Project Summary Toxoplasma tissue cysts form the basis for lifelong chronic infections in humans. In AIDS patients, the immune system can no longer control recrudescent parasites from brain cysts leading to uncontrolled parasite replication and Toxoplasma encephalitis (TE). New drug candidates are needed to treat TE since current drugs have toxic side effects and they do not eliminate the chronic cyst stages. It is not known why current drugs do not eliminate all brain cysts. Because recrudescent parasites from cysts can form new cysts over time, it is unclear if in each cyst there is a fraction of treatment-resistant parasites or if entire cysts are either drug resistant or susceptible. Similarly, when Toxoplasma reactivates during immunosuppression and causes TE it is not known if this is due to only some cysts reactivating or if most cysts reactivate. Because individual cysts cannot be distinguished in a pool, current methods to assess cyst dynamics during drug treatment or reactivation rely on the crude measure of following total brain cyst burden. There is, therefore, a critical need to develop methodology that allows one to follow the within host-dynamics of brain colonization and brain cysts heterogeneity during reactivation and drug treatment. Our preliminary data show that we can use genetically barcoded parasites to follow the fate of individual parasites within a host. Our central hypothesis is that heterogeneity of the parasites inside cysts determines heterogeneity in resistance to therapeutics or reactivation. In our first aim, we will use barcoded parasites expressing luciferase to follow the dynamics of cyst formation in immunocompetent mice and cyst reactivation induced by immunosuppression. This will allow us to determine what fraction of the initial Toxoplasma inoculum disseminates into the brain and subsequent brain cysts dynamics, on the individual cyst level, over time. This is critical information that will allow us to determine the complexity of loss-of-function parasite libraries that can be analyzed in future pooled screens designed to identify parasite genes that are essential for brain colonization, cyst formation, cyst persistence and parasite recrudescence. In this aim we will also determine what fraction of cysts reactivates and disseminates. In our second aim, we will determine if cyst resistance to drug treatment is due to a fraction of individual cysts being resistant or to a fraction of parasites within each cyst being resistant. These results are expected to have an important positive impact because they are expected to allow 1) a more refined assessment of the efficacy of drugs designed to eliminate chronic tissue cysts; 2) the design of future pooled loss-of-function screens to identify parasite genes that are essential for brain colonization, cyst formation, cyst persistence, or parasite recrudescence.