Analysis of Host Defense Genes to Toxoplasma gondii

描述（由申请方提供）：弓形虫是一种广泛存在于细胞内的人类病原体，与包括人类病原体疟原虫（疟疾的病因）和隐孢子虫的顶复门相同。虽然弓形虫正在成为一个模型系统的研究细胞内寄生虫，我们的理解如何宿主抗性基因控制弓形虫的增长已经落后于我们的理解弓形虫基因如何调节宿主细胞内的病原体生长。对弓形虫细胞内宿主防御机制的清楚理解将为如何在感染患者中控制这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，已经开发了一种新的基于FACS的测定来测量宿主细胞中GFP标记的弓形虫的生长。这种基于FACS的测定比以前的弓形虫生长测定显著更灵敏，并且可以测量由未刺激的宿主细胞中的单个逆转录病毒表达基因介导的寄生虫生长的抑制。 这一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GTP家族，尽管其与多种细胞内病原体的发病机制有关，但其作用机制尚不清楚。在原理研究的证明中，我们还确定了已知的宿主抗性基因可以在设计用于鉴定新的宿主抗性基因的功能筛选中鉴定。因此，在目标2中，将使用逆转录病毒cDNA文库进行功能性遗传筛选，以鉴定控制弓形虫感染的宿主抗性基因，所述宿主抗性基因通过（1）对弓形虫的毒性速殖子形式的生长的主动抗性，和（2）弓形虫的毒性速殖子形式到休眠的缓殖子形式的被动相互转化。

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.