Mechanisms of Giardia Iamblia Excystation

描述(由申请人提供)：蓝氏贾第鞭毛虫，一种重要的人类病原体，必须成功地激活才能感染新的宿主。由于兴奋的速度太快，不能完全依赖新的基因表达，这一提议的统一假设是，第二信使途径可能是调节的中心。虽然我们可以在体外复制整个生命周期，但我们既不知道确切的刺激因素，也不知道调节兴奋作用的第二信使途径。基于大量的初步数据，我们提出了这些特定的目标：特定的目标1是定义关键的生理兴奋刺激，并检验贾第鞭毛虫包囊对这些信号的反应是由胞浆钙和/或环磷酸腺苷的变化所介导或反映的假说。我们会问“笼子”钙离子和/或cAMP的药物释放到胞浆中是否可以绕过相应的兴奋刺激。具体目标2是确定钙调蛋白(CaM)在调节兴奋中的作用(S)，并确定执行其功能的特定“下游”效应因子。GCAM与蛋白激酶A共同定位于鞭毛基底体-中心体，表明GCAM在协调这些途径中发挥关键作用。我们将使用体内的交联物和基因组数据来从功能上识别在过度结晶过程中与CaM特异相互作用的下游效应器。具体目的3是验证这样一种假设，即与从头合成的蛋白质相比，蛋白质磷酸化的变化可能在调节兴奋性反应中起重要作用。我们将比较蛋白质磷酸化(“磷酸蛋白质组”)的变化，以及Exying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激活蛋白。这种蛋白质组学方法将检验我们的整体假设，并补充AIMS 1和AIMS 2的细胞和分子数据。我们拟议的研究将揭示对G lamblia过度结晶调控的重要见解。许多其他寄生虫都有囊状寄生虫，传播时需要排泄。贾第鞭毛虫可能是了解肠道寄生虫分化的有价值的模型。在基础水平上，贾第鞭毛虫激发是细胞从休眠中唤醒的独特模式，以响应环境信号在早期分化的真核生物。

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.