Effect of H₂O₂ on the ability of the parasite to maintain an acidic DV.

<p>Digitonin-permeabilised 3D7 trophozoites in which the DV was preloaded with fluorescein-dextran were suspended at a density of ∼5×10⁶ cells/ml at 37°C. The fluorescence measurements were not calibrated; an increase in the fluorescence ratio is indicative of an increase in pH_DV. The addition of either 2 mM ATP or 0.5 mM PPi to the external medium at the point indicated by the black triangle caused a rapid acidification of the vacuole (the trace shown is that obtained following the addition of PP_i; a very similar trace was observed on addition of ATP). On addition of H₂O₂ (10 mM, at the point indicated by the white triangle) to the permeabilised parasites (in the continued presence of ATP or PP_i) there was an immediate alkalinisation in those parasites in which the DV was acidified by the addition of ATP (light grey trace), whereas in those parasites in which the DV was acidified by the addition of PP_i the pH_DV was largely unaffected (dark grey trace). These data are consistent with H₂O₂ inhibiting the parasite's V-type H⁺-ATPase while not inhibiting the H⁺-PPase. The traces shown are from a single experiment and are representative of results obtained from at least three similar experiments.</p

Effect of H₂O₂ on pH_DV in isolated parasites.

<p>(<b>A</b>) Effect of H₂O₂ (2 mM) and the V-type H⁺ ATPase inhibitor concanamycin A (75 nM) on pH_DV in suspensions of isolated D10 trophozoites in which the DV was preloaded with the membrane-impermeant pH-sensitive dye, fluorescein-dextran. The cells were suspended at a density of ∼5×10⁶ cells/ml at 37°C and the reagents were added at the point indicated by the white triangle. The fluorescence measurements were not calibrated; an increase in the fluorescence ratio is indicative of an increase in pH_DV (i.e. an alkalinisation). The traces shown are from a single experiment and are representative of those obtained in five similar experiments. (<b>B</b>) Single cell measurements showing the effect of H₂O₂ (2 mM, added at the point indicated by the white triangle) on pH_DV of isolated D10 <i>P. falciparum</i> trophozoites in which the DV was preloaded with the membrane-impermeant pH-sensitive dye, fluorescein-dextran. An increase in the fluorescence ratio is indicative of an increase in pH_DV. The data are averaged from 48 individual parasites carried out at 22°C on three different days, and are shown±S.D. (<b>C</b>) Effect of H₂O₂ (8 mM) and concanamycin A (75 nM) on pH_DV in suspensions of Dd2 transfectant parasites expressing a pH-sensitive GFP-PM2 fusion protein in the DV, and suspended at a density of 7×10⁷ cells/ml at 37°C. The reagents were added at the point indicated by the white triangle. An increase in the fluorescence ratio is indicative of an increase in pH_DV.</p

Concentration-dependent effects of H₂O₂ on (A) cytosolic pH (pH_i) and (B) [ATP]_I in isolated parasites.

<p>Isolated 3D7 <i>P. falciparum</i> trophozoites were loaded with the pH-sensitive fluorescent dye BCECF. H₂O₂ was either absent (black circles) or added at time-zero at a concentration of either 2 mM (triangles) or 10 mM (open circles). The data are averaged from three separate experiments. In (A) the error bars were omitted for clarity; in the control experiment the S.E.M. ranged from 0.003–0.013 pH units, in the 2 mM H₂O₂ experiment the S.E.M. ranged from 0.003–0.092 pH units, and in the 10 mM H₂O₂ experiment the S.E.M. ranged from 0.008–0.109 pH units. In (B) the error bars denote S.E.M.</p

The parasite DV remains intact in parasites subjected to oxidative stress.

<p>(<b>A</b>) and (<b>B</b>) are confocal micrographs showing the redistribution of acridine orange fluorescence in intact parasitized erythrocytes subjected either to (<b>A</b>) 1 min illumination with the microscope's laser, or (<b>B</b>) 10 min exposure to 30 mM H₂O₂. Prior to the treatment the DV fluoresces red and the parasite cytosol fluoresces green. Both treatments resulted in a loss of red fluorescence from the region of the DV. (<b>C</b>) and (<b>D</b>) show the retention of fluorescein-dextran within the DV of mature, isolated D10 trophozoites (there are two visible in the image) following exposure to 100 mM H₂O₂ for 1 hour (<b>C</b>), followed by excitation with a 488 nm laser line at full power for 1 min (<b>D</b>). Neither the high concentration of H₂O₂ alone, nor the subsequent additional intense light exposure resulted in any redistribution of the fluorescein-dextran from the DV of the parasites (visible as dark regions, coinciding with the hemozoin crystals, in the bright-field images), from which it may be concluded that the DV remained intact. All scale bars (shown in white) are 5 µm.</p

Effect of the oxidising agent H₂O₂ on pH_i in isolated parasites.

