Developing tools for genetic manipulation in Entamoeba invadens

开发内阿米巴入侵基因操作工具

基本信息

批准号：
8018169
负责人：
UPINDER SINGH
金额：
$ 7.92万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-02-01 至 2013-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8018169
关键词：
Amebiasis Amoeba genus Area Award Biological Assay Biological Models Biological Process Biology C-terminal Candidate Disease Gene Clinical Communities Cyst Data Development Developmental Biology Disease Double-Stranded RNA Down-Regulation Entamoeba Entamoeba histolytica Entamoeba invadens Etiology Gastrointestinal Diseases Gene Expression Gene Proteins Gene Silencing Genes Genetic Health Human Immunofluorescence Immunologic Immunoprecipitation In Vitro Infection Intestinal Diseases Life Cycle Stages Measures Methods Modeling Molecular Nucleic Acid Regulatory Sequences Parasites Pathway interactions Pattern Protein Overexpression Proteins Reagent Regulatory Pathway Reptiles Research Research Personnel Resources Staging Study models System Techniques Testing Time Tissues Transfection Western Blotting Work base disease transmission excystation genetic manipulation genome sequencing genome wide association study human disease insight interest member method development novel diagnostics novel therapeutic intervention novel therapeutics overexpression pathogen prevent promoter public health relevance small hairpin RNA tool tool development vector

项目摘要

DESCRIPTION (provided by applicant): Human infections with Entamoeba parasites are common; an estimated 500 million people are colonized with ameba resulting in 50 million cases of invasive amebiasis annually. The parasite's life cycle involves interconversion from a trophozoite to a cyst stage, a developmental pattern which is essential for pathogen propagation and disease causation. However, the factors regulating development of the parasite are poorly understood. The current model for studying Entamoeba stage conversion is E. invadens, a parasite which causes clinical disease in reptiles that is similar to human intestinal disease caused by E. histolytica. In E. invadens, both high level regulated encystation and excystation are possible. However, no genetic tools have been developed for E. invadens, significantly limiting the use of this system for characterizing the molecular basis of stage conversion. In this R03 application, we propose to develop genetic tools for E. invadens. In Aim 1, we will develop methods for stable transfection in E. invadens and approaches for overexpression and regulated expression of candidate genes. In Aim 2, we will develop methods for gene downregulation in E. invadens, including an approach that utilizes regulated downregulation. Successful development of genetic tools in E. invadens will allow us to dissect key aspects of the developmental biology of this parasite including identification of the pathways that trigger encystation. Tools and reagents generated by us will (i) be freely distributed, (ii) provide a valuable resource to the Entamoeba community, and (iii) increase the attractiveness of the model and thus the number of investigators studying this essential biological process in an important human pathogen. PUBLIC HEALTH RELEVANCE: Entamoeba histolytica is an important pathogen with an impact on human health on a global scale. Conversion between the two life cycle stages is crucial to disease transmission; however, this area of parasite biology is poorly understood. We are interested in understanding the molecular mechanisms that the parasite uses to convert between life cycle stages.

描述（由申请人提供）：具有Entamoeba寄生虫的人类感染很常见；估计有5亿人被殖民时，每年有5000万例侵入性阿米巴病。寄生虫的生命周期涉及从滋养体到囊肿阶段的相互转化，这是病原体传播和疾病因果关系至关重要的发育模式。但是，调节寄生虫发展的因素知之甚少。当前研究Entamoeba阶段转化率的模型是E. Invadens，它是一种寄生虫，该寄生虫在爬行动物中引起临床疾病，类似于溶血性大肠杆菌引起的人类肠道疾病。在E. Invadens中，可以进行高水平的监管百科全书和脱叶术。但是，尚未开发出用于E.侵袭的遗传工具，从而显着限制了该系统来表征阶段转化的分子基础。在此R03应用中，我们建议为E. Invadens开发遗传工具。在AIM 1中，我们将开发用于稳定转染的方法。在AIM 2中，我们将开发用于E. Invadens基因下调的方法，包括一种利用调节下调的方法。在E. Invadens中成功开发遗传工具将使我们能够剖析该寄生虫的发育生物学的关键方面，包括识别触发百家的途径。我们生成的工具和试剂将（i）自由分布，（ii）为entamoeba社区提供宝贵的资源，（iii）提高模型的吸引力，从而在重要的人类病原体中研究这一基本生物学过程的研究人员数量。公共卫生相关性：Entamoeba Hissolytica是一种重要的病原体，对全球范围内的人类健康产生了影响。两个生命周期阶段之间的conversion依对疾病传播至关重要。但是，该领域的生物学领域知之甚少。我们有兴趣了解寄生虫在生命周期阶段使用的分子机制。