Interferon-mediated control mechanisms in human cells

人类细胞中干扰素介导的控制机制

• 批准号: 10041166
• 负责人: L. David Sibley
• 金额: $ 23.63万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-16 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10041166
• 关键词: Adult; Animals; Anti-Bacterial Agents; Antibiotics; Antiviral Agents; Autophagocytosis; Back; Binding Proteins; Binding Sites; CRISPR screen; CRISPR/Cas technology; Candidate Disease Gene; Cell Line; Cells; Chronic; Complex; Cyst; DNA Binding; Development; Disease; GBP1 gene; Genes; Growth; Hematopoietic; Human; Immune response; Immune signaling; Immune system; Immunity; Immunocompromised Host; Individual; Infection; Infection Control; Interferon Type II; Interferon-beta; Interferons; Intervention; Knock-out; Knowledge; Lead; Libraries; Life; Lymphoid Cell; Maps; Mediating; Mediator of activation protein; Molecular; Monitor; Mus; Natural Immunity; Nitrogen; Nutrient; Nutrient Depletion; Outcome Study; Oxygen; Parasite Control; Parasites; Pathway interactions; Process; Production; Proteins; Resistance; Risk; Role; Rupture; System; T-Lymphocyte; Testing; Tissues; Toxoplasma; Toxoplasma gondii; Toxoplasmosis; Up-Regulation; Vaccines; Vacuole; Zoonoses; acute infection; adaptive immunity; antigen-specific T cells; antimicrobial; base; cell type; chronic infection; effective intervention; genome wide screen; genome-wide; guanylate; high throughput analysis; immune clearance; improved; in vitro Assay; macrophage; monocyte; novel; obligate intracellular parasite; overexpression; pathogen; response; transcription factor

Abstract Toxoplasma gondii is a widespread parasite of animals that causes zoonotic infection in humans. Although acute infections in healthy adults are normally resolved without serious consequences, infection of immunocompromised individuals can lead to serious complications. Toxoplasma gondii is an obligate intracellular parasite, capable of infecting all nucleated cells in the body, including monocytes and macrophages. Control of infection relies on production of IFN-γ, which is essential to upregulate antimicrobial pathways in both hematopoietic and non-hematopoietic cells. Interferons activate STAT transcription factors to upregulate interferon stimulated genes (ISGs), many of which participate in antimicrobial defenses. Compared to studies in the murine system, our knowledge of how these pathways control T. gondii replication in human cells is relatively limited. Treatment of human cells with type I (IFN-β) or type II (IFN-γ) interferon leads to control of T. gondii replication. A variety of different control pathways have been implicated including nutrient limitation, guanylate binding proteins, autophagy, and production of reactive oxygen or nitrogen species. However prior studies have only focused on a few of the components that operate in each pathway, limiting our knowledge of their relative importance. To provide a comprehensive analysis of ISGs that are required for IFN- mediated control of T. gondii, we have developed a FACS-based screen to monitor parasite replication in different human cell types. Genome wide CRISPR/Cas9 screens will be employed to identify genes whose loss results in an inability to control T. gondii replication in IFN-treated cells. In parallel, we will use a lentiviral over- expression strategy to screen libraries of type I and type II ISGs to identify effectors that induce control of parasite replication. Collectively these approaches should define the repertoire of human genes that are necessary and sufficient to control intracellular parasites. Understanding such mechanisms may be important in overcoming the resistance to clearance seen in chronic toxoplasmosis and may also be important in control of other intracellular pathogens.

摘要 弓形虫是一种广泛存在于动物体内的寄生虫，可引起人畜共患病。虽然 健康成年人的急性感染通常会得到解决而不会产生严重后果， 免疫受损的个体可导致严重的并发症。弓形虫是一种专性 细胞内寄生虫，能够感染体内所有有核细胞，包括单核细胞， 巨噬细胞感染的控制依赖于IFN-γ的产生，这对于上调抗菌药物至关重要 造血和非造血细胞中的通路。干扰素激活STAT转录因子， 上调干扰素刺激基因（ISG），其中许多参与抗微生物防御。相比 在小鼠系统的研究，我们的知识，这些途径如何控制T。人体内弓形虫复制 细胞相对有限。用I型（IFN-β）或II型（IFN-γ）干扰素处理人细胞导致 控制T.弓形虫复制各种不同的控制途径已经牵连，包括营养 限制，鸟苷酸结合蛋白，自噬和活性氧或氮物质的产生。 然而，先前的研究只集中在每个通路中的几个组成部分，限制了我们的研究。 了解其相对重要性。为了提供IFN-γ所需的ISG的综合分析， 介导的控制T.我们已经开发了一种基于流式细胞仪的屏幕，以监测寄生虫的复制， 不同的人类细胞类型。将采用全基因组CRISPR/Cas9筛选来鉴定丢失的基因。 导致无法控制T. IFN处理的细胞中的弓形虫复制。与此同时，我们将使用一种慢病毒- 表达策略来筛选I型和II型ISG文库，以鉴定诱导控制 寄生虫复制总的来说，这些方法应该可以定义人类基因的全部功能， 必要和足够的控制细胞内寄生虫。了解这些机制可能很重要 在克服慢性弓形虫病中所见的清除抗性方面， 其他的细胞内病原体。