Figure 1.
Infection of S2 cells with different Leishmania species and life cycle stages.
A. Percentage of S2 cells infected after incubation with either L. donovani amastigotes or L. major late stationary phase promastigotes over a 24 hr time course. B. Comparison of infection levels of S2 cells and mammalian RAW264.7 macrophages incubated with late stationary phase L. major promastigotes. p = 0.0025; error bars in A and B indicate one standard error (SE). C. S2 cells infected with L. donovani amastigotes for 24 hr are stained with 1 µg/ml DAPI, 20 µg/ml propidium iodide; parasites were pre-labelled with 10 µM CFSE. D. S2 cells infected with L. donovani amastigotes (24 hr post infection) stained with anti-ARL8, a late endosomal/lysosomal marker. E. S2 cells expressing LAMP-GFP (green) infected with L. donovani amastigotes for 12 hr. C – E, scale bars, 10 µm. Arrows indicate intracellular parasites.
Figure 2.
Maintenance of L. donovani amastigotes in S2 cells.
S2 cells were incubated with L. donovani amastigotes for 8 hr, external parasites removed by washing and fresh medium returned to the cells prior to fixation at the time points indicated. A. Mean number of parasites per cell at each time point. Data were analysed by two way unpaired Student’s t-test (not significant). B. Distribution of the number of parasites per cell at each time point. C. Transmission electron micrograph of S2 cell following incubation with L. donovani amastigotes for 24 hr. An amastigote in the process of cell division is shown, with the phagosome membrane clearly separated from the dividing parasite (black arrow). The sub-pellicular microtubules and flagella of the dividing parasites are also clearly visible (white arrows).
Figure 3.
Replication of L. donovani amastigotes within S2 cells.
S2 cells were infected with CFSE-labelled parasites for 6 hr, followed by washing to remove external parasites. At the time points indicated, S2 cells were lysed with saponin to release amastigotes. A. Parasites were identified from cell debris by staining with the sera from an infected hamster conjugated to fluorescent allophycocyanin (APC), followed by flow cytometry and gating on APC high, forward scatter low cells. B. Flow cytometry overlay of CFSE staining of parasites identified in A, released after 6, 24 and 48 hr. C. By gating on CFSE staining in B, the percentage of parasites that had replicated at least once was calculated at each time point. Data are representative of two independent experiments.
Figure 4.
Effect of pharmacological inhibitors on S2 cell phagocytosis of L. donovani amastigotes.
S2 cells were treated with inhibitory compounds for 1.5 hr prior to incubation with CFSE-labelled L. donovani amastigotes (at a 10∶1 ratio) for 3 hr in the continuing presence of these drugs. A. Percentage of S2 cells infected with L. donovani after treatment with the compounds indicated. Approximately 300 cells in 3 independent wells were scored by eye for each treatment. Figures were compared to DMSO-treated controls by one-way analysis of variance (Anova) with Dunnett’s Multiple Comparison Test, *** p<0.0001. Bars indicate one standard error of the mean. B. Representative confocal laser scanning microscopy images of cells treated with the compound indicated. Cells were stained with 1 µg/ml DAPI, 20 µg/ml propidium iodide; amastigotes were stained with CFSE; size bar, 50 µm.
Figure 5.
RNAi against Cdc42 reduces phagocytosis of L. donovani amastigotes.
S2 cells were treated with dsRNA against Cdc42 and Rab5 for 4 days and then incubated with amastigotes for 24 hr. A. Three wells from a 96 well plate were analysed for each treatment and at least 100 cells counted in each well. * p<0.05. B. Representative confocal laser scanning microscopy images of cells treated with dsRNA as indicated. Cells were stained with 1 µg/ml DAPI, 20 µg/ml propidium iodide; amastigotes were stained with CFSE; size bar, 10 µm.
Figure 6.
Results from RNAi screen against 1920 Drosophila gene products.
A. Scatter plot of effect of dsRNA on uptake of L. donovani amastigotes. Each point represents an individual dsRNA treatment. dsRNA probes are ordered on the x axis by probe number (1–1920). Hits outside 1.5 standard deviations from the mean (blue dashed line) were chosen for further analysis. Green shading indicates hits that caused a significant increase in phagocytosis. Red shading indicates hits that caused a significant decrease in phagocytosis. B. Predicted GoFunctions of 87 proteins for which RNAi mediated knock-down of gene expression resulted in a significant decrease in phagocytosis of L. donovani amastigotes by S2 cells.
Figure 7.
A. Percentage of cells infected after RNAi with dsRNA validation (Val) and original (Orig, if OTE free) probes (as indicated). Infection rates normalised to dsRNA GFP control treated cells. Error bars indicate one standard error of the mean. Wells were imaged on a Zeiss LSM 510 confocal laser scanning microscope, and infection rates scored manually. A mean of 154 cells were counted per well, with three replicates of each dsRNA treatment and eight replicate GFP dsRNA treatments. Infection rates were normalised to GFP infection rates (100.0% ±2.264 [1 SEM]). Infection rates compared by two way unpaired Student’s t-test *, p<0.05; **, p<0.01; *** p<0.001. B. Representative images of RNAi treated cells. Bar indicates 10 µm.
Figure 8.
Effect of myriocin on RAW264.7 macrophage phagocytosis of L. donovani amastigotes and promastigotes.
RAW264.7 macrophages were pre-treated with 10 µM myriocin or 0.5% methanol (diluent) for 24 hr to inhibit de novo sphingolipid biogenesis. They were then incubated with L. donovani parasites (at a MOI of 10∶1) for 2 hr, external parasites removed and the infected macrophages fixed or incubated with medium containing myriocin or methanol for the periods indicated. A. Infection with L. donovani amastigotes; B. Infection with L. donovani promastigotes. (i) Percentage of cells infected at the timepoints indicated; (ii) Mean number of parasites per infected cell. Infections rate compared by two way unpaired Student’s t-test * p<0.05, ** p<0.01, *** p<0.001. Error bars indicate one standard error of the mean.