Development of Genetic Tools in Entamoeba histolytica

描述（由申请人提供）：原生动物寄生虫溶组织内阿米巴引起痢疾和肝脓肿，是世界范围内寄生虫病最常见的死亡原因之一。一些与发病机制有关的寄生虫遗传决定因素已被确定；然而，对其致病机制尚未完全了解。更深入地了解疾病的分子基础是很重要的，因为溶组织芽胞杆菌被NIAID列为引起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进行遗传分析的基因子集。有几种方法可以对溶组织芽胞杆菌进行基因操作，但这些方法有限，而且时间和劳动密集型，因此缺乏大规模使用的便性，需要高水平的药物选择及其潜在的一面

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.