Evaluation of pH during cytostomal endocytosis and vacuolar catabolism of haemoglobin in Plasmodium falciparum

Abstract

The DV (digestive vacuole) of the malaria parasite, Plasmodium falciparum, is the site of Hb (haemoglobin) digestion and haem detoxification and, as a consequence, the site of action of CQ (chloroquine) and related antimalarials. However, the precise pH of the DV and the endocytic vesicles that feed it has proved difficult to ascertain. We have developed new methods using EGFP [enhanced GFP (green fluorescent protein)] to measure the pH of intracellular compartments. We have generated a series of transfectants in CQ-sensitive and -resistant parasite strains expressing GFP chimaeras of the DV haemoglobinase, plasmepsin II. Using a quantitative flow cytometric assay, the DV pH was determined to be 5.4-5.5. No differences were detected between CQ-sensitive and -resistant strains. We have also developed a method that relies on the pH dependence of GFP photobleaching kinetics to estimate the pH of the DV compartment. This method gives a pH estimate consistent with the intensity-based measurement. Accumulation of the pH-sensitive probe, LysoSensor Blue, in the DV confirms the acidity of this compartment and shows that the cytostomal vesicles are not measurably acidic, indicating that they are unlikely to be the site of Hb digestion or the site of CQ accumulation. We show that a GFP probe located outside the DV reports a pH value close to neutral. The transfectants and methods that we have developed represent useful tools for investigating the pH of GFP-containing compartments and should be of general use in other systems.

Figure 1. Expression of PM2–GFP at different stages of the intra-RBC cycle of P. falciparum transfectants

(A–D) 3D7 and (E, F) Dd2 transfectants expressing PM2–GFP were imaged at the young to mid trophozoite (A, B, E) or mature trophozoite to schizont (C, D, F) stage. The images (left to right) represent a GFP fluorescence image, a DIC (differential interference contrast) image and an overlay of these images. DV-located GFP is indicated with a blue arrowhead. ER-located GFP is indicated with white arrowheads, while cytostomal vesicles are indicated with white arrows. (G) A 3D7-PM2 trophozoite was labelled with BODIPY® ceramide and the GFP (green fluorescence) and BODIPY® (red fluorescence) signals were imaged by confocal microscopy. The images shown represent an average projection obtained from a series of optical sections. The three-dimensional reconstruction of the labelled cell is presented in Supplementary Movie 1. Note: the intensities of the images were adjusted to optimize the fluorescence signal at each parasite stage. Scale bar, 2 μm.

Figure 2. Spot photobleaching measurements reveal the absence of interconnectivity between DV, ER and vesicular structures in 3D7 transfectants

PM2–GFP-containing structures were subjected to photobleaching using a pulse of unattenuated laser light at the position indicated by the white arrows. Shown are (left to right) DIC images, pre-bleach images and post-bleach images. (A) A vesicular structure was subjected to bleaching for 200 ms. Fluorescence was ablated from this structure but other structures were not affected. (B) A 500 ms bleach of the DV produces localized bleaching with no recovery evident after 15 s. (C) The cell was subjected to five bleach events of 200 ms separated by 10 s intervals at the position indicated by the white arrow then imaged (post). The vesicular structure identified by the grey arrow does not exhibit the loss of fluorescence exhibited by the DV. The image on the right is an overexposed post-bleach image illustrating that the DV fluorescence has disappeared, but that the vesicular and ER fluorescence can still be observed. Scale bar, 2 μm.

Figure 3. Flow cytometric analysis of DV-located GFP fluorescence in 7G8-PM2–GFP transfectants

Untransfected 7G8-infected RBCs and 7G8-PM2–GFP transfectants were isolated using a magnetized column and analysed by flow cytometry as described in the Experimental section. (A) Comparison of signals obtained using untransfected 7G8-infected RBCs (dotted curve) and 7G8-PM2–GFP transfectants (solid black curve) in PIGPA buffer (pH 7.5). Also shown is the signal from 7G8-PM2–GFP transfectants in PIGPA in the presence of CCCP (grey curve). (B) DIC (left) and GFP fluorescence (right) images of control and CCCP-treated 7G8-PM2–GFP transfectants. Scale bar, 2 μm. (C) Isolated 7G8-PM2–GFP transfectants were resuspended in Mes saline at pH values of 4.5–7.5, containing 10 μM CCCP and subjected to flow cytometric analysis (pale grey, mid grey and black curves are pH 5, 5.5 and 7). For reference, the signal from the untransfected cell line in Mes saline at pH.5, containing 10 mM CCCP, is also shown (dotted curve). (D) Calibration curve of the cell-weighted mean fluorescence intensity of the results shown in (C). The curve corresponds to best fit obtained with eqn (1) using a pK_a of 5.5.

Figure 4. Flow cytometric analysis of GFP fluorescence in PfLPL1–GFP transfectants

(A) PfLPL1–GFP transfectants isolated using a magnetized column were analysed in PIGPA buffer (solid black curve) and in Mes saline (pH 7.5) in the presence of CCCP (solid grey curve). The solid black and grey curves overlay. (B) The same sample was measured in Mes saline at pH 5, 6 and 7 (solid curves that become progressively darker with increase in pH). The dotted curves in (A, B) correspond to measurements of uninfected RBCs. (C) Bright field (left panels) and GFP fluorescence images (right panels) of PfLPL1–GFP transfectants treated with CCCP at pH 7.5 and 4.5. Scale bar, 2 μm.

Figure 5. Effect of CQ treatment on DV pH in 3D7-PM2–GFP transfectants

Untransfected 3D7-infected RBCs and 3D7-PM2–GFP transfectants were isolated using a magnetized column resuspended in PIGPA buffer (pH 7.5) and incubated at 37 °C for 1.5 h in the absence or presence of 1.6 μM (broken black curve) or 10 μM CQ (solid black curve) before analysis by flow cytometry. The signals obtained using untransfected 3D7-infected RBCs (black dotted curve) are compared with the control (no drug) 3D7-PM2–GFP transfectants in the absence (light grey curve) and presence (broken grey curve) of CCCP.

Figure 6. Kinetics of GFP photobleaching in the DV of 3D7-PM2–GFP transfectants

(A) Theoretical dependence of τ_obs on pH calculated according to eqn (2). (B) The image panel shows a representative example of GFP photobleaching in a transfectant suspended in PIGPA buffer in the absence of CCCP. Shown is a DIC image of an infected RBC (left panel) and GFP fluorescence images obtained after repeated imaging of the cell. Image numbers are indicated. The Figure shows representative decays of DV fluorescence during repeated imaging in PIGPA in the absence of CCCP (circles), and in Mes buffer at pH 5 (triangles) or pH 7 (diamonds) in the presence of CCCP. The curves correspond to the best fits obtained according to a mono-exponential decay model. (C) Comparison of τ_obs for the DV fluorescence in 3D7-PM2–GFP transfectants suspended in PIGPA, or in Mes buffer at pH 5 or 7 in the presence of CCCP. The results represent photobleaching analysis of a number of cells performed on two separate days. The squares correspond to the individual measurements, illustrating the variability in the results. Error bars represent standard deviations. Scale bar, 2 μm.

Figure 7. Labelling of 3D7-PM2–GFP transfectants with LysoSensor Blue

Mixed-stage transfected parasites were incubated with the pH probe, LysoSensor Blue. Shown are bright field, LysoSensor Blue, GFP and merged GFP/LysoSensor Blue images. The LysoSensor Blue fluorescence is largely associated with the DV, while the GFP chimaera is also present in cytostomal vesicles (arrows) that do not stain with the LysoSensor Blue. Scale bar, 2 μm.