<p>(<b>A</b>) Effect of H₂O₂ (2 mM) and the V-type H⁺ ATPase inhibitor concanamycin A (75 nM) on pH_i. The reagents were added at the point indicated by the white triangle. The traces shown are from a single experiment on a suspension of isolated BCECF-loaded 3D7 <i>P. falciparum</i> trophozoites (5×10⁷ cells/ml) at 37°C and are representative of those obtained in five similar experiments. (<b>B</b>) Effect of parasite concentration on the response of pH_i to the oxidising agent H₂O₂ (4 mM, added at the point indicated by the white triangle) in suspensions of isolated BCECF-loaded 3D7 parasites at 37°C. ΔpH_i indicates the deviation from the initial resting pH_i. The traces shown are from a single experiment but are representative of those obtained in three similar experiments. (<b>C</b>) Effect of H₂O₂ (2 mM, added at the point indicated by the white triangle) on pH_i in single isolated SNARF-loaded D10 parasites immobilized on polylysine coated coverslips at 22°C. The data showing cytosolic pH_i are averaged from 79 individual cells carried out on three different days, and are shown±S.D.</p

Fluorescence traces showing the effects of DMSO (solvent control), MMV007839 and MMV000972 on the cytosolic pH (pH_cyt) of 3D7 <i>P</i>. <i>falciparum</i> trophozoites under varying conditions.

<p>Each trace is representative of those obtained in at least three independent experiments. (A-C) The effects of DMSO (0.1% v/v; solvent control; A), MMV007839 (1 μM; B) and MMV000972 (1 μM; C) on the cytosolic pH of parasites suspended in (glucose-containing) Experimental Saline Solution (pH 7.10) at 37°C. (D-F) The effects of DMSO (0.25% v/v; solvent control; D), MMV007839 (2.5 μM; E), and MMV000972 (2.5 μM; F) on the cytosolic pH of parasites suspended (for 10 min prior to commencing recording, and throughout the initial recording period) in glucose-free Experimental Saline Solution (pH 7.10, 37°C), and the effects of the subsequent addition of glucose (22 mM) to the parasite suspension. (G-I) The effects of adding DMSO (0.25% v/v; solvent control; G), MMV007839 (2.5 μM; H), and MMV000972 (2.5 μM; I), followed by sodium L-lactate (10 mM), on the cytosolic pH of parasites suspended at 4°C in Experimental Saline Solution (pH 7.10).</p

L-[¹⁴C]lactate influx into PfFNT^Gly107Ser mutant parasites and Dd2 parental parasites.

<p>(A) The uptake of L-[¹⁴C]lactate by isolated trophozoite-stage PfFNT^Gly107Ser mutant parasites (white bars) and Dd2 parental parasites (black bars) in the absence of compound (0.4% v/v DMSO; solvent control) and in the presence of the non-specific anion transport inhibitor NPPB (100 μM). The mean distribution ratio (i.e., [intracellular L-[¹⁴C]lactate]/[extracellular L-[¹⁴C]lactate]) and SEM from nine (parent) or seven (mutant) independent experiments for the control, and from three independent experiments for NPPB, are shown. The results of unpaired t-tests are shown; ***<i>P</i> < 0.001. (B-C) The effects of MMV007839 (B) and MMV000972 (C) on L-[¹⁴C]lactate uptake into isolated trophozoite-stage PfFNT^Gly107Ser mutant parasites (white symbols) and Dd2 parental parasites (black symbols). L-[¹⁴C]lactate uptake in the presence of each concentration of MMV compound is expressed as a percentage of that observed when there was no inhibition of transport. The data shown are the average ± SEM from <i>n</i> independent experiments performed on different days, where <i>n</i> was seven for the MMV007839 data with parental parasites, three for the MMV007839 data with PfFNT^Gly107Ser mutant parasites, three for the MMV000972 data with parental parasites, and four for the MMV000972 data with PfFNT^Gly107Ser mutant parasites (with the exception of the lowest concentration, for which there was one less experiment in all cases). In all cases (panels A-C), the experiments were performed at 4°C in pH 6.1 Experimental Saline Solution, and parasites were preincubated with the compound (at the concentration indicated) or DMSO (0.4% v/v; solvent control) for 1 min prior to the addition of L-[¹⁴C]lactate (1.3 μM). The concentrations of compound and DMSO after the addition of L-[¹⁴C]lactate were the same as those present during the preincubation. The uptake of L-[¹⁴C]lactate was measured over 20 s.</p

