An Image-Based High-Content Screening Assay for Compounds Targeting Intracellular Leishmania donovani Amastigotes in Human Macrophages

Leishmaniasis is a tropical disease threatening 350 million people from endemic regions. The available drugs for treatment are inadequate, with limitations such as serious side effects, parasite resistance or high cost. Driven by this need for new drugs, we developed a high-content, high-throughput image-based screening assay targeting the intracellular amastigote stage of different species of Leishmania in infected human macrophages. The in vitro infection protocol was adapted to a 384-well-plate format, enabling acquisition of a large amount of readouts by automated confocal microscopy. The reading method was based on DNA staining and required the development of a customized algorithm to analyze the images, which enabled the use of non-modified parasites. The automated analysis generated parameters used to quantify compound activity, including infection ratio as well as the number of intracellular amastigote parasites and yielded cytotoxicity information based on the number of host cells. Comparison of this assay with one that used the promastigote form to screen 26,500 compounds showed that 50% of the hits selected against the intracellular amastigote were not selected in the promastigote screening. These data corroborate the idea that the intracellular amastigote form of the parasite is the most appropriate to be used in primary screening assay for Leishmania

Anti-<i>L. donovani</i> reference drug activity.

<p>Anti-<i>L. donovani</i> reference drug activity.</p

<i>In vitro</i> infection assay optimization for four <i>Leishmania</i> species.

<p><b>A</b>) Images illustrating the infection of THP1 human macrophages by <i>Leishmania</i> species that causes diseases with different clinical manifestations: <i>L. amazonensis</i> (diffuse cutaneous), <i>L. braziliensis</i> (mucocutaneous), <i>L. donovani</i> (visceral) and <i>L. major</i> (cutaneous). <b>B</b>) Dose response curves for the reference drug amphotericin B against all four species.</p

Software interface.

<p><b>A</b>) List of plates in the left and graphical representation of the infection ratio of a 384-well plate by gradient color intensity: white is 0% infection, and blue is 100% infection, with the colors in between indicating an intermediate infection ratio. The first two columns carry controls of amphotericin B EC₁₀₀-treated wells and non-treated wells (1% DMSO), respectively. The last two columns (23 and 24) are non-infected wells and contain non-infected THP-1 cells. <b>B</b>) The window control to set up the software parameters for optimal tuning. <b>C</b>) Table of parameters generated by the software analysis, including, from left to right, “total number of cells,” “number of infected cells” and “infection ratio.” <b>D</b>) A raw image at left and colors highlighting element detection after software analysis: blue (THP-1 cells), red (nuclei of THP-1 detected cells) and green (parasites). <b>E</b>) Close-up of a selected infected THP-1 from (D), highlighting the intracellular <i>Leishmania</i> parasite.</p

Parameters analyzed for automated image analysis and infection level measurement.

<p><b>A</b>) <b>Input image:</b> Raw image input acquired with Opera confocal microscope. <b>B–E</b>) <b>Cell segmentation based on nuclei detection.. </b><b>B</b>) Image denoising by a Gaussian kernel of radius 5. <b>C</b>) Local maxima point detection from B to define nuclei positions. <b>D</b>) Voronoi diagram computation based on the nuclei positions to delineate the inner boundaries of the attached cells. <b>E</b>) Threshold cut-off of pixels below a selected intensity level to make the foreground mask. This image is an example of final cell segmentation. <b>F–I</b>) <b>Parasite detection.. </b><b>F</b>) Calculation of the upper 50% cumulative intensity level of the raw image (A). <b>G</b>) Threshold cut-off of pixels below the intensity level of (F). <b>H</b>) Objects smaller than 4 or larger than 15 pixels are removed to classify parasites. <b>I</b>) Parasite positions are defined. <b>J</b>) <b>Result image:</b> the merged images of cell segmentation (E) and parasite detection (I).</p

High-throughput screening validation.

<p><b>A</b>) Plot of the number of cells (Y-axis) in the controls: yellow (1% DMSO - compound position in the plate), black (1% DMSO – control position in the plate) and red (amphotericin B EC₁₀₀). <b>B</b>) Plot of the infection ratio (Y-axis) using the same color code as in A). The Z' factor of 0.5 demonstrates the statistical confidence of the assay. <b>C</b>) Plot of the infection ratios (Y-axis) obtained from different validation days (red representing day 1 and blue representing day 2) demonstrating low day-to-day variation in the infection ratio and a clear window between non-treated controls (1% DMSO in the upper portion of the plot) and amphotericin B EC₁₀₀-treated controls (lower portion of the plot). <b>D</b>) DRCs of amphotericin B from two independent validation days (red and blue curves representing days 1 and 2, respectively), demonstrating the consistency in the anti-leishmanial activity of the reference drug.</p

THP-1 infection with <i>L. donovani</i>.

<p><b>A</b>) Growth curves of THP-1 and <i>L. donovani</i>, with the optimal development points for infection highlighted by blue and red circles, respectively. <b>B</b>) Image acquired with an Opera confocal microscope showing THP-1 infected with <i>L. donovani</i> after Draq5 (DNA) staining. <b>C</b>) 3-D reconstitution of multiple series confocal pictures illustrating from two different perspectives THP-1 macrophages stained with Syto-60 (red) and infected by <i>L. donovani</i> parasites. Dapi was used to stain the DNA (blue) of both the host cells and the parasites. BrdU incorporation detected by immunofluorescence (green) indicates the replication of intracellular amastigote parasites.</p