Modulation of Retrograde Transport by a Novel Parasite-Derived GTPase

描述（由申请人提供）：迄今为止，很少有天然宿主-寄生虫相互作用得到高度表征，尽管决定寄生虫成功或失败的分子机制为免疫提供了重要见解。我们已经开发了一个模型系统的基础上共同进化的主机-寄生虫对分析的相互作用的免疫果蝇和攻击武库的寄生蜂（Leptopilina属）。这个系统提供了复杂性和微妙的解剖先天免疫激活，沉默和颠覆。D.黑腹遗传学是易处理的，快速的，低影响的。从果蝇到人类，先天免疫功能广泛保守，我们的长期目标是提供翻译结果。纤翼虫属（Leptopilina spp.）是免疫抑制微结构，称为VLP。细胞特异性药物递送和治疗性免疫调节代表了我们对这些独特的寄生虫衍生颗粒的功能的研究的两种可能的结果。VLP的蛋白质组成是这些果蝇寄生虫攻击成功的关键。这项工作标志着第一个单一的VLP蛋白的功能调查。我们已经选择了一个基因产物，表达只有最毒力的Leptopilina黄蜂，是非常丰富的VLP蛋白质组，并具有一个推定的GTP结合和水解的网站。当在全基因组遗传相互作用筛选中分析时，这种GTdR引起与受损的逆行转运相关的合成生长抑制。在目标1中，我们将确认这些遗传相互作用的结果，然后在GTP/GDP锁定突变体以及对正常囊泡运输产生负面影响的突变的背景下对其进行测试。在目标2中，我们将在果蝇血细胞亚群中表达该蛋白，以研究其亚细胞定位和蛋白质-蛋白质相互作用。我们将测试其对蝇幼虫中NF-κ B依赖性信号传导的影响。Toll-NF-κB通路是果蝇先天免疫系统的细胞和体液臂激活的基础，我们相信这种基于GTP酶的逆行转运调节可能抑制正常的免疫信号传导。在这项工作中使用的策略依赖于一个配对的模型系统的方法。我们已经通过一个强大的酵母筛选获得了关于细胞功能和一种新型蛋白质靶向的初步信息，该筛选提供了数千种遗传相互作用的信息。这种方法使我们能够在果蝇中设计适当和有效的实验计划。我们的目标是为PI提供酵母和果蝇遗传学，转基因，高通量全基因组遗传筛选，免疫组织化学和荧光成像方面的深入技能。这项工作将得到一个团队的大力支持，该团队包括跨学科环境中的该领域专家，将为PI提供高水平的培训， 免疫、多模型系统、遗传学和先进分子生物学技术。

DESCRIPTION (provided by applicant): Few natural host-parasite interactions have been highly characterized to date, even though the molecular mechanisms that determine parasite success or failure offer critical insights into immunity. We have developed a model system based on a coevolved host-parasite pair to analyze the reciprocal interactions underlying immunity in Drosophila melanogaster and the attack arsenals of its parasitic wasps (Leptopilina spp.). This system offers the complexity and subtlety to dissect innate immune activation, silencing, and subversion. D. melanogaster genetics are tractable, fast, and low-impact. Innate immune functions are widely conserved from fly to human and our long-term goal is to provide translational results. The most unique venom products of Leptopilina spp. are immunosuppressive microstructures, referred to as VLPs. Cell-specific drug delivery and therapeutic immune modulation represent two possible outcomes our investigations of the function of these unique parasite-derived particles. The protein composition of VLPs is central to the attack success of these parasites of Drosophila. This work marks the first functional investigation into a singular VLP protein. We have selected a gene product that is expressed only by the most virulent of Leptopilina wasps, is highly abundant in the VLP proteome, and possesses a putative site for GTP binding and hydrolysis. When analyzed in a genome-wide genetic interaction screen, this GTPase causes synthetic growth repression associated with impaired retrograde transport. In Aim 1, we will confirm these genetic interaction results and then test them in the context of GTP/GDP-locked mutants, as well as mutations that negatively impact normal vesicular trafficking. In Aim 2, we will express this protein in sub-populations of Drosophila blood cells to examine its subcellular localization and protein-protein interactions. We will test its impact on NF-κB-dependent signaling in fly larvae. The Toll- NF-κB pathway underlies activation of both the cellular and humoral arms of Drosophila's innate immune system and we believe that this GTPase-based modulation of retrograde transport may suppress normal immune signaling. The strategies utilized in this work depend on a paired model system approach. We have obtained preliminary information on the cellular function and targeting of a novel protein via a powerful yeast screen that provides information on thousands of genetic interactions. This approach has allowed us to design appropriate and efficient experimental plans in Drosophila. Our aims will provide the PI with in-depth skills in yeast and Drosophila genetics, transgenics, high-throughput genome-wide genetic screens, immunohistochemistry, and fluorescence imaging. This work will be strongly supported by a team that includes experts in the field in an interdisciplinary environment that will provide high-level training to the PI in immunity, multiple model system, genetics, and advanced molecular biology techniques.