Development of Genetic Tools in Entamoeba histolytica

描述（由申请方提供）：原生动物寄生虫溶组织内阿米巴（Entamoeba histolytica）可引起痢疾和肝脓肿，是全球寄生虫病最常见的死亡原因之一。一些寄生虫的遗传决定因素参与发病机制的特点，但是，一个完整的理解的致病机制尚未实现。对疾病的分子基础有更深入的了解是很重要的，因为E. NIAID将溶组织剂分类为生物恐怖主义关注的B类制剂。然而，由于缺乏可靠和简便的遗传工具，进展受到限制。来自基因组计划、微阵列实验和蛋白质组学研究的数据已经确定了可以优先用于遗传分析的基因子集。目前有几种方法对E.但是工具有限，并且具有时间和劳动密集型的性质，因此缺乏大规模使用的便利性，需要高水平的药物选择及其潜在的副作用。 影响，以及由于潜在的缺乏重组而缺乏在DNA水平上破坏基因的方法。我们的目标是开发几种新的遗传操作工具，这些工具集中在RNA、蛋白质和DNA水平上的操作，以应用于大肠杆菌。溶组织剂首先，我们将使用内源性RNAi机制和反义小RNA优化一种新的基因沉默。我们将通过开发这种方法来沉默（i）使用基因片段，（ii）多个基因，以及（iii）以受调节的方式来推进该工具。第二，我们的目标是开发一种工具，使用去稳定结构域方法（基于FK506结合蛋白和二氢叶酸还原酶）在蛋白质水平上调节表达。我们将开发一种工具，其中以可调控的方式表达蛋白质的显性版本将是产生功能缺失突变体的策略。第三，我们将建立利用piggyBac转座酶将外源DNA整合到基因组中的系统。我们的目标是产生随机的，单一的整合寄生虫，使一个基因可以与一个表型。这种方法虽然风险很高，但在许多不同的细胞类型中取得了成功，值得尝试，因为整合到基因组中将允许采用新的方法（基因破坏，启动子陷阱等）。总之，这些新的遗传工具应该提供更复杂的遗传操作方法，并将是内阿米巴研究界的一个巨大进步。 公共卫生相关性：溶组织内阿米巴是一种重要的病原体，在全球范围内影响人类健康。尽管有丰富的可用基因组数据，但该领域缺乏简单的遗传工具来表征感兴趣的基因和途径。我们有兴趣为溶组织内阿米巴开发新的遗传工具，这将推进对这种寄生虫的研究，并帮助我们更好地了解致病的重要毒力决定因素。

DESCRIPTION (provided by applicant): The protozoan parasite Entamoeba histolytica causes dysentery and liver abscesses and is one of the most common causes of death from parasitic disease worldwide. Some parasite genetic determinants involved in pathogenesis have been characterized; however, a complete understanding of the pathogenic mechanisms has not been achieved. A more thorough understanding of the molecular basis of diseases is important as E. histolytica is classified by NIAID as a class B agent of concern for bioterrorism. However, progress has been limited by the paucity of reliable and easy genetic tools. Data from genome projects, microarray experiments and proteomic studies have identified subsets of genes that can be prioritized for genetic analysis. Several methods to genetically manipulate E. histolytica are available but the tools are limited and suffer from time and labor-intensive nature and thus lack of ease for large-scale use, the need for high levels of drug selection and its potential side effects, and the lack of methods to disrupt genes on the DNA level due to potential lack of recombination. We aim to develop several novel genetic manipulation tools, which focus on manipulation at the RNA, protein, and DNA levels for application in E. histolytica. First, we will optimize a novel gene silencing using the endogenous RNAi machinery and antisense small RNAs. We will advance this tool by developing this approach to silence (i) using gene fragments, (ii) multiple genes, and (iii) in a regulated manner. Second, we aim to develop a tool to regulate expression on the protein level using a destabilization domain approach (based on the FK506 binding protein and dihydrofolate reductase). We will develop a tool where expression of dominant version of a protein in a regulatable fashion will be a strategy to generate a loss- of function mutant. Third, we will establish a system to integrate foreign DNA into the genome using piggyBac transposase. We aim to generate parasites with random, single integration, so that one gene can be associated to one phenotype. This approach, although high-risk, has succeeded in many different cell types and is worth attempting because integration into the genome will allow novel approaches to be undertaken (gene disruption, promoter trap, etc). In conclusion, these new genetic tools should provide more sophisticated methods of genetic manipulation and will be a great advance for the Entamoeba research community. PUBLIC HEALTH RELEVANCE: Entamoeba histolytica is an important pathogen with an impact on human health on a global scale. Despite a wealth of available genomic data, the field suffers from a lack of easy genetic tools to characterize genes and pathways of interest. We are interested in developing novel genetic tools for Entamoeba histolytica, which will advance the study of this parasite and help us better understand the virulence determinants important for disease causation.