Figure 1.
Cytotoxicity of EGCG on Plasmodium sporozoites.
The percentage of living parasites after application of different EGCG concentrations from 12.5 µg/ml to 2000 µg/ml (27 µM - 4400 µM) was determined over 18 h. A) Snap shots of parasites after treatment with 200 µg/ml EGCG at the start of the experiment and after 6 and 12 h. The upper panels show the green, the lower panels the red fluorescent signal. Living parasites are crescent shaped and show green fluorescence. Dying parasites are round and exhibit red fluorescence. Scale bar: 25 µm; numbers indicate time in hours. B) The percentage of living parasites (normalised to the control set to 100%) was plotted over the concentrations of EGCG at 0 h (filled squares), after 6 h (open circles) and 12 h (crosses). Weighted averages of the triplicates (each usually contained between 200 and 300 sporozoites) and standard errors of the mean of the percentage of living parasites for each data point are shown. The dashed lines mark the IC50 values: 502±2 µg/ml (1095±3 µM) after 6 h, 54±2 µg/ml (118±4 µM) after 12 h.
Figure 2.
Inhibition of gliding motility by EGCG.
The percentage of gliding parasites after treatment with EGCG in a range from 3.125 µg/ml to 500 µg/ml (6.75 µM to 1090 µM) was determined. A) Inverted maximum projections of time-lapse movies from untreated sporozoites and sporozoites treated with 100 µg/ml or 500 µg/ml. As sporozoites glide in circles, maximum projections of motile parasites show up as circles. At 100 µg/ml more than 50% of gliding was inhibited; at 500 µg/ml no motility was detected. Scale bar: 25 µm. B) Concentration dependent inhibition of parasite gliding motility after 40 min of incubation with EGCG. Weighted averages of triplicates (each usually contained between 200 and 300 sporozoites) and standard errors of the mean of the percentage of motile parasites were calculated for each concentration point, normalised to the control (set to 100%) and plotted over the applied EGCG concentrations. IC50 value: 63±1 µg/ml (137±1 µM). C) Inhibition of hepatocyte infection of sporozoites in the presence of various concentrations of EGCG. Sporozoites were either pre-incubated for 40 minutes in EGCG containing medium before addition to hepatocytes (light grey) or not pre-incubated (dark grey). The dashed black lines mark the IC50 which is aorund 130 µg/ml after pre-incubation.
Figure 3.
Permeabilisation efficiency of digitonin.
A) Snap shots of non-treated sporozoites and sporozoites treated with 250 and 500 µg/ml digitonin after 40 min of incubation. The upper panels show the green, the lower panels the red fluorescent signal. Scale bar: 25 µm. B) The percentage of living parasites (normalised to the control set to 100%) plotted over the concentrations of digitonin at the beginning of the experiment (filled squares), after 6 h (open circles) and 12 h (crosses). Weighted averages of triplicates (each usually contained between 200 and 300 sporozoites) and standard errors of the mean are shown.
Figure 4.
Cytotoxicity of EGCG combined with digitonin.
The percentage of living parasites (normalised to the control set to 100%) after exposure to a low A) or high B) concentration of EGCG, combined with digitonin in a range from 40 to 70 µg/ml (40 = grey line with filled circle; 50 = grey line with cross; 60 = light grey line with filled rhombi; 70 = light grey line with open triangle) decreases with time. The values were averaged over triplicates (each containing 200 to 300 parasites) and normalised to the cytotoxicty assay of 40 µg/ml to 70 µg/ml digitonin without EGCG application measured over 18 h since digitonin alone showed a lethal effect (not shown, but partially included in Figure 3).
Figure 5.
Synergistic cytotoxicity of EGCG and digitonin.
The percentage of living parasites over (normalised to the control set to 100%) the concentrations of EGCG (dashed grey lines, already shown in Figure 1) and EGCG with 60 µg/ml (115 µM) digitonin was plotted at the beginning of the experiment (straight line on top), after 6 h (without digitonin = dashed black line with filled squares; with digitonin = black line with filled squares) and 12 h (without digitonin = dashed grey line with open circles; with digitonin = grey line with open circles). Weighted averages of triplicates (each usually contained between 200 and 300 sporozoites) and standard errors of the mean are shown normalised to the death curves of digitonin alone. The dashed black lines mark the IC50 values. The IC50 values of the cytotoxic effect of EGCG are reduced from 502±2 µg/ml (1095±3 µM) to 20±1 µg/ml (44±2 µM) after 6 h and from 54±2 µg/ml (118±4 µM) to 16±1 µg/ml (35±2 µM) after 12 h by 60 µg/ml digitonin application.
Figure 6.
Inhibition of gliding motility by EGCG and digitonin.
Concentration dependent inhibition of gliding sporozoites (normalised to the control set to 100%) treated with different EGCG concentrations and permeabilised with 50 (open circles), 60 (filled squares), 65 (crosses) and 70 (filled rhombi) µg/ml digitonin. IC50 value of EGCG and 60 µg/ml digitonin for inhibition of gliding motility: 52±1 µg/ml (113±2 µM). The EGCG curves based on 60 µg/ml and 65 µg/ml are similar, except of a sharper decline of the EGCG curve with 65 µg/ml digitonin.