Secreted effectors of Toxoplasma gondii bradyzoites

弓形虫缓殖子的分泌效应子

• 批准号: 9762554
• 负责人: Joshua Alexander Mayoral
• 金额: $ 3.31万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9762554
• 关键词: Acute; Apicomplexa; Biology; Biotin; Blood; Brain; C-terminal; Cell Nucleus; Cell physiology; Cells; Chimeric Proteins; Chronic; Chronic Phase; Complement; Cryptosporidiosis; Cyst; Cytoplasm; Cytoplasmic Granules; Data; Disease; Encephalitis; Engineering; Enzymes; Epitopes; Exposure to; Future; Genes; Genetic Transcription; Goals; Harvest; Human; IRF1 gene; Image; Immunocompromised Host; In Vitro; Individual; Infection; Interferons; Intervention; Knock-out; Knowledge; Label; Laboratories; Life; Ligase; Light; Malaria; Mass Spectrum Analysis; Mediating; Medical; Membrane; Mus; Muscle; Myrosinase; N-terminal; Neuraxis; Neurons; Optics; Organelles; Organism; Parasites; Peroxidases; Phase; Population; Pregnancy Complications; Protein Export Pathway; Proteins; Proteomics; Public Health; Publishing; Research; Role; STAT1 gene; Serological; Signal Transduction; Site; Structure; Therapeutic; Time; Tissues; Toxoplasma; Toxoplasma gondii; Toxoplasmosis; Up-Regulation; Vacuole; Validation; Work; acute infection; ascorbate; base; chronic infection; congenital infection; fetal; in vivo; novel; obligate intracellular parasite; pregnant; relating to nervous system; small molecule; transcriptomics

Project Summary/Abstract Toxoplasma gondii is an obligate intracellular parasite of the phylum Apicomplexa, a diverse group of parasites that cause various diseases in humans, such as malaria and cryptosporidiosis. T. gondii is ubiquitous worldwide, estimated to infect one-third of the human population, and is a public health concern in immunocompromised and pregnant individuals. The acute phase of infection is characterized by the tachyzoite, a fast-replicating life stage that disseminates throughout the body of a warm-blooded host. The chronic phase of infection is characterized by the bradyzoite, a slowly- replicating life stage which encysts in muscle and neural tissue via the formation of a cyst wall within a host cell. Bradyzoite biology is poorly understood; the exact composition of the cyst wall, how bradyzoites persist within host cells indefinitely, or how bradyzoites manipulate host cell function is unknown. Previous studies have demonstrated the secretion of several protein effectors by tachyzoites into their host cells across the parasitophorous vacuole, the site where parasites replicate within host cells. These include the proteins GRA16, GRA24, GRA28, and TgIST, most of which have been shown to localize to the host cell nucleus and directly interact with host cell proteins, altering host cell signaling cascades and transcription. Whether these proteins, or other unidentified secreted proteins, cross the cyst wall and enter bradyzoite infected host cells has not been previously explored. In this proposal, the first aim will be to determine the localization of GRA16, GRA24, GRA28, and TgIST after bradyzoite differentiation in vitro by epitope tagging secreted effectors at their endogenous loci and in vivo in the mouse brain using an optical clearing approach. In the second aim, the role of TgIST in bradyzoite infected cells will be investigated by knocking out and complementing the TgIST gene in the parasite and subsequently determining the inhibition of STAT1 mediated transcription in bradyzoite infected host cells. In the third aim, proximity-based biotin labeling enzymes will be fused to MYR1, a protein implicated in parasite protein export into the host cell. This will allow the identification of secreted effectors during the bradyzoite stage by mass spectrometry of biotinylated proteins following validation of positive hits. The discovery of secreted effectors by bradyzoites into host cells would challenge a paradigm in the field of Toxoplasma research, where the bradyzoite has classically been viewed as an inert but transmissible stage of the parasite. This will lead to further studies on understanding how bradyzoites alter host cell function and maintain persistency in their hosts.

项目概要/摘要 弓形虫是顶复门的专性细胞内寄生虫，顶复门是一个不同的群体 引起人类各种疾病的寄生虫，例如疟疾和隐孢子虫病。弓形虫是 在世界范围内普遍存在，估计会感染三分之一的人口，并且是一种公共卫生问题 免疫功能低下和孕妇的担忧。感染的急性期是 以速殖子为特征，这是一个快速复制的生命阶段，可传播到整个身体 一个热血的主人。感染的慢性阶段的特点是缓殖子，一种缓慢的 复制生命阶段，通过在肌肉和神经组织内形成囊壁来包囊 宿主细胞。人们对缓殖子的生物学知之甚少；囊肿壁的确切组成，如何 缓殖子无限期地存在于宿主细胞内，或者说缓殖子如何操纵宿主细胞功能 未知。先前的研究已经证明了几种蛋白质效应物的分泌 速殖子穿过寄生液泡进入宿主细胞，寄生液泡是寄生虫复制的场所 宿主细胞内。其中包括蛋白质 GRA16、GRA24、GRA28 和 TgIST，其中大多数具有 已被证明定位于宿主细胞核并直接与宿主细胞蛋白质相互作用，改变 宿主细胞信号级联和转录。无论是这些蛋白质，还是其他不明的分泌物 蛋白质，穿过包囊壁并进入缓殖子感染的宿主细胞，以前从未有过 探索过。在该提案中，首要目标是确定 GRA16、GRA24、 GRA28 和 TgIST 在体外通过表位标记分泌效应器分化缓殖子后 使用光学清除方法在小鼠大脑中研究它们的内源性基因座和体内。在 第二个目标是通过敲除和研究 TgIST 在缓殖子感染细胞中的作用。 补充寄生虫中的 TgIST 基因并随后确定 STAT1 的抑制作用 介导缓殖子感染宿主细胞中的转录。在第三个目标中，基于邻近性的生物素 标记酶将与 MYR1 融合，MYR1 是一种与寄生虫蛋白输出到宿主有关的蛋白质 细胞。这将允许通过质量鉴定缓殖子阶段的分泌效应子 验证阳性命中后，对生物素化蛋白质进行光谱测定。分泌物的发现 缓殖子进入宿主细胞的效应器将挑战弓形虫领域的范例 研究中，缓殖子传统上被视为惰性但可传播的阶段 寄生虫。这将导致进一步研究了解缓殖子如何改变宿主细胞功能和 保持宿主的持久性。