High-content imaging assay to evaluate Toxoplasma gondii infection and proliferation: A multiparametric assay to screen new compounds.

Bastien Touquet,Wei Yi,Delphine Aldebert,Pierre Cavailles,Corinne Mercier,Valeria Bellini,Marie-France Cesbron-Delauw,Léonie Pelissier,Ahcène Boumendjel,Silvia N Moreno

doi:10.1371/journal.pone.0201678

Abstract

Toxoplasma gondii is an intracellular protozoan parasite widely distributed in animals and humans. Infection of host cells and parasite proliferation are essential steps in Toxoplasma pathology. The objective of this study was to develop and validate a novel automatic High Content Imaging (HCI) assay to study T. gondii infection and proliferation. We tested various fluorescent markers and strategies of image analysis to obtain an automated method providing results comparable to those from gold standard infection and proliferation assays. No significant difference was observed between the results obtained from the HCI assay and the standard assays (manual fluorescence microscopy and incorporation of [3H]-uracil). We developed here a robust and time-saving assay. This automated technology was then used to screen a library of compounds belonging to four classes of either natural compounds or synthetic derivatives. Inhibition of parasite proliferation and host cell toxicity were measured in the same assay and led to the identification of one hit, a thiosemicarbazone that allows important inhibition of Toxoplasma proliferation while being relatively safe for the host cells.

Highlights

Severe cases of toxoplasmosis resulting from infection with atypical genotypes of Toxoplasma strains have been recently observed in immune-competent subjects [2]
The most common therapeutic medicines used against the parasite are 1) spiramycin in pregnant women and 2) combination therapies such as pyrimethamine-sulfadoxine, pyrimethamine-sulfadiazine and trimethoprim-sulfamethoxazole in immune-deficient patients or in severe cases of toxoplasmosis
Validation of High Content Imaging assay in comparison with gold standard assays Human foreskin fibroblasts are currently used as a host model to study Toxoplasma infection in vitro

Summary

Introduction

Toxoplasma gondii is an obligate intracellular protozoan parasite responsible for toxoplasmosis, an infection which is asymptomatic in more than 80% of immune-competent subjects [1]. Severe cases of toxoplasmosis resulting from infection with atypical genotypes of Toxoplasma strains have been recently observed in immune-competent subjects [2]. The most common therapeutic medicines used against the parasite are 1) spiramycin in pregnant women and 2) combination therapies such as pyrimethamine-sulfadoxine, pyrimethamine-sulfadiazine and trimethoprim-sulfamethoxazole in immune-deficient patients or in severe cases of toxoplasmosis. These treatment regimens cannot always be used due to diverse reasons, such as intolerance, problems related to drug absorption or resistance of the parasite. Drug toxicity due to sulfadiazine hypersensitivity further complicates the life-long prophylactic treatment of immunocompromised patients as well as treatment during pregnancy [3]

Objectives

Methods

Results

Discussion

Conclusion