In vitro reactivation of chronic-stage Toxoplasma gondii parasites

In the intracellular apicomplexan parasite Toxoplasma gondii, differentiation from proliferative to chronic, cyst-forming life stages facilitates stress tolerance, immune evasion, and environmental persistence. Differentiation is accompanied by global changes in parasite gene expression and regulation, but tissue cysts retain the capacity to reactivate and resume rapid growth in immunocompromised hosts, which largely drives the morbidity associated with Toxoplasma infection. Our lab previously demonstrated that BFD1, a Myb-like transcription factor required for differentiation, is upregulated under stress via a positive feedback loop involving one of its targets, the RNA-binding protein BFD2. However, the molecular pathways underlying reactivation, including the role of the BFD1-BFD2 circuit, remain poorly understood. We established a genetic model to study Toxoplasma reactivation in vitro by introducing an additional, conditionally stabilizable copy of BFD2 (DD-BFD2) into differentiation-competent ME49 parasites. Similar to conditional BFD1 stabilization (DD-BFD1) in Δbfd1 parasites, this new strain reliably differentiates and expresses canonical markers of early and late bradyzoite development. Using stage-specific fluorescent reporters, we demonstrate that DD-BFD2 stabilization induces bradyzoite reporter expression to a higher magnitude and with greater heterogeneity compared to DD-BFD1 stabilization, suggesting that DD-BFD2 parasites explore a broader range of cellular states during differentiation. Critically, DD-BFD2 destabilization in chronic-stage parasites produces a subpopulation of cysts that remain differentiated over time, consistent with long-term maintenance of the endogenous BFD1-BFD2 positive feedback loop. Under these conditions, we simultaneously observe the emergence of a rapidly growing tachyzoite population distinct from mature cysts. Conversely, DD-BFD1 destabilization in Δbfd1 cysts leads to a rapid switch in stage-specific reporter expression. These observations highlight the role of heterogeneity in bradyzoite development and imply that reactivating parasites may originate from an as-yet-undefined parasite subpopulation.