Automated high-content assay for compounds selectively toxic to Trypanosoma cruzi in a myoblastic cell line

Abstract

Background: Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, represents a very important public health problem in Latin America where it is endemic. Although mostly asymptomatic at its initial stage, after the disease becomes chronic, about a third of the infected patients progress to a potentially fatal outcome due to severe damage of heart and gut tissues. There is an urgent need for new drugs against Chagas disease since there are only two drugs available, benznidazole and nifurtimox, and both show toxic side effects and variable efficacy against the chronic stage of the disease.

Methodology/principal findings: Genetically engineered parasitic strains are used for high throughput screening (HTS) of large chemical collections in the search for new anti-parasitic compounds. These assays, although successful, are limited to reporter transgenic parasites and do not cover the wide T. cruzi genetic background. With the aim to contribute to the early drug discovery process against Chagas disease we have developed an automated image-based 384-well plate HTS assay for T. cruzi amastigote replication in a rat myoblast host cell line. An image analysis script was designed to inform on three outputs: total number of host cells, ratio of T. cruzi amastigotes per cell and percentage of infected cells, which respectively provides one host cell toxicity and two T. cruzi toxicity readouts. The assay was statistically robust (Z´ values >0.6) and was validated against a series of known anti-trypanosomatid drugs.

Conclusions/significance: We have established a highly reproducible, high content HTS assay for screening of chemical compounds against T. cruzi infection of myoblasts that is amenable for use with any T. cruzi strain capable of in vitro infection. Our visual assay informs on both anti-parasitic and host cell toxicity readouts in a single experiment, allowing the direct identification of compounds selectively targeted to the parasite.

Fig 1. Image analysis development.

Representative images of the T. cruzi infected control, non-infected control, and 30 μM benznidazole treated infected cells (BNZ) for: (a) raw image acquisition (at 635 nm); (b) nuclei selection (selected in green and discarded in red; as described in Methods) and their cytoplasm boundaries identification (green lines); (c) discrimination of true amastigotes (green) from other non-parasitic cytoplasmic spots (red) amongst all cytoplasmic spots detected; (d) identification of infected cells (those with >1 amastigote inside, green) and non-infected cells (red); (e) scatter plots of all detected spots used to discriminate true (green dots) from false amastigote spots (red dots) based on their area and intensity (‘Spot area’ and ‘Corrected intensity’ thresholds lines are shown; ‘Corrected Intensity’ is expressed in arbitrary units). Bar is 30 μm.

Fig 2. High resolution image of controls.

Representative T. cruzi infected and uninfected H9c2 pictures at 40X. Kinetoplastid kDNA (circle) and nDNA (bean-like) can be distinguished at this magnification. Bar is 30 μm.

Fig 3. Discrimination of spots using ‘Corrected intensity’ as parameter.

A total of 1.18×10⁶ spots were acquired from infected and non-infected control wells (five fields per well from 96 wells for each control) from 6 independent plates each. Spots were distributed in 9 groups and classified accordingly to their ‘Corrected intensity’ parameter based on the Hartigan-Wong algorithm [34]. The three parametric regions (low, intermediate and high) are indicated by red threshold lines in the box plot. Percentage of spots from infected and non-infected controls in each of the nine classes is shown below

Fig 4. Determination of thresholds.

‘True amastigote’ discrimination thresholds based on their (a) ‘Corrected intensity’, and (b) ‘Spot area’ parameters were defined from the spots populations within both, infected and non-infected control wells (lognormal adjustments, blue lines; lower and upper ‘Corrected intensity’ thresholds (respectively at 48 and 274 a.u.), and the upper ‘Spot area’ cut off (51 px²), green lines). Inset in panel (a) zooms in at the spots distribution in the intermediate ‘Corrected intensity’ region (true amastigotes).

Fig 5. Individual analysis.

Controls for T. cruzi infected (I) and non-infected (N) cells of six independent plates (P1–P6) are represented: (a) box plot of the number of amastigotes per cell for each control: I (infected untreated cells) and N (non-infected cells) (16 wells of each per plate), and BNZ treated infected cells (30 μM; 16 wells in one plate) is shown. The distribution of the populations is not normal, so the median value per plate (black line) is shown. The second (white box under median), third (white box over media) and forth (dashed line) quartiles and population outliers (dots) are represented. (b) Scatter plot correlating the number of host cells to the number of amastigotes per cell. Infected cells control wells of each plate (blue lines) and BNZ treated infected cells plate (red line).

Fig 6. Drugs dose-response curves.

Representative curves of three independent experiments of BNZ, NFX, posaconazole and cyclohexymide normalized activity (%) against T. cruzi amastigotes per compound concentration (M) for each of the three assay outputs: ‘Am/Cell’ (blue), ‘%Infected’ (green) were normalized as described in [15]; ‘Cells’ (cytotoxic impact of the drugs; yellow) is expressed as percentage of live cells, considering non-treated T. cruzi infected wells as 100%.