Analysis of Host Defense Genes to Toxoplasma gondii

弓形虫宿主防御基因分析

• 批准号: 6849714
• 负责人: William C. Sha
• 金额: $ 30.12万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-02-15 至 2009-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6849714
• 关键词: Toxoplasma gondii; astrocytes; complementary DNA; fibroblasts; flow cytometry; gene expression; genetic library; host organism interaction; macrophage; microglia; microorganism immunology; polymerase chain reaction; protozoal genetics; southern blotting

DESCRIPTION (provided by the applicant): Toxoplasma gondii is a widespread intracellular human pathogen in the same phylum Apicomplexa that includes the human pathogens Plasmodium (the cause of malaria) and Cryptosporidium. Although Toxoplasma is emerging as a model system for the study of intracellular parasitism; our understanding of how host resistance genes control Toxoplasma growth has lagged behind our understanding of how Toxoplasma genes regulate pathogen growth within host cells. A clear understanding of the mechanisms responsible for intracellular host defenses to Toxoplasma will provide useful insights into how this important class of human pathogens can be controlled in infected patients. A major block impeding the functional analysis of how host-cell gene expression controls intracellular Toxoplasma growth is the tack of sensitivity of in vitro assays that measure intracellular parasite growth. To address this issue, a novel FACS-based assay has been developed to measure growth of GFP-tagged Toxoplasma in host cells. This FACS-based assay is significantly more sensitive than previous Toxoplasma growth assays, and can measure the inhibition of parasite growth mediated by individual retroviral-expressed genes in un-stimulated host cells. This new assay permits examination of host-cell responses to Toxoplasma in greater detail than previously possible, and will be used in Aim 1 to determine the mechanism of action of known host resistance genes, particularly the IGTP family of interferon-induced genes whose mechanism of action is unknown, despite having been implicated in the pathogenesis of multiple intracellular pathogens. In proof of principle studies, we have also established that known host resistance genes can be identified in functional screens designed to identify novel host resistance genes. Thus, in Aim 2, functional genetic screens will be conducted using retroviral cDNA libraries to identify host resistance genes that control Toxoplasma infection by (1) active resistance of growth of the virulent tachyzoite form of Toxoplasma, and by (2) passive inter-conversion of the virulent tachyzoite form to the dormant bradyzoite form of Toxoplasma.

描述（由申请人提供）：弓形虫是一种广泛存在的细胞内人类病原体，属于顶复门，包括人类病原体疟原虫（疟疾的病因）和隐孢子虫。尽管弓形虫正在成为研究细胞内寄生的模型系统；我们对宿主抗性基因如何控制弓形虫生长的理解落后于对弓形虫基因如何调节宿主细胞内病原体生长的理解。清楚地了解细胞内宿主对弓形虫的防御机制，将为了解如何在感染患者中控制这一类重要的人类病原体提供有用的见解。 阻碍宿主细胞基因表达如何控制细胞内弓形虫生长的功能分析的一个主要障碍是测量细胞内寄生虫生长的体外测定的灵敏度。为了解决这个问题，开发了一种新的基于 FACS 的测定法来测量宿主细胞中 GFP 标记的弓形虫的生长。这种基于 FACS 的检测方法比之前的弓形虫生长检测方法更加灵敏，并且可以测量未刺激宿主细胞中个体逆转录病毒表达基因介导的寄生虫生长抑制情况。 这种新的检测方法可以比以前更详细地检查宿主细胞对弓形虫的反应，并将用于目标1以确定已知宿主抗性基因的作用机制，特别是干扰素诱导基因的IGTP家族，其作用机制尚不清楚，尽管与多种细胞内病原体的发病机制有关。在原理证明研究中，我们还确定可以在旨在识别新型宿主抗性基因的功能筛选中识别已知的宿主抗性基因。因此，在目标 2 中，将使用逆转录病毒 cDNA 文库进行功能遗传筛选，以通过以下方式鉴定控制弓形虫感染的宿主抗性基因：(1) 主动抵抗弓形虫的有毒速殖子形式的生长，以及 (2) 有毒速殖子形式与弓形虫的休眠缓殖子形式的被动相互转化。