Loss of pH control in Plasmodium falciparum parasites subjected to oxidative stress

Abstract

The intraerythrocytic malaria parasite is susceptible to oxidative stress and this may play a role in the mechanism of action of some antimalarial agents. Here we show that exposure of the intraerythrocytic malaria parasite to the oxidising agent hydrogen peroxide results in a fall in the intracellular ATP level and inhibition of the parasite's V-type H(+)-ATPase, causing a loss of pH control in both the parasite cytosol and the internal digestive vacuole. In contrast to the V-type H(+)-ATPase, the parasite's digestive vacuole H(+)-pyrophosphatase is insensitive to hydrogen peroxide-induced oxidative stress. This work provides insights into the effects of oxidative stress on the intraerythrocytic parasite, as well as providing an alternative possible explanation for a previous report that light-induced oxidative stress causes selective lysis of the parasite's digestive vacuole.

Figure 1. Effect of the oxidising agent H₂O₂ on pH_i in isolated parasites.

(A) 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 P. falciparum trophozoites (5×10⁷ cells/ml) at 37°C and are representative of those obtained in five similar experiments. (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. (C) 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.

Figure 2. Concentration-dependent effects of H₂O₂ on (A) cytosolic pH (pH_i) and (B) [ATP]_I in isolated parasites.

Isolated 3D7 P. falciparum 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.

Figure 3. Effect of H₂O₂ on pH_DV in isolated parasites.

(A) 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) 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 P. falciparum 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. (C) 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.

Figure 4. Effect of H₂O₂ on the ability of the parasite to maintain an acidic DV.

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.

Figure 5. The parasite DV remains intact in parasites subjected to oxidative stress.

(A) and (B) are confocal micrographs showing the redistribution of acridine orange fluorescence in intact parasitized erythrocytes subjected either to (A) 1 min illumination with the microscope's laser, or (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. (C) and (D) 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 (C), followed by excitation with a 488 nm laser line at full power for 1 min (D). 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.