Development of Genetic Tools in Entamoeba histolytica

描述(申请人提供)：原生动物寄生虫溶组织内阿米巴引起痢疾和肝脓肿，是全世界最常见的寄生虫病死亡原因之一。一些寄生虫参与发病的遗传决定因素已经被表征，然而，对致病机制还没有完全的了解。更透彻地了解疾病的分子基础是重要的，因为NIAID将溶组织埃希菌列为生物恐怖主义关注的B类病原体。然而，由于缺乏可靠和简单的遗传工具，进展受到了限制。来自基因组计划、微阵列实验和蛋白质组研究的数据已经确定了可以优先进行遗传分析的基因亚组。有几种方法可用来对组织裂解杆菌进行遗传操作，但这些方法有限，而且存在时间和劳动力密集型的特点，因此不容易大规模使用，需要高水平的药物选择及其潜在的一面。 影响，以及由于潜在的缺乏重组而缺乏在DNA水平上干扰基因的方法。我们的目标是开发几种新型的遗传操作工具，这些工具专注于在RNA、蛋白质和DNA水平上的操作，以应用于溶组织埃希氏菌。首先，我们将利用内源RNAi机制和反义小RNA来优化一种新的基因沉默。我们将通过开发这一方法来推动这一工具的发展，以沉默(I)使用基因片段，(Ii)多基因，以及(Iii)以受调控的方式。其次，我们的目标是开发一种工具，使用不稳定结构域方法(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，其中以可调节的方式表达蛋白质的显性版本将成为产生功能缺失突变体的策略。第三，我们将建立一个使用猪Bac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、单一整合的寄生虫，以便一个基因可以与一种表型相关联。这种方法虽然风险很高，但在许多不同的细胞类型中都取得了成功，值得尝试，因为整合到基因组中将允许采取新的方法(基因破坏、启动子陷阱等)。总而言之，这些新的基因工具应该会提供更复杂的基因操作方法，并将是内阿米巴研究界的一大进步。 公共卫生相关性：溶组织内阿米巴是一种在全球范围内对人类健康产生影响的重要病原体。尽管有丰富的可用基因组数据，但该领域缺乏简单的基因工具来表征感兴趣的基因和途径。我们有兴趣开发新的溶解组织内阿米巴的遗传工具，这将促进对这种寄生虫的研究，并帮助我们更好地了解致病原因中重要的毒力决定因素。

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.