CELL-CELL SIGNALLING IN MICROBE-HOST INTERACTIONS

微生物-宿主相互作用中的细胞-细胞信号传导

• 批准号: 7104190
• 负责人: Stephen Kendall Farrand
• 金额: $ 33.23万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-05-01 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7104190
• 关键词: Agrobacterium tumefaciens; DNA directed RNA polymerase; X ray crystallography; bacterial proteins; biological signal transduction; heat shock proteins; host organism interaction; ligands; plasmids; protein degradation; protein protein interaction; protein purification; protein structure function; proteolysis; quorum sensing; serine proteinases; transcription factor

DESCRIPTION (provided by applicant): Interactions between microbes and their hosts, including man, depend on specific communication systems. Thus bacteria, including pathogens, perceive their hosts by sensing chemical signals and respond appropriately. Understanding these signaling pathways, and how the microbe and its host respond, could lead to strategies for preventing pathogenesis or fostering beneficial relationships. Agrobacterium tumefaciens, which interacts with plants, provides an excellent model for such studies. As part of this interaction, the bacterium responds to a plant signal by eliciting a second signal that is then perceived by the entire bacterial population. This second quorum-sensing signal controls transfer of the Ti plasmid, a virulence element, to other bacteria. The long term goal is to understand this hierarchical signaling process including how the quorum-sensing signal, called AAI triggers activation of the positive transcription factor, TraR, how TraR retains its activity, and how a specific antiactivator, TraM, interferes with TraR activity. There are three goals for the project period. First we will examine the effect of signal loss on the structure of TraR using genetic screens and biochemical and spectral technologies. We will probe the structure of monomer TraR using a mutant that can bind signal but cannot form dimers. In the second goal we will examine the interaction between TraR and components of RNA polymerase including RpoA and RpoD. Our goal is to identify the amino acids of TraR, of RpoA, and of RpoD that contribute to stable complexes. We will establish conditions for isolating stable complexes of TraR and a C-terminal fragment of RpoD preparatory to efforts to determine the crystal structure of the complexes. In the second goal, we will assess the nature of the interaction between TraR and the antiactivator TraM. We have established a collaboration to determine the crystal structure of TraM and also the crystal structure of the complex formed by TraM and TraR. We also will probe the role of TraM and Lon protease in determining the stability of TraR using physiological tests to measure rates of TraR turnover. We also will establish an in vitro system using purified proteins to characterize the Lon-mediated degradation of TraM. In the third goal we propose to explore the evolutionary diversity of the Ti plasmid quorum-sensing systems by isolating and characterizing plasmids that induce transfer genes in response to novel plant signals.

描述（由申请人提供）：微生物及其主机（包括人）之间的交互取决于特定的通信系统。因此，包括病原体在内的细菌通过传感化学信号并做出适当反应来感知其宿主。了解这些信号通路，以及微生物及其宿主的反应，可能会导致预防发病机理或促进有益关系的策略。与植物相互作用的农杆菌Tumefaciens为此类研究提供了一个极好的模型。作为这种相互作用的一部分，细菌通过引起第二个信号对植物信号的反应，然后由整个细菌种群感知到第二个信号。第二个法定感应信号控制了Ti质粒（毒力元件）向其他细菌的转移。长期目标是了解这种层次信号传导过程，包括如何触发正规传感信号（称为AAI）触发正转录因子的激活，Trar，Trar如何保留其活性以及特定的抗激活器，TRAM，如何干扰Trar活动。项目期间有三个目标。首先，我们将使用遗传筛选以及生化和光谱技术检查信号丢失对TRAR结构的影响。 我们将使用可以结合信号但不能形成二聚体的突变体探测单体TRAR的结构。在第二个目标中，我们将检查RNA聚合酶（包括RPOA和RPOD）的TRAR与组件之间的相互作用。我们的目标是确定有助于稳定复合物的Trar，RPOA和RPOD的氨基酸。我们将建立隔离Trar的稳定复合物和RPOD的C末端片段的条件，以确定复合物的晶体结构的努力。在第二个目标中，我们将评估Trar与抗激活器电车之间相互作用的性质。我们已经建立了一个协作，以确定电车和TRAR形成的配合物的晶体结构以及晶体结构。我们还将探测电车和LON蛋白酶在使用生理测试来测量TRAR更新速率的TRAR稳定性中的作用。我们还将使用纯化的蛋白质建立一个体外系统，以表征Tram的LON介导的降解。在第三个目标中，我们建议通过隔离和表征响应新型植物信号诱导转移基因的质粒来探索Ti质粒群体感应系统的进化多样性。