Summary Toxoplasma gondii is an obligate intracellular apicomplexan parasite causing severe opportunistic infections. Current drugs are prone to induce hypersensitivity, especially upon long-term use. Infection comprises a short, acute stage (tachyzoite) followed by a dormant stage (bradyzoites in tissue cysts) for the life of the host. However, an impaired or suppressed immune response (e.g., AIDS, organ transplant, cancer treatment) can lead to reactivation of a dormant infection that was acquired years ago, leading to clinical toxoplasmosis. Interference with the tachyzoite-bradyzoite interconversion is an attractive target for novel therapeutic strategies. A key factor in stage interconversion is the ability to respond to stress: the immune response ‘stress’ maintains the bradyzoite stage, but if it wanes, the parasite defaults back to the acute stage. Reprogramming toward the bradyzoite is facilitated by the competitive interplay of about 10 transcription factors (TFs) balancing sequential, cooperative, and opposing roles. The research team recently discovered that the TF set associated with bradyzoite differentiation overlaps by 2/3 with the TFs expressed in parasites exposed to the extracellular (e.c.) environment. This provided the first glimpse of how the stress response is integrated into the transcriptional program leading to the bradyzoite. Indeed, the transcriptome of e.c. and bradyzoite parasites shares many genes, which positions the extracellular stage in between tachyzoites and bradyzoites. Here, the stress-related transcriptional program will be further dissected by extending on this observation with innovative CUT&RUN, single cell expression profiling (scRNA-seq), and the application of a novel in vitro generated myotube bradyzoite differentiation system. Excitingly, this myotube system demonstrated that spontaneous bradyzoite differentiation occurs in this cell type, but to reach and maintain a mature bradyzoite state, an exogenous, alkaline stress needs to be applied. Hence, this provides the perfect platform to dissect the contribution of stress-related transcriptional regulation to bradyzoite maturation. In short, the transcriptional network of stress will be dissected by 1. identifying the genes controlled by the 4-6 TFs shared between e.c. stress and bradyzoite conversion through CUT&RUN experiments; 2. scRNA-seq of mixed cell populations representing the transcriptional transitions of extracellular stress over the span of 6 hrs, and myotube-induced bradyzoite differentiation with and without alkaline stress. An advanced computational pipeline will be established to model the transitional programs that will permit answers to several open questions, such as: is there indeed a two-step bradyzoite differentiation pathway, and what are the genes driving these?; is the myotube seen as a stress by the parasites or not?; is there a specific stage in the cell cycle when bradyzoite differentiation occurs (a G2-like cell cycle stag...