More than 30% of the world’s population harbors a chronic parasitic infection that cannot be eliminated with current treatments. While chronic infection with Toxoplasma gondii has typically been considered asymptomatic growing evidence suggests that the parasite can have long term consequences to human health, including increased risk of neurological disorders and muscular dysfunction. The presence of T. gondii cysts within organs including the brain, eyes, heart and skeletal muscles likely underpin these pathologies. The long-term goal of this work is to identify novel therapeutics to eliminate all the cysts in a chronically infected host. However, very little is known about T. gondii cysts outside the brain and how they are able survive within these diverse tissues. The goal of this project is to better understand how the parasite survives within these tissues and how the host responds to their presence to identify a core set of proteins likely essential for survival within all clinically relevant tissues. This will allow us to strategically pursue targets able to eliminate cysts throughout the host not just a subset. We will use single cell transcriptional profiling of parasites isolated from different tissues and spatial transcriptomics of these tissues to achieve this goal. Finally, we will tests a set of proteins known to be important for cyst formation in vitro and/or in the brains of mice for their essentiality in cyst development and maintenance across all chronically infected tissues. Combined these data will provide (1) valuable, specific, candidates that are required for chronic parasite survival and (2) proof of concept for methods to tests the candidates for their function in vitro. This will be used to support an R01 grant application to identify specific vulnerabilities of the cysts that could be exploited in drug intervention strategies.