Despite their critical role in chronic toxoplasmosis, the biology of Toxoplasma gondii bradyzoites is poorly understood. In an attempt to address this gap, we optimized approaches to purify tissue cysts and analyzed the replicative potential of bradyzoites within these cysts. In order to quantify individual bradyzoites within tissue cysts, we have developed imaging software, BradyCount 1.0, that allows the rapid establishment of bradyzoite burdens within imaged optical sections of purified tissue cysts. While in general larger tissue cysts contain more bradyzoites, their relative “occupancy” was typically lower than that of smaller cysts, resulting in a lower packing density. The packing density permits a direct measure of how bradyzoites develop within cysts, allowing for comparisons across progression of the chronic phase. In order to capture bradyzoite endodyogeny, we exploited the differential intensity of TgIMC3, an inner membrane complex protein that intensely labels newly formed/forming daughters within bradyzoites and decays over time in the absence of further division. To our surprise, we were able to capture not only sporadic and asynchronous division but also synchronous replication of all bradyzoites within mature tissue cysts. Furthermore, the time-dependent decay of TgIMC3 intensity was exploited to gain insights into the temporal patterns of bradyzoite replication in vivo. Despite the fact that bradyzoites are considered replicatively dormant, we find evidence for cyclical, episodic bradyzoite growth within tissue cysts in vivo. These findings directly challenge the prevailing notion of bradyzoites as dormant nonreplicative entities in chronic toxoplasmosis and have implications on our understanding of this enigmatic and clinically important life cycle stage.

SUMMARY The endothelial specific receptor tyrosine kinase Tie2, and the orphan receptor tyrosine kinase, Tie1, are essential for endothelial cell proliferation, migration, and survival during angiogenesis. Despite their considerable similarity, experiments with Tie1 or Tie2 deficient mice highlight distinct functions for these two receptors in vivo. However, Tie1 cooperates with Tie2 during Angiopoietin signaling, demonstrating a degree of functional overlap between the two receptor-systems. Tie2 is further unique among receptor tyrosine kinases with respect to its structurally homologous ligands. Angiopoietin-2 and -3 can function as agonists or antagonists depending upon the local environment, while Angiopoietin-1 and -4, are constitutive agonists. To address the role of Tie1 in Angiopoietin-mediated Tie2 signaling, and determine the basis for the unique behavior of the individual Angiopoietins, we used an in vivo FRET-based proximity assay to monitor Tie1 and Tie2 localization and association in the presence and absence of ligands. Here, we provide direct evidence for Tie1-Tie2 complex formation on the endothelial cell surface and identify molecular surface areas essential for receptor-receptor recognition. We further demonstrate that the Tie1-Tie2 interactions are dynamic, inhibitory, and differentially modulated by Angiopoietin-1 and -2, thereby providing a discrete molecular mechanism for the observed variations in Angiopoietin function. Based upon the available data, we propose a unified model for Angiopoietin-induced Tie2 signaling which highlights the essential role of Tie1 in vascular homeostasis.

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