Mechanisms of Giardia Iamblia Excystation

贾第鞭毛虫排泄机制

• 批准号: 7048563
• 负责人: FRANCES D. GILLIN
• 金额: $ 33.4万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-01 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7048563
• 关键词: Giardia; binding proteins; calcium flux; calmodulin; cyclic AMP; enzyme linked immunosorbent assay; exocytosis; host organism interaction; intracellular parasitism; mass spectrometry; northern blottings; nucleic acid sequence; phosphorylation; proteomics; second messengers; two dimensional gel electrophoresis

DESCRIPTION (provided by the applicant): Giardia lamblia, an important human pathogen, must successfully excyst in order to infect a new host. Since excystation is too rapid to rely entirely on new gene expression, the unifying hypothesis of this proposal is that second messenger pathways may be central for regulation. While we can reproduce the entire life cycle in vitro, neither the precise stimuli nor the second messenger pathways that regulate excystation are known. Based on extensive Preliminary Data, we propose these Specific Aims: Specific Aim 1 is to define the key physiological excystation stimuli, and to test the hypothesis that Giardia cysts' responses to these signals are mediated by or reflected in changes in cytosolic calcium and/or cyclic AMP. We will ask whether pharmacological release of "caged" Ca 2v and/or cAMP into the cytosol can bypass the corresponding excystation stimulus. Specific Aim 2 is to define the role(s) of calmodulin (CAM) in regulating excystation and to identify specific "downstream" effectors that carry out its function. gCaM co-localizes with protein kinase A to the flagellar basal bodies-centrosomes, suggesting a key role in coordinating these pathways. We will use in vivo cross-linking and genomic data to functionally identify downstream effectors that specifically interact with CaM during excystation. Specific Aim 3 is to test the hypothesis that changes in protein phosphorylation may be important in regulating excystation, compared with de novo protein synthesis. We will compare changes in protein phosphorylation (the "phosphoproteome"), with overall changes in the protein expression in excysting cells. Sequence analyses and genomic data will permit us to identify key excystation proteins. This proteomic approach will test our overall hypothesis and complement the cellular and molecular data of Aims 1 and 2. Our proposed studies will reveal important insights into regulation of G lamblia excystation. Many other parasites have cystic forms whose excystation is required for transmission. Giardia may be a valuable model for understanding parasite differentiation in the intestinal tract. On a basic level, Giardia excystation is a unique model for cellular awakening from dormancy in response to environmental signals in an early-diverging eukaryotes.

描述（由申请人提供）：贾第鞭毛虫是一种重要的人类病原体，必须成功排出才能感染新宿主。由于排泄速度太快而不能完全依赖新的基因表达，因此该提议的统一假设是第二信使途径可能是调节的核心。虽然我们可以在体外重现整个生命周期，但调节排泄的精确刺激和第二信使途径均未知。基于广泛的初步数据，我们提出以下具体目标： 具体目标 1 是定义关键的生理排泄刺激，并检验贾第鞭毛虫包囊对这些信号的反应是由胞质钙和/或胞质的变化介导或反映的假设。环磷酸腺苷。我们将询问“笼中的”Ca 2v 和/或 cAMP 进入细胞质的药物释放是否可以绕过相应的排泄刺激。具体目标 2 是确定钙调蛋白 (CAM) 在调节排泄中的作用，并确定执行其功能的特定“下游”效应器。 gCaM 与蛋白激酶 A 共定位于鞭毛基体中心体，表明在协调这些途径中发挥着关键作用。我们将使用体内交联和基因组数据来功能性地识别在排泄过程中与 CaM 特异性相互作用的下游效应子。具体目标 3 是检验以下假设：与从头蛋白质合成相比，蛋白质磷酸化的变化可能对调节排泄作用很重要。我们将比较蛋白质磷酸化（“磷酸化蛋白质组”）的变化与排泄细胞中蛋白质表达的总体变化。序列分析和基因组数据将使我们能够识别关键的排泄蛋白。这种蛋白质组学方法将测试我们的整体假设，并补充目标 1 和 2 的细胞和分子数据。我们提出的研究将揭示对 Gambalia 排泄调节的重要见解。许多其他寄生虫具有囊状形式，其传播需要其排泄。贾第鞭毛虫可能是了解肠道寄生虫分化的有价值的模型。在基本层面上，贾第鞭毛虫排泄是一种独特的模型，用于响应早期分化真核生物中的环境信号，将细胞从休眠状态唤醒。