Secreted effectors of Toxoplasma gondii bradyzoites

弓形虫缓殖子的分泌效应子

• 批准号: 9855290
• 负责人: Joshua Alexander Mayoral
• 金额: $ 2.66万
• 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/9855290
• 关键词: Secreted; effectors; Toxoplasma; gondii; bradyzoites

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.

项目总结/摘要 弓形虫是一种专性细胞内寄生虫的门顶复门，一个多样化的群体， 引起人类各种疾病的寄生虫，如疟疾和隐孢子虫病。T.弓形虫是 在世界范围内普遍存在，估计感染三分之一的人口，是一个公共卫生 免疫功能低下和怀孕的人的关注。感染的急性期是 以速殖子为特征，速殖子是一种快速复制的生命阶段， 一个热血的宿主感染的慢性期以缓殖子为特征，缓殖子是一种缓慢的- 通过在肌肉和神经组织内形成囊壁而包囊的复制生命阶段 宿主细胞缓殖子生物学知之甚少;囊壁的确切组成，如何 缓殖子在宿主细胞内无限期地存在，或者缓殖子如何操纵宿主细胞功能， 未知以前的研究已经证明了几种蛋白质效应物的分泌， 速殖子穿过寄生虫的空泡进入宿主细胞，空泡是寄生虫复制的地方 在宿主细胞内。这些包括蛋白质GRA 16、GRA 24、GRA 28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，还是其他未知的分泌物 蛋白质，穿过囊壁并进入缓殖子感染的宿主细胞，以前没有 探讨了在这个建议中，第一个目标将是确定GRA 16，GRA 24， GRA 28和TgIST在通过表位标记分泌的效应子体外分化后， 它们的内源性位点和在小鼠脑中的体内使用光学清除方法。在 第二个目的，将通过敲除和转染研究TgIST在缓殖子感染细胞中的作用。 补充寄生虫中的TgIST基因，随后确定STAT 1的抑制 在缓殖子感染的宿主细胞中介导转录。在第三个目标中，基于邻近的生物素 标记酶将与MYR 1融合，MYR 1是一种与寄生虫蛋白质输出到宿主中有关的蛋白质 cell.这将允许通过质量鉴定缓殖子阶段期间分泌的效应物 在确认阳性命中后，生物素化蛋白的光谱分析。秘密的发现 缓殖子进入宿主细胞的效应器将挑战弓形虫领域的范式 研究，其中缓殖子已被经典地视为一个惰性的，但传输阶段的 寄生虫这将导致进一步的研究，了解缓殖子如何改变宿主细胞的功能， 在它们的宿主中保持持久性。