Bradyzoite subtypes rule the crossroads of Toxoplasma development

Reactivation of toxoplasmosis is a significant health threat to chronically infected individuals, especially those who are or become immunocompromised. An estimated one-third of the world population is infected with Toxoplasma, placing millions at risk. The Toxoplasma cyst is the foundation of disease with its ingestion leading to infection and its reactivation, from slow replicating bradyzoites to fast replicating tachyzoites, leading to cell lysis in tissues such as the brain. There are no treatments that prevent or eliminate cysts in part due to our poor understanding of the mechanisms that underlie cyst formation and recrudescence. In this study, we aimed to understand the biology of bradyzoites prior to recrudescence and the developmental pathways they initiate. We have discovered ME49EW cysts from infected mice harbor multiple bradyzoite subtypes that can be identified by their expression of distinct proteins. Sorting of these subtypes revealed they initiate distinct developmental pathways in animals and in primary astrocyte cell cultures. Single bradyzoite RNA sequencing indicates 5 major bradyzoite subtypes occur within these cysts. We further show that a crucial subtype comprising the majority of bradyzoites in chronically infected mice is absent from conventional in vitro models of bradyzoite development. Altogether this work establishes new foundational principles of Toxoplasma cyst development and reactivation that operate during the intermediate life cycle of Toxoplasma.