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First published online 21 February 2006
doi: 10.1242/jcs.02795

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The pH of the digestive vacuole of Plasmodium falciparum is not associated with chloroquine resistance

¹ School of Biochemistry and Molecular Biology, Faculty of Science, The Australian National University, Canberra ACT 0200, Australia
² Medical School, The Australian National University, Canberra ACT 0200, Australia

View larger version (9K): [in a new window]   Fig. 1. Estimated digestive vacuole pH (pHDV) of mature trophozoite-stage D10' (CQS, open bars) and 7G8' (CQR, filled bars) parasites as determined using either (A) Fluorescein or (B) Oregon Green 514 calibrated with the nigericin/high-K+ method. The pHDV of 7G8' was significantly higher than the pHDV of D10' when measured with either Fluorescein (P<0.001) or Oregon Green 514 (P<0.01). However, for both strains, the pHDV estimated using Fluorescein was significantly higher than that estimated using Oregon Green 514 (P<0.001). The concentration of dextran in each treatment was 28 µM, resulting in a Fluorescein concentration of 40 µM and an Oregon Green 514 concentration of 70 µM as a consequence of the different number of fluorophore molecules per dextran molecule (Haughland, 2002) (see Materials and Methods). The data for each dye represent the means of at least nine (Fluorescein) or at least four (Oregon Green 514) independent experiments, with at least three replicate measurements per experiment. The error bars show s.e.m.

View larger version (14K): [in a new window]   Fig. 2. pHDV of mature trophozoite-stage D10' parasites as reported by BCECF (pKa 7.0), Fluorescein (pKa 6.4), Oregon Green 514 (pKa 4.7) and CL-NERF (pKa 3.8), all at a loading concentration of 28 µM dextran. The three different pH calibration methods described in Materials and Methods each report a consistent linear relationship between dye pKa and pHDV (R2>0.99, P<0.05 in all cases), with the reported pHDV ranging from 3.7 to 6.5. The data represent the means (error bars show s.e.m.; where not shown, error bars fall within the symbol) of at least three independent experiments. The dotted line indicates data that would be obtained if pHDV were equal to dye pKa.

View larger version (21K): [in a new window]   Fig. 3. The normalised ratiometric pH-dependent change in fluorescence of either Fluorescein (open symbols) or Oregon Green 514 (closed symbols). In situ measurements (squares) were made using isolated mature trophozoite-stage D10' parasites suspended in high-K+ saline and exposed to 50 µM nigericin. The concentration of dextran in the loading solution was 28 µM. Cell-free measurements (circles) were made as described in Materials and Methods. The pH range over which the Fluorescein response is linear was 5.0-7.5, whereas the linear response of Oregon Green 514 was in the pH range 3.5-5.5.

View larger version (13K): [in a new window]   Fig. 4. Comparison of pHDV in D10' parasites at the early trophozoite stage (16-22 hours post-invasion) and at the mature trophozoite stage (32-38 hours post-invasion) as determined using either 40 µM Fluorescein (open symbols) or 70 µM Oregon Green 514 (closed symbols) (i.e. 28 µM dextran in the loading solution in each case), calibrated using the nigericin/high-K+ method. For both dyes, the reported pHDV changed significantly as the intraerythrocytic parasites developed from the early to the mature stage (P<0.05). The data represent the means (± s.e.m.) of at least three independent experiments, each derived from at least three replicate measurements per experiment.

View larger version (21K): [in a new window]   Fig. 5. Fluorometer traces of mature trophozoite-stage D10' parasites isolated from erythrocytes loaded with (A) 150 µM Fluorescein, (B) 10 µM Fluorescein, (C) 70 µM Oregon Green 514, or (D) 4 µM Oregon Green 514. Cells were suspended in minimal saline solution at pH 7.1. Black arrowheads indicate the point of addition of concanamycin A (75 nM), a potent and specific inhibitor of the digestive vacuole V-type H+ pump (Saliba et al., 2003). Numbered traces indicate the pH of calibration samples (nigericin/high-K+ method). The traces are from single experiments, and are representative of those obtained in at least three separate experiments for each dye concentration. Note that the dynamic range of Oregon Green 514 (see Fig. 3) precluded accurate measurement of the endpoint of digestive vacuole alkalinisation by concanamycin A.

View larger version (20K): [in a new window]   Fig. 6. pHDV of mature trophozoite-stage D10' (circles) and 7G8' (squares) parasites as determined using either Fluorescein (open symbols) or Oregon Green 514 (closed symbols) over a range of concentrations and calibrated with the nigericin/high-K+ method. For both D10' and 7G8' parasites, the pHDV reported by Fluorescein was significantly lower (P<0.01) at the lowest concentration tested than at the highest (10 µM and 156 µM, respectively). However, there was no significant change in reported pHDV for either strain over the range of concentrations of Oregon Green 514 tested (4-70 µM). At Fluorescein loading concentrations of 40 µM and above, the reported pHDV of 7G8' parasites was significantly higher than that of D10' parasites (P<0.01). Similarly, at Oregon Green 514 loading concentrations of 35 µM and above, the reported pHDV of 7G8' parasites was significantly higher than that of D10' parasites (P<0.05). The data represent the means (± s.e.m.; where not shown, error bars fall within the symbol) of at least three independent experiments, each derived from at least three replicate measurements per experiment. Curves (rectangular hyperbolic and simple linear) were fitted by least-squares regression with SigmaPlot 2001 (SPSS).

View larger version (17K): [in a new window]   Fig. 7. Total uptake of [3H]CQ into parasites in intact normal erythrocytes (i.e. not subjected to the lysing/resealing process; filled bars) and in intact lysed/resealed erythrocytes (open bars). For both types of erythrocytes [3H]CQ uptake by cells infected with D10' (CQS) parasites was significantly (>eightfold) higher than [3H]CQ uptake by cells infected with and 7G8' (CQR) parasites (P<0.05). The data represent the means (+ s.e.m.) of four independent experiments, each carried out in triplicate.

View larger version (97K): [in a new window]   Fig. 8. Dextran-linked pH indicator fluorescence localises to the Plasmodium falciparum digestive vacuole, as demonstrated by this representative set of fluorescence and brightfield images. (a) False-colour image of the fluorescence emitted at >515nm when dextran-Fluorescein-loaded mature trophozoite-stage parasitised erythrocytes, together with dye-loaded uninfected erythrocytes, were excited at 420-490 nm. (b) Brightfield image of the same field, collected immediately after the fluorescent image. (c,d) Composite images with uninfected red blood cells (uRBC), infected red blood cell (iRBC), parasite cytosol (cyt) and digestive vacuole, as indicated. As described previously, fluorescence within the dye-loaded parasite emanates predominantly from a restricted area that coincides with the haemozoin crystals within the digestive vacuole (Bray et al., 2002a; Spiller et al., 2002). Bar, 5 µm.