Centrosomal Proteins in Giardia Differentiation

描述(由申请人提供)：蓝氏贾第鞭毛虫对外界刺激的分化能力是其发病机制的核心。贾第鞭毛虫的细胞骨架在包囊和排泄过程中发生了剧烈的变化。然而，人们对这些关键转变的中介途径知之甚少。统一这一修订更新的总体假设是，中心体是细胞的“控制中心”，而贾第虫特有的细胞骨架元件具有特定的效应功能，这些功能反映在它们的蛋白质组成中。我们将用这些特定的目标来检验我们的假设：在胞化过程中，运动装置被内化。在特定的目标1 A中，我们检验了这样的假设，即包囊化导致贾第鞭毛虫中心体/基底的蛋白质组成发生数量和质量的变化，这指向了新的调节水平。我们将通过多维蛋白质鉴定技术(MudPIT)比较滋养体生长和包囊的中心体的蛋白质组。在特定的目标1B中，我们将在贾第鞭毛虫中对选定的蛋白进行表位标记和表达，以验证它们的定位，并在目标2和目标3中进行功能分析。细胞生物学研究的一个主要目标是将蛋白质定位的变化与分化的生理刺激联系起来。到目前为止，大多数中心体蛋白也定位于贾第鞭毛虫特有的细胞骨架元件。具体目标2是测试某些蛋白质定位到中心体或贾第鞭毛虫特定结构对生理信号有反应的假设。具体目标3是分析选定的中心体信号蛋白和贾第虫特有的蛋白的生物学作用。在目标3A中，我们将识别与信号蛋白形成复合体的蛋白质，以测试它们通过与特定的靶向或调节亚基相关联来定位和发挥功能的假设。在目标3B中，我们将使用转录后基因沉默(PTGS)和过度表达来进一步验证这一假说，通过定义选定的信号和独特蛋白质在生长和分化中的功能。与公共卫生相关：蓝氏贾第鞭毛虫是美国和世界各地水源性腹泻疾病的主要原因，也是B类生物恐怖主义病原体。这些研究将极大地促进我们对兰氏革兰氏菌生物学的理解，它的发病依赖于它通过包囊或外囊对环境变化的信号做出快速反应的能力。贾第鞭毛虫是其他生活周期尚未完成的寄生虫的宝贵模式。

DESCRIPTION (provided by applicant): The ability of Giardia lamblia to differentiate in response to external stimuli is central to its pathogenesis. The Giardia cytoskeleton undergoes drastic changes during encystation and excystation. However, the pathways mediating these key transformations are poorly understood. The overall hypothesis unifying this revised renewal is that the centrosomes are cellular "control centers", while cytoskeletal elements unique to Giardia have specific effector functions that are reflected in their protein compositions. We will test our hypothesis with these Specific Aims: During encystation, the motility apparatus is internalized. In Specific Aim 1 A, we test the hypothesis that encystation entails quantitative and qualitative changes in the protein composition of Giardia centrosomes/basal bodies that point to new levels of regulation. We will compare the proteomes of centrosomes from growing and encysting trophozoites by Multidimensional Protein Identification Technology (MudPIT). In Specific Aim 1B, we will epitope-tag and express selected proteins in Giardia to validate their localization and for functional analyses in Aims 2 and 3. A major goal of cell biology research is to relate changes in protein localization to physiologic stimuli of differentiation. Most centrosomal proteins identified so far also localize to cytoskeletal elements that are unique to Giardia. Specific Aim 2 is to test the hypothesis that the localization of certain proteins to centrosomes or Giardia-specific structures is responsive to physiologic signals. Specific Aim 3 is to analyze the biological roles of selected centrosomal signaling proteins and proteins unique to Giardia. In Aim 3A, we will identify proteins that form complexes with signaling proteins to test the hypothesis that they localize and function by association with specific targeting or regulatory subunits. In Aim 3B, we will use post-transcriptional gene silencing (PTGS) and over- expression to further test this hypothesis by defining the functions of selected signaling and unique proteins in growth and differentiation. Relevance to public health: Giardia lamblia is a major cause of water-borne diarrheal disease in the US and worldwide and a Class B bioterrorism agent. These studies will greatly advance our understanding of the biology of G. lamblia, whose pathogenesis depends on its ability to respond rapidly to changing signals from its environment by encysting or excysting. Giardia is a valuable model for other parasites whose life cycles have not been completed.