Secreted effectors of Toxoplasma gondii bradyzoites

项目摘要/摘要 弓形虫是尖端复合门的一种专性细胞内寄生虫，它是一种不同的 引起人类各种疾病的寄生虫，如疟疾和隐孢子虫病。刚地弓形虫是 在世界范围内普遍存在，估计感染三分之一的人类人口，是一种公共卫生 对免疫功能受损和怀孕的人的担忧。感染的急性期是 以速殖子为特征的，这是一种快速复制的生命阶段，在人体内传播 一个热血的主人。慢性期感染的特征是缓殖子，一种缓慢的- 通过形成囊壁在肌肉和神经组织中包囊的复制生活期 一个宿主细胞。对慢殖子的生物学了解甚少；囊壁的确切成分，如何 缓殖体在宿主细胞内无限期地存在，或者缓殖体如何操纵宿主细胞的功能 未知。以前的研究已经证明了几种蛋白质效应物的分泌是通过 速殖子穿过寄生虫复制的液泡进入宿主细胞 在宿主细胞内。这些蛋白质包括GRA16、GRA24、GRA28和TgIST，其中大多数都有 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 宿主细胞信号传递级联和转录。无论是这些蛋白质，还是其他不明原因的分泌物 蛋白质，穿过囊壁，进入缓殖子感染的宿主细胞，这是以前没有的 探索过了。在这项提案中，第一个目标将是确定GRA16、GRA24、 GRA28和TgIST在体外通过表位标记分泌效应物诱导缓殖子分化 他们的内源性基因位点和体内的小鼠大脑中使用光学清除方法。在 第二个目的，将通过敲除和敲除TgIST来研究TgIST在慢殖子感染细胞中的作用。 在寄生虫中补充TgIST基因并随后确定STAT1的抑制作用 介导慢殖子感染宿主细胞的转录。在第三个目标中，基于接近的生物素 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 手机。这将允许在缓殖子阶段按质量识别分泌的效应物。 确认阳性结果后生物素化蛋白质的光谱分析。隐秘基因的发现 缓虫进入宿主细胞的效应器将挑战弓形虫领域的一种范式 研究中，缓殖子被经典地视为一个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是如何改变宿主细胞功能和 保持对主人的坚持不懈。

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.