New reporter system for spatiotemporal visualization of Toxoplasma gondii growth and development

项目摘要 弓形虫是最成功的机会性寄生虫之一，估计感染了世界上三分之一的 人口。虽然复制速殖子引起的临床弓形虫病是可以治疗的，但目前还没有 慢性期消灭囊状缓殖子的治疗方法。尽管在T. 关于弓形虫的发病机制，人们对缓殖子复制知之甚少。由于缺乏技术，缓殖子是 主要作为异质组织内的一个种群进行研究。这种方法掩盖了错综复杂的 寄生虫复制的动力学。深入了解慢性和复活型弓形虫病，并发现新的 对于未来抗寄生虫药物的脆弱过程，我们建议构建一种新的时空分析工具 寄生虫生长发育的可视化。在当前的应用程序中，我们将把唯一的T。 刚地用强大的细胞周期技术应用于多细胞真核生物中，基于荧光泛素化 细胞周期指标(Fucci)。Fucci方法依赖于关键细胞周期调节因子的受控蛋白质分解。 在目标1中，为了可视化速殖子的细胞周期阶段，我们将建立一个三色弓形虫-FUCCITz探针融合蓝色， 绿色和红色荧光蛋白与动态细胞周期报告TgRRM1(G1)，TgMcm5(S期和 有丝分裂)和TgIMC3(萌芽)。为了便于检测到 缓殖子，我们将创建一个基本相同配置的Toxo-FUCCIBz菌株，只用TgRRM1替换 缓殖子特异性标记TgLDH2。我们将使用我们的弓形虫Fucci探针观察速殖子是如何进入的 药物诱导的细胞周期阻滞(AIM 2.1)。使用时间推移显微镜和流式细胞术，我们将定义基本的 体外分化过程中缓殖子细胞周期的参数(目标2.2)。总而言之，建议的 实验将建立一种先进的技术来研究寄生虫的生长和发育动态， 解剖复杂的寄生虫种群，并筛选抗寄生虫药物。我们的研究提供了另一种选择 是剖析慢性弓形虫病发病机制的有效途径。

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.