Rac: a critical regulator of the cytoskeleton and membrane trafficking in Giardia

 描述(申请人提供)：贾第鞭毛虫病是由真核寄生虫贾第鞭毛虫引起的疾病，是美国最常见的肠道寄生虫病，也是全球儿童发病的主要原因；估计每年有2.8亿例贾第虫病例。事实证明，新出现的耐药菌株很难治疗，一线治疗甲硝唑的副作用发生率很高。因此，迫切需要针对新的分子途径的抗贾第虫药物。贾第鞭毛虫属于真核生物中最早的发散类群之一，因此与后生动物非常不同。在这种不同的生物学中，存在着开发靶向治疗的机会。这项建议探索了贾第虫中Rho家族GTP酶的生物学。这些蛋白质起着分子开关的作用，控制着基本的细胞过程。贾第虫含有单一的Rho家族GTPase同源物Grac，以前被证明在调节极性、膜运输和细胞骨架方面发挥着保守的作用，所有这些都是生存和致病所必需的。功能分析表明，GRAC在包囊形成过程中发挥了作用。这一过程需要脉冲式生产、加工和分泌囊壁蛋白(CWP)来产生耐环境的包囊，是贾第鞭毛虫唯一已知的调节分泌途径。已知Rho GTP酶调节动植物体内的内质网高尔基体运输和分泌活动。贾第鞭毛虫如何触发CWP的分泌仍然是个谜，但Grac可能是调节这一过程的候选者。这项建议的目标1通过在分泌过程中跟踪囊壁标记物来确定感染性囊肿形成的特殊作用。GRAC信号是如何与下游过程联系在一起的也是未知的，以前未知的下游效应器(贾第虫特有的或可能是古老的和未被发现的)被假设将GRAC信号与细胞骨架和膜运输联系起来。通过亲和纯化和质谱分析，鉴定了199个可能的GRAC相互作用元件。AIM 2建议通过显微镜和基因敲除研究，确认其中四个GRAC相互作用分子是膜运输和/或细胞骨架的下游效应因子。GRAC的上游是一组六个GRAC调制器，其作用是激活或抑制GRAC信号。目的3，建议确定它们对GRAC信号的贡献，并使用耗竭研究和检测细胞增殖、寄生虫附着和形成感染性包囊的能力相结合的方法来确定每个调节器的特定生物学作用。拟议的实验有望确定Grac在贾第虫生物学中的核心作用，并为发现新的治疗靶点铺平道路。此外，由于贾第虫基因组只包含一个Rho GTP酶和少数上游调节子，该系统非常容易处理，并可能揭示被模型真核生物中更复杂的信号系统所掩盖的基本生物学。

 DESCRIPTION (provided by applicant): Giardiasis, the disease caused by the eukaryotic parasite Giardia lamblia, is the most common intestinal parasitic disease in the U.S. and a major cause of morbidity in children throughout the world; estimates indicate 280 million cases of Giardia annually. Newly emerging drug-resistant strains are proving difficult to treat, and the front-line treatment, metronidazole, has a high incidence of side effects. Therefore, there is a critical need for anti-Giardia drugs that target novel molecular pathways. Giardia belongs to one of the earliest diverging groups of eukaryotes and is therefore quite divergent from metazoans. Opportunities for developing targeted therapeutics exist within this divergent biology. This proposal explores the biology of Rho family GTPases in Giardia. These proteins act as molecular switches that control essential cellular processes. Giardia contains a single Rho family GTPase homolog, gRac, previously demonstrated to play a conserved role in regulating polarity, membrane trafficking, and the cytoskeleton, all of which are essential to viability and pathogenesis. Functional assays have demonstrated that gRac plays a role in cyst formation. This process requires pulsed production, processing, and secretion of cyst wall protein (CWP) to make environmentally resistant cysts and is the only known regulated secretory pathway in Giardia. Rho GTPases are known to regulate ER-Golgi transport and secretory events in plants and animals. How CWP secretion is triggered in Giardia remains enigmatic, yet gRac is a likely candidate for regulating this process. Aim 1 of this proposal identifies the specific role of gRac n infectious cyst formation by following cyst wall markers through the secretory process. Also unknown is how gRac signaling is linked to downstream processes, previously unidentified downstream effectors (Giardia-specific or potentially ancient and undiscovered) are hypothesized to link gRac signaling to the cytoskeleton and membrane trafficking. Using affinity purification and mass spectroscopy, 199 putative gRac interactors were identified. Aim 2 proposes to validate four of these gRac interactors as downstream effectors of membrane trafficking and/or the cytoskeleton through microscopy and knockdown studies. Upstream of gRac is a set of six gRac modulators that act to activate or inhibit gRac signaling. Aim 3, proposes to determine their contribution to gRac signaling and identify specific biological roles for each modulator using a combination of depletion studies and assays to measure cell proliferation, parasite attachment, and the ability to form infectious cysts. The proposed experiments are expected to define the central role of gRac in Giardia biology and pave the way toward discovery of novel therapeutic targets. Furthermore, because the Giardia genome contains only a single Rho GTPase and a handful of upstream modulators, the system is highly tractable and may uncover fundamental biology that has been obscured by the more complex signaling systems found in model eukaryotes.