CELL-CELL SIGNALING IN MICROBE-HOST INTERACTIONS

微生物-宿主相互作用中的细胞-细胞信号传导

基本信息

批准号：
6627195
负责人：
Stephen Kendall Farrand
金额：
$ 32万
依托单位：
UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
依托单位国家：
美国
项目类别：
财政年份：
1995
资助国家：
美国
起止时间：
1995-05-01 至 2004-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6627195
关键词：
Agrobacterium tumefaciens DNA footprinting bacterial genetics biological signal transduction gene deletion mutation gene expression gene induction /repression genetic regulatory element host organism interaction lactones proteolysis serine transcription factor

项目摘要

Interactions between microbes and their hosts, including man, depend upon 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. As a part of this interaction, the bacterium responds to a plant signal by eliciting a second signal 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, an acyl- homoserine lactone called AAI regulates the activity of TraR, the quorum-sensing transcriptional activator required for expression of the Ti plasmid conjugation system. There are three goals for the project period. First, primer extension, and DNA footprinting will be used to determine how AccR, the plant signal-responsive repressor regulates a divergent promoter system responsible for expression of TraR. This region contains several regulated promoters; one controlled by phosphorus availability and two or more controlled by AccR. Deletions will be used to identify cis acting elements responsible for these activities. Second, using wild-type and mutant alleles, novel genetic screens as well as in vitro assays with purified protein will be employed to examine the multimeric nature of TraR, the role played by AAI in converting TraR into an active activator, for forming multimers, for binding promoters, and for activating transcription. The nature of the interaction between unactivated TraR and inner membrane will be determined, as well as the role of AAI in converting TraR into a cytoplasmic protein. The role of Agrobacterium RpoA in gene activation by TraR will be assessed. If they interact regions of the two proteins involved in this interaction will be identified. Mutants of TraR affected in affinity for AAI will be isolated and used to probe the mechanism by which AAI activates the protein. Third, genetic screens, as well as in vitro interaction assays will be used to determine how the antiactivator, TraM, interacts with TraR. Using physiological and biochemical assays the role of TraM in targeting TraR for proteolytic degradation will be determined. The pathways for this proteolysis will be identified by isolating and characterizing Agrobacterium mutants that are unable to degrade TraR in the presence and absence of AAI.

微生物及其主机（包括人）之间的相互作用取决于特定的通信系统。因此，包括病原体在内的细菌通过感测化学信号来感知其宿主并做出适当反应。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，细菌通过引起整个细菌种群感知的第二个信号来响应植物信号。第二个，群体感应信号控制了Ti质粒（一种毒力元件）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，包括如何调节Ti质粒共轭系统表达的群体感应转录激活剂的偶联传感信号如何调节TRAR的活性。项目期间有三个目标。首先，将使用底漆延伸和DNA足迹来确定植物信号响应阻遏物如何调节负责TRAR表达的发散启动子系统。该区域包含几个受管制的启动子；一种由磷的可用性控制，两个或多个由ACCR控制。删除将用于识别负责这些活动的CIS表演元素。其次，使用野生型和突变等位基因，将使用新型的遗传筛选以及具有纯化蛋白质的体外测定法检查TRAR的多聚体性质，TRAR的多聚体性质，AAI在将TRAR转换为活性激活剂中，用于形成多聚体，用于结合启动子和激活转录。将确定未激活的Trar和内膜之间相互作用的性质，以及AAI在将TRAR转化为细胞质蛋白中的作用。将评估农杆菌RPOA在TRAR基因激活中的作用。如果它们相互相互作用的区域，则将确定参与这种相互作用的蛋白质。将分离出对AAI亲和力的Trar的突变体将被分离，并用于探测AAI激活蛋白质的机制。第三，将使用遗传筛选以及体外相互作用测定法来确定抗激活因子如何与TRAR相互作用。使用生理和生化测定，将确定电车在靶向Trar进行蛋白水解降解中的作用。这种蛋白水解的途径将通过分离和表征在存在和不存在AAI的情况下无法降解Trar的农业突变体来识别。