Analysis of Host Defense Genes to Toxoplasma gondii

描述(由申请人提供)：弓形虫是一种广泛存在于尖端复合体同一门中的细胞内人类病原体，包括人类病原体疟疾(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式系统，但我们对宿主抗性基因如何控制弓形虫生长的理解落后于我们对弓形虫基因如何调控宿主细胞内病原体生长的理解。对弓形虫细胞内宿主防御机制的清楚了解将为如何在感染患者中控制这一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检测的敏感性。为了解决这个问题，一种新的基于流式细胞仪的检测方法已经被开发出来，用来检测GFP标记的弓形虫在宿主细胞中的生长。这种基于FACS的检测方法比以前的弓形虫生长检测方法更敏感，并且可以测量单个逆转录病毒表达的基因在未刺激的宿主细胞中对寄生虫生长的抑制作用。 这种新的检测方法可以比以前更详细地检测宿主细胞对弓形虫的反应，并将在目标1中用于确定已知的宿主耐药基因的作用机制，特别是干扰素诱导的IGTP基因家族，其作用机制尚不清楚，尽管与多种细胞内病原体的发病有关。作为原理研究的证据，我们还证实了已知的寄主抗性基因可以在旨在鉴定新的寄主抗性基因的功能筛选中得到鉴定。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(1)主动抵抗强毒力的弓形虫速殖子形式的生长，以及(2)通过(2)强毒力的速殖子形式到休眠的慢速殖子形式的被动相互转化来识别控制弓形虫感染的宿主抗性基因。

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.