Identification of Transcriptional Regulatory Networks in Entamoeba Histolytica

描述(由申请人提供)：转录控制允许在相似的过程或生命阶段中涉及的基因的协调表达。溶组织内阿米巴是寄生虫死亡的主要原因，但人们对这种原生寄生虫用来导致侵袭性结肠或肝脏疾病的毒力程序知之甚少。尽管有大量关于模式生物转录调控机制的信息，但对大多数寄生虫系统中的这种机制知之甚少。我们实验室的长期目标是了解寄生虫的遗传学和基因表达如何影响毒力。我们的目标是确定转录调控网络，以控制毒力相关基因亚群在溶组织性肠杆菌中的表达。对溶组织埃希氏菌转录调控机制的鉴定将有助于深入了解该寄生虫的基础生物学，并为研究阿米巴基因的调控提供途径。在目标1中，我们将确定允许毒力相关基因协调表达的启动子基序。此外，我们将在3‘非编码区和感兴趣基因的下游基因组区域寻找保守的基序。我们对毒力的定义包括半胱氨酸蛋白酶基因(参与组织侵袭)，参与阿米巴应激反应的基因(Ehssp基因家族)，在强毒和非强毒菌株中差异表达的基因，在结肠炎动物模型中体内调节的基因，以及寄生虫发育过程中调节的基因。在目标2中，我们将使用报告基因和电泳迁移率改变分析来鉴定半胱氨酸蛋白酶Ehssp和菌株特异性基因家族的潜在调控基序。这些目标的成功将使我们能够证明我们的方法是有效的。在结肠炎动物模型和发育过程中控制基因表达调控的基序将在晚些时候描述，因为这些基序在技术上更具挑战性，需要在溶组织性肠杆菌中开发新技术。这项工作将确定全球转录调控网络，控制与毒力相关的阿米巴基因的表达，并为表征溶组织内阿米巴的转录机制提供一个框架。 溶组织内阿米巴是一种重要的病原菌，在全球范围内对人类健康产生影响。疾病的主要表现是痢疾和肝脓肿。尽管大多数疾病发生在发展中国家，但这种寄生虫可以在水净化系统受到不利影响的任何地方引起感染。诸如海啸或一个区域的政治和社会基础设施的剧变等事件可能会导致疾病的出现。我们对了解寄生虫致病的分子机制很感兴趣。

DESCRIPTION (provided by applicant): Transcriptional control allows coordinated expression of genes involved in similar processes or life stages. Entamoeba histolytica is a leading cause of parasitic death, but little is known about the virulence program that this protozoan parasite uses to cause invasive colonic or hepatic disease. Although a great deal of information is available on transcriptional regulatory mechanisms in model organisms, there is limited knowledge about such mechanisms in most parasite systems. The long-term goals of our lab are to understand how parasite genetics and gene expression affect virulence. Our goal is to identify transcriptional regulatory networks that control expression of virulence associated gene subsets in E. histolytica. The identification of transcriptional regulatory mechanisms in E. histolytica will give an insight into the basic biology of this parasite, as well as provide avenues for studying the regulation of amebic genes. In Aim 1 we will identify promoter motifs that allow coordinated expression of genes involved in virulence. Additionally, we will look for conserved motifs in the 3'UTR and downstream genomic regions of the gene sets of interest. Our definition of "virulence" includes the cysteine proteinase genes (involved in tissue invasion), genes involved in the stress response in ameba (Ehssp gene family), genes differentially expressed in virulent and non-virulent E. histolytica strains, genes regulated in vivo in an animal model of colitis, and genes regulated during parasite development. In Aim 2, we will functionally characterize potential regulatory motifs from the cysteine proteinase, Ehssp, and the strain-specific gene families using reporter gene and electrophoretic mobility shift assays. Success in these aims will allow us to show proof of concept that our approach is valid. Motifs that control expression of genes regulated during an animal model of colitis and during development will be characterized at a later date as those are technically much more challenging and will require the development of novel technologies in E. histolytica. This work will identify global transcriptional regulatory networks that control expression of amebic genes associated with virulence and provide a framework for characterization of the transcriptional machinery in Entamoeba histolytica. Entamoeba histolytica is an important pathogen and has an impact on human health on a global scale. The main disease manifestations are dysentery and liver abscesses. Although the majority of disease is in developing countries, this parasite can cause infections anywhere that water purification systems get adversely affected. Events such as the Tsunami or upheaval in the political and social infrastructure of a region can allow disease to emerge. We are interested in understanding the molecular mechanisms that the parasite uses to cause disease.