A kinetic fluorescence assay reveals unusual features of Ca⁺⁺ uptake in Plasmodium falciparum-infected erythrocytes

Abstract

Background: To facilitate development within erythrocytes, malaria parasites increase their host cell uptake of diverse solutes including Ca++. The mechanism and molecular basis of increased Ca++ permeability remains less well studied than that of other solutes.

Methods: Based on an appropriate Ca++ affinity and its greater brightness than related fluorophores, Fluo-8 was selected and used to develop a robust fluorescence-based assay for Ca++ uptake by human erythrocytes infected with Plasmodium falciparum.

Results: Both uninfected and infected cells exhibited a large Ca++-dependent fluorescence signal after loading with the Fluo-8 dye. Probenecid, an inhibitor of erythrocyte organic anion transporters, abolished the fluorescence signal in uninfected cells; in infected cells, this agent increased fluorescence via mechanisms that depend on parasite genotype. Kinetic fluorescence measurements in 384-well microplates revealed that the infected cell Ca++ uptake is not mediated by the plasmodial surface anion channel (PSAC), a parasite nutrient channel at the host membrane; it also appears to be distinct from mammalian Ca++ channels. Imaging studies confirmed a low intracellular Ca++ in uninfected cells and higher levels in both the host and parasite compartments of infected cells. Parasite growth inhibition studies revealed a conserved requirement for extracellular Ca++.

Conclusions: Nondestructive loading of Fluo-8 into human erythrocytes permits measurement of Ca++ uptake kinetics. The greater Ca++ permeability of cells infected with malaria parasites is apparent when probenecid is used to inhibit Fluo-8 efflux at the host membrane. This permeability is mediated by a distinct pathway and may be essential for intracellular parasite development. The miniaturized assay presented here should help clarify the precise transport mechanism and may identify inhibitors suitable for antimalarial drug development.

Figure 1

Kinetics of fluorescence development. (A) Fluorescence kinetics in 96 well format. Symbols represent means ± S.E.M. of replicate wells for Dd2-infected and uninfected cells (blue and red symbols, respectively) in 1 mM free external Ca⁺⁺ or 1 mM EGTA (circles and triangles, respectively). Notice greater fluorescence with Ca⁺⁺ than EGTA for both cell types. A difference between infected and uninfected cells is apparent. (B) Identical experiment using 384 well format. While fluorescence development is specific for Ca⁺⁺, the greater permeability of infected cells is not clearly resolved in this format. (C) Mean ± S.E.M. fluorescence after 12 hours with 1 mM free Ca⁺⁺ for Dd2-infected and uninfected cells (blue and red bars, respectively) in indicated formats. n =4-8 independent trials each; P = 0.01 and 0.7 for comparisons of infected vs. uninfected cells in 96- and 384-well formats, respectively.

Figure 2

Effects of probenecid on transport kinetics in 384-well format. (A) Uninfected cells have a large fluorescence signal in 1 mM Ca⁺⁺ (circles), which is abolished by addition of 5 mM probenecid (triangles). (B, C, D) Fluorescence kinetics for erythrocytes infected with indicated parasite lines in the absence or presence of probenecid (circles and triangles, respectively). Note that parasite genotype influences the magnitude of probenecid-associated increase in fluorescence. (E) Mean ± S.E.M. effects of probenecid addition at 12 h, normalized to 0% for no effect. n = 5-7 trials each; P < 10^-5 and < 0.02 for comparisons of Dd2 to uninfected and HB3, respectively. (F) Kinetics of Fluo-8 efflux from uninfected cells in the absence or presence of 5 mM probenecid (circles and triangles, respectively). Error bars, S.E.M. of replicates; representative of two experiments.

