Development of an in vitro system to study the developmental stages of Toxoplasma gondii using a genetically modified strain expressing markers for tachyzoites and bradyzoites.

Toxoplasma gondii, the agent of toxoplasmosis, is an intracellular parasite that can infect a wide range of vertebrate hosts. Toxoplasmosis causes severe damage to immunocompromised hosts and its treatment is mainly based on the combination of pyrimethamine and sulfadiazine, which causes relevant side effects primarily observed in AIDS patients, including bone marrow suppression and hematological toxicity (pyrimethamine) and/or hypersensitivity and allergic skin reactions (sulfadiazine). Thus, it is important to investigate new compounds against T. gondii, particularly those that may act on bradyzoites, which are present in cysts during the chronic disease phase. We propose an in vitro model to simultaneously study new candidate compounds against the two main causative stages of Toxoplasma infection in humans, using the EGS-DC strain that was modified from a type I/III strain (EGS), isolated from a case of human congenital toxoplasmosis in Brazil and engineered to express markers for both stages of development. One feature of this strain is that it presents tachyzoite and bradyzoite in the same culture system and in the same host cell under normal culture conditions. Additionally, this strain presents stage-specific fluorescent protein expression, allowing for easy identification of both stages, thus making this strain useful in different studies. HFF cells were infected and after 4 and 7 days post infection the cells were treated with 10 μM of pyrimethamine or atovaquone, for 48 or 72 h. We used high-throughput screening to quantify the extent of parasite infection. Despite a reduction in tachyzoite infection caused by both treatments, the atovaquone treatment reduced the bradyzoite infection while the pyrimethamine one increased it. Ultrastructural analysis showed that after treatment with both drugs, parasites displayed altered mitochondria. Fluorescence microscopy of cells labeled with MitoTracker CMXRos showed that the cysts present inside the cells lost their mitochondrial membrane potential. Our results indicate that this experimental model is adequate to simultaneously analyze new active compounds against tachyzoite and bradyzoite forms.