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

用于弓形虫生长和发育时空可视化的新报告系统

• 批准号: 10307135
• 负责人: Elena Suvorova
• 金额: $ 7.48万
• 依托单位: UNIVERSITY OF SOUTH FLORIDA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-11-23 至 2023-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10307135
• 关键词: Alveolar; Antiparasitic Agents; Biology; CRISPR/Cas technology; Cell Cycle; Cell Cycle Stage; Cell Separation; Chronic; Chronic Disease; Clinical; Complex; Cyan Fluorescent Protein; Cyst; Daughter; Detection; Development; Disease; Disease Progression; Dissection; Drug Targeting; Drug resistance; Eukaryota; Flow Cytometry; Fluorescence; Future; G1 Phase; Growth; Growth and Development function; Health; Human; Immune response; Immunocompromised Host; In Vitro; Individual; Infection; Kinetics; Length; Life; Light; Link; Maps; Masks; Microscopy; Mitosis; Mitotic; Monitor; Organism; Parasites; Pathogenesis; Pharmaceutical Preparations; Phase; Phosphotransferases; Population; Population Heterogeneity; Process; Proteins; Proteolysis; RNA Splicing; Reporter; Research; Resistance; Resolution; Role; S phase; Source; Stress; Surface; System; Technology; Testing; Therapeutic; Time; Tissues; Toxoplasma gondii; Toxoplasmosis; Ubiquitination; Visualization; alkalinity; base; experimental study; insight; new technology; novel; opportunistic pathogen; phase change; preservation; pyrrolidine dithiocarbamate; red fluorescent protein; spatiotemporal; time use; tool

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.

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 人口虽然由复制速殖子引起的临床弓形虫病可以治疗，但目前还没有治疗方法。 治疗以消除慢性阶段的包囊缓殖子。尽管在T. 弓形虫的发病机制，对缓殖子复制知之甚少。由于缺乏技术， 大多数研究是作为异质组织囊肿内的群体。这种做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再活化弓形虫病， 脆弱的过程，为未来的抗寄生虫药物，我们建议建立一个新的工具， 寄生虫生长和发育的可视化。在当前的应用中，我们将耦合唯一的T。 用一种功能强大的技术将弓形虫细胞周期应用于多细胞真核生物，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水解。 在目标1中，为了可视化速殖子中的细胞周期阶段，我们将构建融合蓝色的三色Toxo-FUCCITz探针， 绿色和红色荧光蛋白与动态细胞周期报告基因TgRRM 1（G1）、TgMcm 5（S期和 有丝分裂）和TgIMC 3（出芽）。为了便于检测 我们将以基本相同的构型创建Toxo-FUCCIBz菌株，仅用 缓殖子特异性标记TgLDH 2。我们将使用T。弓形虫FUCCI探针观察速殖子如何进入 药物诱导的细胞周期阻滞（目标2.1）。使用延时显微镜和流式细胞术，我们将定义基本的 在体外分化过程中缓殖子细胞周期的参数（目标2.2）。总的来说， 实验将建立一种先进的技术来研究寄生虫生长和发育的动力学， 解剖复杂的寄生虫种群，筛选抗寄生虫药物。我们的研究提供了一个替代方案 是剖析慢性弓形虫病发病机制的有效途径。