Novel Dense Granule Protein Function in the Chronic Toxoplasma Infection

慢性弓形虫感染中的新型致密颗粒蛋白功能

• 批准号: 9141001
• 负责人: Peter John Bradley
• 金额: $ 23.1万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-02-10 至 2018-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9141001
• 关键词: Acquired Immunodeficiency Syndrome; Acute; Binding; Biochemical; Biotin; Biotinylation; CD4 Lymphocyte Count; CRISPR/Cas technology; Cells; Chronic; Cyst; Cytoplasmic Granules; Data Set; Development; Disease; Effectiveness; Ensure; Foundations; Gene Deletion; Genes; Growth; Guide RNA; HIV; Human; Immune; Immunocompromised Host; Immunologic Surveillance; In Vitro; Infection; Invaded; Knowledge; Label; Life; Life Cycle Stages; Ligase; Maintenance; Mass Spectrum Analysis; Mediating; Membrane; Methods; Modification; Names; Neuraxis; Opportunistic Infections; Organelles; Parasites; Patients; Persons; Phenotype; Play; Process; Proteins; Proteome; Resistance; Role; Staging; Streptavidin; System; Techniques; Tissues; Toxoplasma; Toxoplasma gondii; Vacuole; XRCC5 gene; effective therapy; follow-up; gene function; immune clearance; improved; in vivo; knockout gene; loss of function; new technology; new therapeutic target; novel; novel strategies; pathogen; protein function; public health relevance; research study; rhoptry

 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.

 描述(申请人提供)：弓形虫是一种广泛传播的人类病原体，是艾滋病毒-艾滋病患者的一种重要的机会性感染。艾滋病患者的疾病通常是由维持艾滋病的寄生虫的缓殖子包囊形式重新激活的结果 宿主一生中的慢性感染。尽管慢殖子在这种慢性感染中起着核心作用，但人们对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。由于提纯含有寄生虫的液泡以分离和研究组成包囊的分泌蛋白的生化方法的局限性，这些过程的阐明一直受到阻碍。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)，它使用了一种混杂的生物素连接酶，可以标记弓形虫亚细胞间的蛋白质。我们对致密颗粒(GRA)蛋白进行了优化，这些蛋白被结构性地分泌到寄生虫的空泡中，并表明我们可以标记寄生虫快速生长的速殖子阶段的一系列空泡成分，这些成分介导了急性感染。在这一应用中，我们利用BioID对缓殖子中的空泡进行可靠的标记，从而建立在这一坚实的基础上，使我们能够填补我们对分泌到缓殖子包囊中的寄生虫成分的了解空白。我们还将把这一进展与一种改进的CRISPR/CAS9方法结合起来，该方法有助于在∆ku80株弓形虫中快速删除基因，以确定由BioID鉴定的新型缓殖子GRAs的功能。总之，这些方法将使我们能够识别并从功能上评估包囊成分，这些成分有望揭示弓形虫如何在宿主体内持续存在并在这一独特的生命周期阶段导致疾病。