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）：人类感染内阿米巴寄生虫很常见；据估计，有5亿人感染阿米巴原虫，每年导致5 000万例侵袭性阿米巴病病例。寄生虫的生命周期涉及从滋养体到包囊阶段的相互转化，这是一种对于病原体繁殖和疾病致病至关重要的发育模式。然而，人们对调节寄生虫发育的因素知之甚少。目前研究内阿米巴阶段转换的模型是侵入阿米巴，这是一种在爬行动物中引起临床疾病的寄生虫，其与溶组织阿米巴引起的人类肠道疾病类似。在入侵大肠杆菌中，高水平调节的包囊和脱囊都是可能的。然而，尚未开发出针对入侵大肠杆菌的遗传工具，这极大地限制了该系统用于表征阶段转换的分子基础的用途。在此 R03 应用中，我们建议开发入侵大肠杆菌的遗传工具。在目标 1 中，我们将开发在入侵大肠杆菌中稳定转染的方法以及候选基因过表达和调节表达的方法。在目标 2 中，我们将开发入侵大肠杆菌基因下调的方法，包括利用调节下调的方法。在入侵大肠杆菌中成功开发遗传工具将使我们能够剖析这种寄生虫发育生物学的关键方面，包括识别触发成囊的途径。我们生成的工具和试剂将（i）免费分发，（ii）为内阿米巴社区提供宝贵的资源，以及（iii）增加模型的吸引力，从而增加在重要领域研究这一重要生物过程的研究人员数量人类病原体。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。两个生命周期阶段之间的转换对于疾病传播至关重要；然而，人们对寄生虫生物学的这一领域知之甚少。我们有兴趣了解寄生虫在生命周期阶段之间转换的分子机制。