Project Summary Toxoplasma gondii is one of the most successful opportunistic parasites, estimated to infect a third of the world’s population. While clinical toxoplasmosis caused by replicating tachyzoites can be treated, there are no current therapies to eliminate encysted bradyzoites in the chronic stage. Despite the central role in T. gondii pathogenesis, little is known about bradyzoite replication. Due to the lack of technologies, bradyzoites are mostly studied as a population within heterogeneous tissue cysts. Such an approach masks the intricate dynamics of parasite replication. To gain insights into chronic and reactivated toxoplasmosis and to uncover new vulnerable processes for future antiparasitic drugs, we propose to build a new tool for spatiotemporal visualization of parasite growth and development. In the current application, we will couple the unique T. gondii cell cycle with a powerful technology used in multicellular eukaryotes, Fluorescence Ubiquitination-based Cell Cycle Indicator (FUCCI). The FUCCI approach relies on controlled proteolysis of key cell cycle regulators. In Aim 1, to visualize cell cycle stages in tachyzoites, we will build a tricolor Toxo-FUCCITz probe fusing blue, green, and red fluorescent proteins with dynamic cell cycle reporters TgRRM1 (G1), TgMcm5 (S-phase and mitosis), and TgIMC3 (budding) in the developmentally competent ME49 strain. To facilitate the detection of bradyzoites, we will create a Toxo-FUCCIBz strain in largely the same configuration, replacing only TgRRM1 with a bradyzoite-specific marker TgLDH2. We will use our T. gondii FUCCI probes to observe how tachyzoites enter a drug-induced cell cycle block (Aim 2.1). Using time-lapse microscopy and flow cytometry, we will define basic parameters of the bradyzoite cell cycle during in vitro differentiation (Aim 2.2). Altogether, the proposed experiments will establish an advanced technology to study the dynamics of parasite growth and development, to dissect complex populations of parasites, and to screen for antiparasitic drugs. Our studies offer an alternative and effective approach to dissect the mechanisms of chronic toxoplasmosis.