Translational Control of Encystation in the Entamoebae

 描述（由申请方提供）：溶组织内阿米巴是阿米巴痢疾和肝脓肿的病原体，被归类为B类生物恐怖剂。潜伏的食物和水传播的包囊是这种寄生虫的传染形式。E.溶组织菌在无菌培养中不容易包囊。因此，E. invadens是一种在体外包囊的爬行动物寄生虫，已被用作模型。不能重复诱导E.体外溶组织杆菌包囊形成是研究工具集的一个重大空白，阻碍了对E.溶组织潜伏期在其他寄生虫中，潜伏期部分受翻译控制。应激可以激活磷酸化真核起始因子-2（eIF 2a）α亚基的激酶。eIF 2a是将Met-tRNA递送至核糖体以进行翻译起始的复合物的一部分。eIF 2a的磷酸化是抑制性的，并导致蛋白质合成的下降。这允许细胞保存资源并重新配置基因表达以诱导潜伏期。基因组分析表明，内阿米巴具有这种应激反应系统的组成部分。此外，初步数据显示在E.侵入细胞。因此，中心假设是内阿米巴包囊化部分地通过eIF 2a的磷酸化来调节，eIF 2a的磷酸化反过来下调翻译。磷酸-eIF 2a在包囊形成中的作用将使用两种物种来定义（E. invadens和E.组织溶解）在两个特定的目标。在第一个目标中，将在E.表达eIF 2a的非磷酸化和磷酸模拟突变体形式的侵袭细胞系。这将揭示磷酸-eIF 2a是否是阶段转化所必需和足够的。两个推测的E.还将表征侵袭菌eIF 2a激酶。在第二个目标中，磷酸化eIF 2a的水平将在E.在人类病原体中诱导包囊形成和脱囊样事件。如果成功的话，这将是第一个 在体外重现这种寄生虫的生命周期。鉴于E.尽管可以在培养物中诱导侵袭，但是可能可以定义磷酸化-eIF 2a在内阿米巴潜伏期中的作用。包括E.组织溶解性代表了高风险/高回报的方面， 该项目建立了大肠杆菌体外成囊和脱囊体系。histolytica将为研究人员提供一个创新的工具，将激发许多新的研究E。溶组织潜伏期

 DESCRIPTION (provided by applicant): Entamoeba histolytica is the causative agent of amoebic dysentery and liver abscess, and is classified as a Category B bioterrorism agent. Latent food- and water-borne cysts are the infectious form of this parasite. E. histolytica does not readily encyst in axenic culture. Thus, E. invadens, a reptilian parasite that encysts in vitro has been used as a model. The inability to reproducibly induce E. histolytica encystation in vitro represents a significant gap in the research tool-set and has hampered studies of E. histolytica latency. In other parasites latency is regulated, in part, by translational control. Stress can activate kinases that phosphorylate the a subunit of eukaryotic initiation factor-2 (eIF2a). eIF2a is part of a complex that delivers Met-tRNA to ribosomes for translation initiation. Phosphorylation of eIF2a is inhibitory and leads to a decline in protein synthesis. This allows cells to conserve resources and reconfigure gene expression to induce latency. Genome analyses indicate that the Entamoebae possess the components of this stress response system. Furthermore, preliminary data show an increase in the level of phospho-eIF2a during encystation in E. invadens cells. Thus, the central hypothesis is that Entamoeba encystation is regulated, in part, by phosphorylation of eIF2a, which in turn, down-regulates translation. The role of phospho-eIF2a in encystation will be defined using both species (E. invadens and E. histolytica) in two specific aims. In the first aim, encystation will be tracked in E. invadens cel lines expressing non-phosphorylatable and phosphomimetic mutant versions of eIF2a. This will reveal if phospho-eIF2a is necessary and sufficient for stage conversion. Two presumptive E. invadens eIF2a kinases will also be characterized. In the second aim, the levels of phospho-eIF2a will be manipulated genetically in E. histolytica in an attempt to induce encystation- and excystation-like events in the human pathogen. If successful, it would represent the first protocol for reproducibly recapitulating the life cycle of this parasite in vitro. Given that encystation ofE. invadens can be induced in culture, it is likely that a role for phospho-eIF2a in Entamoeba latency can be defined. Inclusion of E. histolytica represents the high-risk/high- reward aspect of the project. Establishing an in vitro system for encystation and excystation of E. histolytica would provide an innovative tool to the community of researchers that would inspire numerous novel studies of E. histolytica latency.