Figure 3

Effects of EGTA and SDS. (A, B) Addition of EGTA to chelate extracellular Ca⁺⁺ 4 hours after initiating uptake (arrow in each panel; final [EGTA], 3.33 mM); the free Ca⁺⁺ was buffered at 1.0 mM prior to EGTA addition. Panel A shows uninfected cells in the absence of probenecid. Here, EGTA addition abruptly reduces the fluorescence and prevents further increases (black circles); control kinetics are shown for resuspension of cells without addition of chelator (red circles). Panel B shows matched experiments for Dd2-infected cells in both the presence and absence of 5 mM probenecid (triangles and circles, respectively). With infected cells, the initial reduction with EGTA addition is smaller (black symbols), suggesting a lesser contribution of exported Fluo-8. EGTA prevents subsequent increases in fluorescence; probenecid-induced augmentation is also abolished (compare black triangles to black circles). These processes depend on extracellular Ca⁺⁺ and uptake at the host membrane. (C, D) Addition of SDS to lyse cells 6 hours after initiating uptake with uninfected and Dd2-infected cells in the absence of probenecid (arrows; final [SDS], 0.05%). Note the large increases in fluorescence after addition of SDS (black circles), when compared to resuspension only kinetics (red or blue circles). (E) Effect of probenecid on plateau fluorescence after SDS addition, calculated by normalization of matched controls without probenecid to 100%. Bars represent mean ± S.E.M. of replicates from 2-3 independent experiments.

Figure 4

Localization using confocal microscopy. Bright field, fluorescence, and overlay DIC images of uninfected and infected cells loaded with Fluo-8 AM (top and bottom pairs of rows, respectively). Cells were loaded in the presence or absence of probenecid (Pr) and imaged in media containing 1 mM Ca⁺⁺. Cell-associated fluorescence is not detected in uninfected cells in the absence of probenecid, consistent with exported Fluo-8; probenecid reveals a weak intracellular signal. With infected cells, a clear signal is detected within both host and parasite compartments. Cell-to-cell variability prevented quantitative assessment of probenecid effect.

Figure 5

Effects of known transport inhibitors. (A) Mean ± S.E.M. Ca⁺⁺ uptake by Dd2-infected cells in the presence of 15 μM ISPA-28, 10 μM verapamil, 100 μM CdCl₂, or 10 μM nifedipine, normalized to 100% for control measurements over 12 hours without inhibitor. Cd⁺⁺ increases parasite-associated fluorescence, but none of the agents block the parasite-induced erythrocyte Ca⁺⁺ permeability. (B) Mean ± S.E.M. fluorescence change associated with 100 μM CdCl₂ for uninfected cells in media without probenecid. At these concentrations, extracellular Cd⁺⁺ does not contribute significantly to the signal.

Figure 6

Conserved parasite requirement for extracellular Ca⁺⁺. (A) Normalized growth of divergent parasite lines over 72 hours as a function of measured free external [Ca⁺⁺], achieved by EGTA addition to standard media. Absissca is shown on log scale. Symbols represent mean ± S.E.M. growth of HB3, 3D7A, and Dd2 lines (green, white, and blue circles, respectively). Solid line represents the best fit to a sigmoidal decay. (B) Mean ± S.E.M normalized growth of each line when 2 mM CaCl₂ is added to the medium with 2 mM EGTA, the highest used in panel (A).

Figure 7

Schematic showing proposed Ca⁺⁺and Fluo-8 transport mechanisms. (A) Uninfected human erythrocytes maintain a low intracellular Ca⁺⁺ through a low passive Ca⁺⁺ permeability and an ATP-dependent extrusion pump (Ca⁺⁺ pump). Fluo-8 AM enters cells by diffusion and is hydrolyzed to yield activated Fluo-8, which fluoresces upon Ca⁺⁺ binding. Fluo-8 may be exported via organic anion transporters (OAT). (B) Infected cells have a higher intracellular Ca⁺⁺ through activation of a distinct Ca⁺⁺ uptake pathway. Ca⁺⁺ that enters the cell may be exported via the Ca⁺⁺ pump, may remain in the host cytosol, or may enter parasite compartments by crossing the parasitophorous vacuolar membrane (PVM) through nonselective PVM channels (PVMC) [39,40] and the parasite plasma membrane (PPM) via unknown mechanisms. Fluo-8 AM that enters infected cells may be hydrolyzed to the active form in various compartments; the activated dye may be exported via the host cell OAT and possibly via PSAC. Transport of the dye across parasite membranes may also occur, but is not illustrated.