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Identification of novel Toxoplasma genes involved in host-parasite interactions

鉴定参与宿主-寄生虫相互作用的新弓形虫基因

基本信息

批准号：
9203042
负责人：
Sebastian Lourido
金额：
$ 24.38万
依托单位：
WHITEHEAD INSTITUTE FOR BIOMEDICAL RES
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-01-06 至 2018-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9203042
关键词：
Accounting Affect Biological Assay Biological Models Blood CRISPR library CRISPR/Cas technology Categories Cell Line Cells Chromosome Mapping Clustered Regularly Interspaced Short Palindromic Repeats Code Cryptosporidiosis Development Disease Drug resistance Embryo Epitopes Family Fibroblasts Future Genes Genetic Genetic Variation Genome Genome engineering Growth Human Image Analysis Inbred Mouse Inbred Strains Mice Inbreeding Individual Infection Infection prevention Innate Immune Response Interferon Type II Laboratory mice Libraries Life Cycle Stages Lytic Phase MAP Kinase Gene Malaria Measures Mediating Metabolism Methods Microscopy Morbidity - disease rate Mouse Strains Mus National Institute of Allergy and Infectious Disease Organelles Outcome Parasites Peptide Signal Sequences Phenotype Process Proteins Recombinants Regulation Resources Role Testing Toxoplasma Toxoplasma gondii Toxoplasmosis Variant Viral Tumor Antigens Work base biodefense cell type cost effective experimental study fitness genome-wide genome-wide analysis high throughput screening member microscopic imaging mortality next generation sequencing novel obligate intracellular parasite parasite genome parasitism pathogen public health relevance

项目摘要

DESCRIPTION (provided by applicant): Apicomplexans are among the most common parasites of humans, accounting for significant mortality and morbidity through diseases like malaria, toxoplasmosis and cryptosporidiosis. As obligate intracellular parasites, they establish intimate interactions with their hosts. Toxoplasma gondii is an extreme example of this adaptation, able to replicate within nearly every cell type in any warm-blooded host. Genome-scale analyses will be needed to systematically explore the genetics of host-parasite interactions. We present preliminary evidence for the use of CRISPR/Cas9 to target all predicted protein-coding genes in the parasite. This advance enables rapid and cost-effective assessment of the relative contribution of each parasite gene to growth under different conditions. Using this platform we can identify genes important for parasite survival in genetically diverse hosts. Coevolution between T. gondii and mice is evident in the variable parasite growth observed in cell lines derived from different mouse strains. We therefore propose to measure the effect of host variation on the parasite genes required for optimal growth in fibroblasts. To maximize the resources available to downstream applications we will focus our study on the founder strains for two recombinant inbred (RI) families, the Collaborative Cross (CC) and the BXD family, which together account for more than 90% of the genetic diversity in laboratory mice. The first aim of this study will extend our CRISPR/Cas9-mediated genome-wide analysis to cell lines derived from the nine inbred mouse strains. This aim will define two sets of parasite genes: those important for parasitism across different hosts, and those that only contribute to growth in certain mouse strains. The second aim will examine the phenotypes associated with disrupting specific parasite genes, leveraging the efficiency of CRISPR/Cas9, automated microscopy, and image analysis. Based on our preliminary work, we have selected 14 genes to study, which strongly contribute to fitness in human fibroblasts, are conserved among apicomplexans, and are predicted to be secreted. Study of these important parasite factors will extend our understanding of host-parasite interactions, and establish the methods for high-throughput phenotyping of parasite genes. These methods will be valuable to investigate the phenotypes of other relevant genes identified in the first aim. The proposed work will be the first systematic analysis of the apicomplexan genome in the context of natural host variation. Defining the categories of genes that broadly or specifically affect infection will helpus understand the cellular basis of parasitism.

描述（由申请人提供）：顶复门是人类最常见的寄生虫之一，通过疟疾、弓形体病和隐孢子虫病等疾病导致显著的死亡率和发病率。作为专性细胞内寄生虫，它们与宿主建立密切的相互作用。刚地弓形虫是这种适应的一个极端例子，它能够在任何温血宿主的几乎所有细胞类型中复制。需要进行基因组规模的分析，以系统地探索宿主-寄生虫相互作用的遗传学。我们提出了使用CRISPR/Cas9靶向寄生虫中所有预测的蛋白质编码基因的初步证据。这一进展使得能够快速和具有成本效益的评估每个寄生虫基因在不同条件下对生长的相对贡献。利用这个平台，我们可以识别对寄生虫在遗传多样性宿主中生存重要的基因。T.弓形虫和小鼠之间的差异在来自不同小鼠品系的细胞系中观察到的可变寄生虫生长中是明显的。因此，我们建议测量宿主变异对成纤维细胞最佳生长所需的寄生虫基因的影响。为了最大限度地利用下游应用的资源，我们将把我们的研究重点放在两个重组近交系（RI）家族的创始人菌株上，这两个家族是协作杂交（CC）和BXD家族，它们共同占实验室小鼠遗传多样性的90%以上。这项研究的第一个目的是将我们的CRISPR/Cas9介导的全基因组分析扩展到来自9个近交系小鼠品系的细胞系。这个目标将定义两组寄生虫基因：那些对不同宿主的寄生很重要的基因，以及那些只对某些小鼠品系的生长有贡献的基因。第二个目标将研究与破坏特定寄生虫基因相关的表型，利用CRISPR/Cas9，自动显微镜和图像分析的效率。基于我们的初步工作，我们选择了14个基因进行研究，这些基因在人类成纤维细胞中对适应性有很大贡献，在顶复门中是保守的，并且预测是分泌的。对这些重要寄生虫因子的研究将有助于我们深入了解宿主-寄生虫相互作用，并建立高通量的寄生虫基因表型分析方法。这些方法对于研究第一个目标中鉴定的其他相关基因的表型具有重要意义。拟议的工作将是第一个系统的分析apicomplexan基因组的背景下，自然宿主的变化。定义广泛或特异性影响感染的基因类别将有助于理解寄生的细胞基础。