Developing tools for genetic manipulation in Entamoeba invadens

描述(申请人提供)：人类感染内阿米巴寄生虫很常见；估计有5亿人在阿米巴中定居，每年导致5000万侵袭性阿米巴病病例。寄生虫的生命周期包括从滋养体到包囊阶段的相互转换，这是病原体繁殖和疾病原因所必需的一种发育模式。然而，调节这种寄生虫发展的因素却知之甚少。目前研究内阿米巴阶段转换的模型是侵袭性内阿米巴，这是一种在爬行动物身上引起临床疾病的寄生虫，类似于由溶组织性内阿米巴引起的人类肠道疾病。在入侵的E.inadens中，高水平调节的囊化和超量结晶都是可能的。然而，还没有开发出针对入侵E.inadens的遗传工具，这极大地限制了该系统在表征阶段转化的分子基础方面的使用。在这个R03应用中，我们建议开发入侵E.inadens的遗传工具。在目标1中，我们将开发在入侵E.inadens中稳定转染的方法以及候选基因的过表达和调控表达的方法。在目标2中，我们将开发入侵E.inadens基因下调的方法，包括一种利用受调控下调的方法。入侵棘球绦虫基因工具的成功开发将使我们能够剖析这种寄生虫发育生物学的关键方面，包括识别触发囊化的途径。我们产生的工具和试剂将(I)免费分发，(Ii)为内阿米巴群落提供宝贵的资源，以及(Iii)增加模型的吸引力，从而增加研究这一重要人类病原体的基本生物学过程的研究人员的数量。 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。这两个生命周期阶段之间的转换对疾病传播至关重要；然而，人们对这一领域的寄生虫生物学知之甚少。我们感兴趣的是了解寄生虫在生命周期阶段之间转换的分子机制。

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.