The Malaria Parasite's Lactate Transporter PfFNT Is the Target of Antiplasmodial Compounds Identified in Whole Cell Phenotypic Screens

<div><p>In this study the ‘Malaria Box’ chemical library comprising 400 compounds with antiplasmodial activity was screened for compounds that perturb the internal pH of the malaria parasite, <i>Plasmodium falciparum</i>. Fifteen compounds induced an acidification of the parasite cytosol. Two of these did so by inhibiting the parasite’s formate nitrite transporter (PfFNT), which mediates the H⁺-coupled efflux from the parasite of lactate generated by glycolysis. Both compounds were shown to inhibit lactate transport across the parasite plasma membrane, and the transport of lactate by PfFNT expressed in <i>Xenopus laevis</i> oocytes. PfFNT inhibition caused accumulation of lactate in parasitised erythrocytes, and swelling of both the parasite and parasitised erythrocyte. Long-term exposure of parasites to one of the inhibitors gave rise to resistant parasites with a mutant form of PfFNT that showed reduced inhibitor sensitivity. This study provides the first evidence that PfFNT is a druggable antimalarial target.</p></div

PfFNT mutation and growth inhibition by various compounds in parasites generated by exposure to increasing concentrations of MMV007839.

<p>PfFNT mutation and growth inhibition by various compounds in parasites generated by exposure to increasing concentrations of MMV007839.</p

The effect of MMV007839 on the metabolite profile of parasitised erythrocytes.

<p>(A) Relative amounts of intracellular metabolites in MMV007839-treated parasitised erythrocytes (infected with the 3D7 strain) and untreated parasitised erythrocytes. The data shown are the mean log₂ ratios of the level of each metabolite in the MMV007839-treated samples relative to its level in the untreated samples (+ MMV007839/- MMV007839), determined 1 h, 3 h and 6 h after the addition of MMV007839, obtained from three independent experiments. The concentration of MMV007839 used (6 μM) corresponds to approximately 20× the IC₅₀ value for growth inhibition of 3D7 parasites. (B) The log₂ ratios of metabolite levels in MMV007839-treated samples relative to those in untreated samples for all metabolites of known identity for which a statistically significant difference in abundance was observed between MMV007839-treated and untreated infected erythrocytes (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006180#ppat.1006180.s009" target="_blank">S1 Data</a> for statistical analysis). The data shown are the mean + SEM from three independent experiments. GL3P, sn-glycerol-3-phosphate; Pyr, pyruvate; Lac, lactate; R5P, ribose-5-phosphate; AcCoA, acetyl coenzyme A; Gln, glutamine; VD, Val-Asp; PE, Pro-Glu; PVQ, Pro-Val-Gln; PDAV, Pro-Asp-Ala-Val; TNVK, Thr-Asn-Val-Lys; Oro, orotate; O5P, orotidine-5-phosphate; DC, deoxycytidine; GGPP, geranylgeranyl-pyrophosphate. (C) Relative amounts of metabolites in the extracellular medium in suspensions containing MMV007839-treated and untreated parasitised erythrocytes. The data shown are the mean log₂ ratios (+ MMV007839/- MMV007839) + SEM from three independent experiments. There was a statistically significant difference in the concentrations of 3-phosphoglycerate and phosphoenolpyruvate in the extracellular medium between MMV007839-treated and untreated cell suspensions (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006180#ppat.1006180.s009" target="_blank">S1 Data</a>). DHAP, glycerone phosphate; PEP, phosphoenolpyruvate; 3PG, 3-phosphoglycerate; Cit, citrate; αKG, 2-ketoglutarate; Mal, malate; Pip, pipecolate.</p