Figure 1.
Leishmania amazonensis lodge and multiply within giant acidic vacuoles.
Six days-old macrophages were seeded into 384-well microplates and incubated with L. amazonensis DsRed2 expressing amastigotes for 48 h. (A) Phase contrast image demonstrating the lodging of the amastigotes within giant vacuoles. Images of living cells were acquired with a 100× immersion objective on a Zeiss Axiovert 200 M microscope. Scale bar corresponds to 5 µm. (B–D) Images of living infected macrophage cultures incubated 60 min with fluorescent reporters were acquired on a confocal plate reader (Opera QEHS) after 3 days of incubation with the compounds. 20× confocal images acquired from samples treated with (B) 1% DMSO (C-), (C) 0.5 µM Amphotericin B (C+) and (D) 180 µM Cycloheximide (C†) are depicted. Scale bars represent 20 µm. Macrophage nuclei and PV were stained with Hoechst 33342 (Blue) and LysoTracker DND-26 (Green), respectively. Amastigotes expressing DsRed2 are represented in red.
Figure 2.
Assay Workflow chart and outputs.
(A) Experimental pipeline. Bone marrow cells recovered from BALB/c mice were cultured in presence of 50 ng/mL rmCSF-1 in hydrophobic flasks at 37°C allowing CSF-1 responsive progenitors to further develop along the macrophage lineage. Fresh medium was added at day 3 (black triangle) and differentiated adherent macrophages (MΦ) were harvested at day 6 and seeded into 384-well culture-treated optical bottom plates in the presence of a lower concentration of rmCSF-1 to prevent cell death. Macrophage cultures were then maintained at 34°C until the end of the experiment. Amastigotes (Am) freshly isolated from nude mouse footpads were added (ratio of 3 amastigotes per macrophage) to macrophages 5 hours after macrophages plating. Compounds are added 19 hours later for a contact period of 3 days. One hour before image acquisition on the confocal plate reader, the fluorescent reporters Hoechst 33342 and LysoTracker DND-26 were added to the macrophage cultures (diamond). All dispensing procedures in the 384-well plates were performed using an automated liquid handling robot without any washing step. (B) Boxplots with whiskers from minimum to maximum of Percent Of negative Control (POC) for the amastigote (Am), parasitophorous vacuoles (PV) and Viability Index (VI) after the following treatments: no compound added (Media), 1% DMSO, 180 µM cycloheximide, 2 mM Leu-oMe and 1 µM Amphothericin B (AmpB) leishmanicidal drug. Sets of 96 data points for each are issued from a representative validation 384-well plate. (C) AmphoB dose-response curves were expressed as POC for (VI) (black circle), (PV) (black square) and (Am) (black triangle). Each concentration has been tested in quadruplicates; error bars represent the data range. IC50 curve fitting and the Spearman correlation coefficient (r, for n = 5) between the IC50 dose curves for the PV and Am outputs were calculated using GraphPad Prism 6 software (GraphPad Software Inc., San Diego, CA).
Table 1.
QC metrics by variables (Am, PV/HM and VI) expressed as robust Z′factor and (SSMD) values.
Table 2.
Pairwise comparison for replicates within P1 to P4.
Table 3.
Pairwise comparison for all plate combinations.
Figure 3.
Merged images for nucleus, PV and amastigote fluorescence of one acquisition field representing different phenotypes are displayed. Scale bar corresponds to 50 µm for all images. Each compound has been categorized into High, Low, ∼Toxic and Cytotoxic based on its SSMD values and following the decision tree described in Figure S3. The Medium class has been introduced to define compounds that were classified as “High” and “Low” in independent replicate experiments. Percentages per category are indicated.
Figure 4.
Effect of various anti-leishmanial molecules on the viability of L. amazonensis promastigotes.
Procyclic promastigotes (2×104/well) were incubated with different concentrations of compounds (0.0125 to 25 µM). Parasites cultured in medium alone (C−, 0% growth inhibition) or in the presence of 1 µM Amphotericin B (C+, 100% growth inhibition) were used as negative and positive controls, respectively. After 64 h incubation with compounds, resazurin (2.5 µg/ml) was added to each well and parasites were incubated for an additional 8 h before measuring resofurin fluorescence (λex = 550 nm, bandwidth = 9 nm; λem = 590 nm, bandwidth = 20 nm). Each compound concentration was tested in quadruplicates and the average ± standard deviation is displayed. EC50 values were calculated using SigmaPlot (SPSS, SSI, San Jose,CA, USA) 4-parameter logistic nonlinear regression analysis (values in parenthesis in the upper right panel).