK in parasite lysates. A, the effect of 100 µM furosemide on the phosphorylation of [14C]pantothenol (0.35 µM, measured over 20 min) in the presence and absence of 400 µM CoA. B and C, the effect of pH on the phosphorylation of [14C]pantothenol (0.35 µM, measured over 20 min) in the absence (B) and presence (C) of 400 µM CoA. The pH values shown (7.07, 7.30, 7.72) correspond to the predicted intracellular pH values upon exposure of parasites to the extracellular pH values 6.1, 7.1, and 8.1, respectively. The data are averaged from three separate experiments, each performed in duplicate, and are shown + S.E.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Fig. 10 shows a schematic representation of the processes involved in the uptake of pantothenol in P. falciparum-infected erythrocytes. Pantothenol gains access into the infected erythrocyte rapidly via the broad-specificity furosemide-sensitive NPP induced by the parasite in the host cell membrane (Fig. 10, step A) and also, at a slower rate, via an additional pathway (Fig. 10, step B). Because pantothenate uptake essentially relies on the NPP (4), whereas pantothenol can enter via an additional pathway, NPP inhibition may result in an increase in the intraerythrocytic [pantothenol]/[pantothenate] ratio. Having entered the erythrocyte, pantothenol is taken up by the intracellular parasite via a mechanism that differs in pH dependence and inhibitor sensitivity from the parasite pantothenate transport mechanism (Fig. 10, step C). Thus, the combination of pantothenol with a pantothenate transport inhibitor is likely to be synergistic. Unfortunately, the pantothenate transport inhibitors identified to date (5) lack the necessary specificity to allow this hypothesis to be tested directly. The observations that the transport mechanism of pantothenol is not coupled to H⁺ equivalents (Fig. 5B), is insensitive to sulfhydryl reagents (NEM and pCMBS, Fig. 6), and is not dependent on ATP (Fig. 4B), the membrane potential (Fig. 5C), Na⁺, or Cl^– (not shown) are consistent with pantothenol crossing the parasite plasma membrane via a process of simple diffusion through the lipid bilayer. However, the activation energy for pantothenol transport across the uninfected erythrocyte membrane (Fig. 1B, inset) and its low LogD value are consistent with a carrier-mediated process (26, 31, 32), at least across this membrane. Further work needs to be carried out to determine the exact mechanism of pantothenol transport across the various membranes of the P. falciparum-infected erythrocyte.

On reaching the parasite cytosol, pantothenol is phosphorylated by PfPanK (Fig. 10, step D). It is not yet known whether phosphopantothenol continues to be metabolized by downstream enzymes to form CoA antimetabolites, as is the case for N-pentylpantothenamide in Escherichia coli (12). Furosemide and cytosol alkalinization partially alleviate the inherent feedback inhibition of PfPanK by CoA (Fig. 10, step E), thereby increasing pantothenol phosphorylation and consequently its uptake. Under similar conditions, furosemide does not have the same effect on pantothenate uptake (Fig. 4A, inset); furosemide might, therefore, be expected to increase the [pantothenol + derivatives]/[pantothenate] ratio inside the parasite.

View larger version (14K): [in this window] [in a new window]   FIGURE 9. Kinetic analysis of the effect of furosemide on PfPanK activity in the presence or absence of CoA. A, the effect of increasing the concentration of furosemide on [14C]pantothenol phosphorylation by PfPanK in parasite lysate in the absence of CoA. Dixon plots showing the effect of increasing furosemide concentrations on the CoA-mediated inhibition of pantothenate (B) and pantothenol (C) phosphorylation by parasite lysates. The data are averaged from three independent experiments, each performed in duplicate. The linearity of the phosphorylation reaction over the time course of the experiments was verified in each experiment (to control for variability in activity from different batches of lysate; data not shown). Error bars represent S.E. and, where not shown, fall within the symbols. For clarity, only positive error bars are shown in B and C. DMSO,Me2SO.

View larger version (27K): [in this window] [in a new window]   FIGURE 10. Schematic representation of pantothenol uptake and phosphorylation by the intraerythrocytic malaria parasite. A, about 86% of the pantothenol (filled circles) that enters into the infected erythrocyte does so via the furosemide-sensitive broad-specificity NPPs induced by the parasite in the host cell membrane. B, the remaining 14% enters infected erythrocytes at a slower rate via a furosemide-insensitive pathway. C, pantothenol is taken up by the intracellular parasite via a mechanism that differs in pH dependence and inhibitor sensitivity from the pantothenate transport mechanism. D, pantothenol is phosphorylated by pantothenate kinase (PfPanK). Furosemide reduces the CoA-mediated negative feedback inhibition of PfPanK (E). By this mechanism furosemide increases the accumulation of phosphorylated pantothenol derivatives within the parasite despite inhibiting its initial entry into the parasitized erythrocyte via the NPP.

Several endogenous compounds have been shown previously to interfere with the feedback inhibition of PanKs. L-Carnitine was found to activate PanK from rat heart and reverses CoA-mediated feedback inhibition by an unknown mechanism (33). Very recently it was demonstrated that palmitoylcarnitine (and not L-carnitine) activated purified human PanK2 by competitively reversing the inhibition observed by acetyl-CoA (30). The authors suggested that both positive and negative regulation of PanK2 play a role in the functioning of the enzyme (30). The demonstration in our study that an unrelated exogenous compound (furosemide) is also able to competitively antagonize the inhibitory activity of the endogenous regulator (CoA) of PfPanK opens up the possibility that PanK regulation might be amenable to pharmacological intervention.

This study highlights the potential of targeting a pathway from two angles; (i) by impeding the access of a nutrient while allowing the access of analogs of the nutrient via an alternative route and (ii) by selectively increasing the uptake of an analog and its derivatives. Despite the very close structural similarity between pantothenate and pantothenol, their mechanisms of uptake are significantly different, providing opportunities for increasing the [pantothenol + derivatives]/[pantothenate] ratio in the intraerythrocytic malaria parasite with a second compound. Whether such a strategy is feasible in vivo remains to be seen.

	FOOTNOTES

 
^* This work was supported by the Australian National Health and Medical Research Council Grant 224245 and by a grant from the UNICEF/UNDP/World Bank/World Health Organization Special Programme for Research and Training in Tropical Diseases (TDR). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: School of Biochemistry and Molecular Biology, The Australian National University, Canberra, ACT 0200, Australia. Tel.: 61-2-6125-7549; Fax: 61-2-6125-0313; E-mail: kevin.saliba{at}anu.edu.au.

² The abbreviations used are: PanK, pantothenate kinase; PfPanK, P. falciparum PanK; BCECF, 2',7'-bis-(2-carboxyethyl)-5,6-carboxyfluorescein; NPP, new permeation pathways; ESI, electrospray ionization; MS, mass spectrometry; pCMBS, p-chloromercuribenzene sulfonate; NEM, N-ethylmaleimide.

	ACKNOWLEDGMENTS

 
We are grateful to the Canberra Branch of the Australian Red Cross Blood Service for the provision of blood and to John Allen, Gordon Lockhart, and Anitha Jeyasingham of The Australian National University Mass Spectroscopy Facility for assistance with mass spectrometry.

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