Interferon-mediated control mechanisms in human cells

摘要 弓形虫是一种广泛存在的动物寄生虫，可引起人畜共患传染病。虽然 健康成年人的急性感染通常会得到缓解，不会有严重的后果，感染 免疫功能受损的人可能会导致严重的并发症。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，包括单核细胞和 巨噬细胞。感染的控制依赖于干扰素-γ的产生，而干扰素-DNA是上调抗菌药物的关键 在造血细胞和非造血细胞中的通路。干扰素激活STAT转录因子以 上调干扰素刺激基因(ISGs)，其中许多基因参与抗菌防御。相比较 对小鼠系统的研究，我们对这些途径如何控制人类弓形虫复制的了解 细胞是相对有限的。用I型(干扰素-β)或II型(干扰素-γ)干扰素处理人细胞可导致 弓形虫复制的控制。包括营养在内的多种不同的调控途径都已被涉及。 限制，鸟苷结合蛋白，自噬，以及产生活性氧或氮物种。 然而，以前的研究只集中在每条通路中的几个组件上，限制了我们的 了解它们的相对重要性。提供干扰素所需的ISGs的全面分析- 为了控制弓形虫，我们已经开发了一种基于FACS的屏幕来监控寄生虫的复制 不同的人类细胞类型。全基因组CRISPR/Cas9筛查将被用于识别其丢失的基因 导致无法控制经干扰素处理的细胞中的弓形虫复制。同时，我们将使用慢病毒- 筛选I型和II型ISGs文库的表达策略以识别诱导控制的效应器 寄生虫复制。总而言之，这些方法应该定义人类基因的全套 对控制细胞内寄生虫来说是必要的和充分的。了解这些机制可能很重要 在克服慢性弓形虫病对清除的抵抗力方面，在控制方面也可能是重要的 其他细胞内病原体。

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.