Automated High-Content Assay for Compounds Selectively Toxic to Trypanosoma cruzi in a Myoblastic Cell Line

Abstract

BackgroundChagas 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 FindingsGenetically 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/SignificanceWe 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.

A flow cytometer-based method to simultaneously assess activity and selectivity of compounds against the intracellular forms of Trypanosoma cruzi

“…In this sense, image based approaches have been developed and refined in recent years that allow to screen thousands of molecules in short term informative assays that are fully automated and allow to test diverse T. cruzi clinical isolates (Moon et al, 2014, Engel et al, 2010, Alonso-Padilla et al, 2015. Some of them have been designed to simultaneously evaluate the toxicity of candidates based on counting total host cell nuclei using DNA intercalating dyes on previously fixed cells.…”

Uncertainty in cell confluency measurements

“…Assays are constantly under development to improve, make faster and simplify the data analysis. As clearly stated by Xia and Wong [2], HCS is considered a "cellular image-based high-throughput screening" (HTS): the combination of HTS and microscopy led to the HCS technology which allows automated acquisition and analysis of data coming from cell behaviors which are highly informative about what it is happening in the cell culture [2,1,3]. In a HCS, each single cell is specifically and fluorescently stained to be analyzed at a sub-cellular level (micrometer resolution) [4].…”

“…In addition, it is a promising methodology for personalized medicine and stem cells research [4,2,3]. HCS allow quick evaluation of the effects of thousands of molecules on cells and provide starting points for drug design and for understanding the interaction or role of particular biochemical processes [5].…”

“…Cell confluency is measured in cell cultures to monitor the cell growth during a treatment and to modulate the subculture in 2D and 3D cell culture [16,17,18,19], to perform toxicity tests with chemical or drug treatment at a certain cell confluency % [20,21], to measure tumor cell proliferation in vitro [22], in drug development to find cytoplasm abnormalities discriminating between areas with cells (occupied by cells) and areas no cells (background) [3]. Cell confluency has been found to affect the expression of cell surface markers in studies on murine stem cells [23], to have effect on the cell stiffness [24], intracellular forces [25] and on the cell cycle [26], to have a critical impact in gene expression in adipose stromal cells [27].…”

“…The confluency measurement is obtained through analysis of images acquired via microscopy techniques [3,9]. HCS integrates fluorescence microscopy and algorithms for image analysis to automate cell analysis [6].…”