Novel Dense Granule Protein Function in the Chronic Toxoplasma Infection

 描述（由申请人提供）：弓形虫是一种广泛存在的人类病原体，是HIV/AIDS患者的重要机会性感染。艾滋病患者的疾病通常是由维持免疫原性的寄生虫的缓殖子包囊形式重新激活的结果。 慢性感染贯穿宿主的一生。尽管缓殖子在建立这种慢性感染中的中心作用，但关于介导包囊形成和抵抗宿主免疫清除的分泌型寄生虫因子知之甚少。阐明这些过程已受到限制的生化方法纯化含寄生虫的液泡分离和研究分泌的蛋白质，包括囊肿。为了克服这一障碍，我们最近采用了一种体内生物素化方法（称为BioID），该方法采用了一种混杂的生物素连接酶，可以标记T.刚地。我们已经优化了这种技术的致密颗粒（GRA）蛋白组成型分泌到寄生虫空泡和表明，我们可以标记一系列空泡组件从快速增长的速殖子阶段的寄生虫介导的急性感染。在这个应用程序中，我们建立在这个坚实的基础上，利用BioID在缓殖子中的液泡的强大的标签，使我们能够填补差距，我们的知识分泌到缓殖子包囊的寄生虫成分。我们还将把这一进展与一种改进的CRISPR/Cas9方法结合起来，这种方法有助于在弓形虫的Ranku 80菌株中快速基因缺失，以确定BioID鉴定的新型缓殖子GRA的功能。总之，这些方法将使我们能够识别和功能评估包囊成分，这些成分有望揭示弓形虫寄生虫如何在宿主体内持续存在并在这个独特的生命周期阶段引起疾病。

 DESCRIPTION (provided by applicant): Toxoplasma gondii is a widespread human pathogen that is an important opportunistic infection in HIV-AIDS patients. Disease in AIDS patients is typically the result of reactivation of the bradyzoite cyst form of the parasite that maintains the chronic infection throughout the life of the host. In spite of the central role of bradyzoites in establishing this chronic infection, remarkably little is known regarding the secreted parasite factors that mediate cyst formation and resistance to host immune clearance. Elucidating these processes has been hampered by limitations in the biochemical approaches for purifying the parasite-containing vacuole to isolate and study the secreted proteins that comprise the cyst. To overcome this barrier, we have recently adapted an in vivo biotinylation approach (called BioID) that employs a promiscuous biotin ligase that can label proteins in subcellular compartments in T. gondii. We have optimized this technique for dense granule (GRA) proteins that are constitutively secreted into the parasitophorous vacuole and shown that we can label a wide array of vacuolar components from the fast-growing tachyzoite stage of the parasite that mediates the acute infection. In this application, we build on this strong foundation by utilizing BioID for robust labeling of the vacuole in bradyzoites, enabling us to fill the gap in our knowledge of the parasite constituents secreted into the bradyzoite cyst. We will also couple this advance with an improved CRISPR/Cas9 approach that facilitates rapid gene deletions in ∆ku80 strains of Toxoplasma for ascertaining the function of the novel bradyzoite GRAs identified by BioID. Together, these methods will allow us to identify and functionally evaluate cyst components that promise to reveal how Toxoplasma parasites persist within their host and cause disease during this unique life-cycle stage.