Toxoplasma gondii is an apicomplexan protozoan parasite that can infect all warm blooded animals ranging from reptiles, birds to mammals including humans. T. gondii causes disease ranging from asymptomatic to influenza-like in adults, to severely morbid or fatal in the developing fetus and immunocompromised patients. In addition to candidiasis and kaposi sarcoma, toxoplasmosis is considered as prominent opportunistic infection in HIV patients leading to their death. During its complex life cycle, T. gondii exhibits three morphologically distinct infectious stages: tachyzoïtes, bradyzoïtes, and sporozoïtes. The tachyzoïtes are slender, crescentic types that represent the rapidly multiplying forms responsible for the acute phase of infection leading to the tissue damage. The bradyzoïtes are shorter and slow-growing forms leading to tissue cysts in the brain and skeletal muscles, and represent the dangerous form that reactivates back to tachyzoites once the immune system is affected. And lastly the sporozoïtes are the infective forms found in sporulated oocysts. The back and forth switch between the tachyzoïte and bradyzoïte stages is a key modulator of the status of toxoplasmosis and its progression between acute and chronic phases. However, this switch remains very poorly understood. In this current thesis, we focus on three bradyzoite markers (P18, P21 and P34). On one hand, the gene encoding for P18 has been already sequenced and we aim at establishing the construct harboring the 3’ and 5’ flanking regions of the gene to knock it out. On the other hand, the genes encoding for P21 and P34 are not yet identified. We aim at characterizing them in a retrospective manner going from the protein back to the gene. In more details, our approach consists of immunoprecipitating both proteins, each on its appropriate monoclonal antibody in order to prepare them for mass spectrometry analysis. This later will allow identifying their encoding genes on Toxoplasma database www.toxoDB.org.
Toxoplasma gondii (T.gondii) is an obligate intracellular protozoan parasite that can infect all warm blooded animals and humans and that exhibits three morphologically distinct infectious stages: tachyzoïtes, bradyzoïtes, and sporozoïtes. T. gondii causes disease ranging from asymptomatic to influenza-like in adults, to severely morbid or fatal in the developing fetus and immunocompromised patients. In addition to candidiasis and kaposi sarcoma, toxoplasmosis is considered as prominent opportunistic infection in HIV patients leading to their death. A 21 KDa late bradyzoite specific surface protein has an unknown function. One of our aims is to understand its role and unravel its encoding gene. In order to achieve this objective, we first aimed at optimizing the best bradyzoite producing murine model to get the greatest number for immunopurification. To do so, we first started by reproducing a successful murine model of chronic toxoplasmosis. We then optimized the purification of cysts from different types of mice (Swiss Webster, balb-c and black-6) upon injection of two doses of type II strains of T.gondii (PRU and PRU ΔKU80). We then quantified the number of cysts in these three mice strains in order to determine the best animal model of chronic toxoplasmosis that leads to the best yield of cysts in the brain and therefore to the best amount of bradyzoites to be used for immunoprecipitation of p21. All of this resulted in reproducing a murine model for toxoplasmosis and revealed several studies to be done regarding the immune response of the host and prepared the path to start purifying p21 for its characterization.
Key words: Toxoplasma gondii; bradyzoites; cysts formation; Swiss Webster; Balb-c, Black-6 mice, BAG-1.