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Abstract 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 withToxoplasma, placing millions at risk. TheToxoplasmacyst 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 ofToxoplasmacyst development and reactivation that operate during the intermediate life cycle ofToxoplasma. 

ABSTRACT The recrudescence ofToxoplasmacysts is the cause of clinical disease in the immunocompromised. AlthoughToxoplasmahas been a useful parasite model for decades because it is relatively easy to genetically modify and culture, attempts to generate and study the recrudescence of tissue cysts have come up short with cell culture-adapted strains generating low numbers of tissue cystsin vivo. Taking advantage of a newex vivomodel ofToxoplasmarecrudescence that uses a Type II ME49 strain unadapted to cell culture, we determined the cell biology, gene expression, and host cell dependency that define bradyzoite-cyst reactivation. Bradyzoite infection of fibroblasts and astrocytes produced sequential tachyzoite growth stages with pre-programmed kinetics; thus, an initial fast-growing stage was followed by a slow-growing replicating form.In vivoinfections demonstrated that only fast growth tachyzoites, and not parasites post-growth shift, led to successful parasite dissemination to the brain and peripheral organs. In astrocytes, cells that reside in the central nervous system (CNS), bradyzoites initiated an additional recrudescent pathway involving brady-brady replication, which is a pathway not observed in fibroblasts. To investigate the molecular basis of growth and cell-dependent reactivation pathways, single-cell mRNA sequencing was performed on recrudescing parasites, revealing distinct gene signatures of these parasite populations and confirming multifunctionality of the originalex vivobradyzoite population. This revised model ofToxoplasmarecrudescence uncovers previously unknown complexity in the clinically important bradyzoite stage of the parasite, which opens the door to further study these novel developmental features of theToxoplasmaintermediate life cycle. IMPORTANCEThe classical depiction of theToxoplasmalifecycle is bradyzoite excystation conversion to tachyzoites, cell lysis, and immune control, followed by the reestablishment of bradyzoites and cysts. In contrast, we show that tachyzoite growth slows independent of the host immune response at a predictable time point following excystation. Furthermore, we demonstrate a host cell-dependent pathway of continuous amplification of the cyst-forming bradyzoite population. The developmental plasticity of the excysted bradyzoites further underlines the critical role the cyst plays in the flexibility of the lifecycle of this ubiquitous parasite. This revised model ofToxoplasmarecrudescence uncovers previously unknown complexity in the clinically important bradyzoite stage of the parasite, which opens the door to further study these novel developmental features of theToxoplasmaintermediate life cycle.