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（由适用提供）：弓形虫Gondii是一种广泛的人类病原体，是HIV-AIDS患者的重要机会感染。艾滋病患者的疾病通常是寄生虫的胸甲囊肿形式重新激活的结果，该形式保持了 宿主一生中的慢性感染。尽管Bradyzoites在建立这种慢性感染中的核心作用，但对于介导囊肿形成和抗宿主免疫清除耐药性的分泌寄生虫因子而言，鲜为人知。阐明这些过程的生化方法的局限性阻碍了净化含寄生虫的液泡以分离和研究构成囊肿的分泌蛋白。为了克服这一障碍，我们最近改编了一种体内生物素化方法（称为生物类），该方法采用了混杂的生物素连接酶，该酶可以在T. gondii的亚细胞隔室中标记蛋白质。我们已经优化了该技术，可用于始终分泌到寄生虫液泡中的致密颗粒（GRA）蛋白，并表明我们可以从寄生虫快速生长的tachyzoite阶段的多种液泡成分标记，从而介导急性感染。在此应用程序中，我们通过使用生物剂来在Bradyzoites中对真空吸尘器的稳健标记来建立强大的基础，从而使我们能够填补对寄生虫构成的寄生虫的差距。我们还将将此前进与改进的CRISPR/CAS9方法相结合，以促进Δku80毒素菌株中快速基因缺失，以确定由生物体鉴定的新型Bradyzoite Gras的功能。这些方法将共同确定并在功能上评估囊肿成分，这些囊肿成分有望揭示如何在其宿主中持续存在的弓形虫寄生虫，并在这个独特的生命周期阶段引起疾病。