Effect of caspase inhibition on the infection of S2 cells with <i>Pa. acanthamoebae</i>.

<p>S2 cells were infected with <i>Pa. acanthamoebae</i> (MOI 5) and were incubated for the indicated time periods in the absence or presence of the pan caspase inhibitor Z-VAD-FMK (10 µM). Bacteria were detected with the FISH probes UV7-763 (Cy3, red) and Chls-0523 (Fluos, green), host cells with the probe EUK516 (Cy5, blue). Representative confocal images of the infection cycle (in the absence of Z-VAD-FMK) are shown in (A). The bar indicates 10 µm. The percentage of infected cells observed in the absence (blue) or presence (red) of Z-VAD-FMK over the course of infection is depicted in (B). Black stars indicate statistically significant differences between both curves (t-test) and colored stars indicate significant differences to the respective 0 h p.i. time point (ANOVA & Scheffé). Numbers of intracellular bacteria per infected cell were determined and are shown in (C). Infected cells were classified into 5 groups according to the number of intracellular bacteria (1–3, blue; 4–10, rose; 11–30, green; 31–100, orange; >100, red). Stars indicate statistically different distributions among these classes at a given time point between infections carried out in the absence or presence of Z-VAD-FMK (χ² test). In (B) and (C) mean values and standard deviations of three replicates are shown (***, p≤0.001; **, p≤0.01; *, p≤0.05). The gray boxes indicate time points that were analyzed after cells had been passaged. The microscopic images in (D) illustrate the enhanced infection of S2 cells with <i>Pa. acanthamoebae</i> in presence of Z-VAD-FMK at 48 h p.i. Bacteria were detected with the FISH probe UV7-763 (Cy3, red). The bar corresponds to 20 µm.