Structural and Functional Analysis of a Quorum-Sensing Inhibition Mechanism

群体感应抑制机制的结构和功能分析

• 批准号: 0416447
• 负责人: Lingling Chen
• 金额: $ 41.97万
• 依托单位: Indiana University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-04-01 至 2010-03-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0416447&HistoricalAwards=false
• 关键词: Structural; Functional; Analysis; Quorum; Sensing

Bacteria can exhibit social behaviors, one of which is the process known as quorum sensing. Quorum sensing regulates a range of functions, often those involved with host interactions such as pathogenesis and symbiosis, in response to bacterial population density via signal molecules. Once the signal molecule (e.g. acylated homoserine lactones or AHLs for the Proteobacteria) reaches a threshold concentration, it effectively binds and activates a transcription factor(s), which regulates expression of genes responsible for collective bacterial activities. Although the role of AHL quorum sensing in microbial systems is relatively well accepted, mechanistic studies of the regulatory circuitry, including production and diffusion of AHLs, activation and inhibition of AHL-responsive transcription factors, and control of gene expression, are still at an early stage. The aim of this project is to examine the structural and biochemical basis of quorum sensing in Agrobacterium tumefaciens, a model plant pathogen and a premier system for mechanistic studies of quorum sensing, using a range of genetic, biochemical and structural methods particularly protein X-ray crystallography. In A. tumefaciens quorum sensing requires the AHL-responsive TraR transcription factor, and under non-inducing conditions this protein is inhibited by the TraM anti-activator through formation of a highly stable heterocomplex. The objectives of this research are as follows. First, structural and functional studies of TraM, based on the PIs' recently solved crystal structure of TraM, will be continued. These efforts will dissect the biochemical and functional complexity of TraM activity. Second, the TraM inhibitory mechanism will be studied by determining the structure of the anti-activator heterocomplex (TraM-TraR-AHL) using X-ray crystallography, complemented with biochemical analysis of this complex using biophysical and kinetic techniques. Third, functional studies will be performed to test the predictions revealed through the co-crystal structure on TraM inhibition of TraR function. These studies will represent the first structural studies on mechanistic aspects of transcription factor inhibition in a quorum sensing pathway, and the first kinetic characterizations on the molecular events of TraM antagonism on TraR. Results from this work will provide a comprehensive structure-function understanding of TraM-TraR interaction. These findings could lead to novel approaches for limiting the spread of infective A. tumefaciens in agricultural situations by the deliberate intervention into their quorum sensing. These studies will also have significant impact in the area of microbial cell-to-cell communication, and may help develop novel approaches such as the design of chemical compounds that interfere with bacterial communication, to control and combat microbial invasion. This work will also provide abundant opportunities for the involvement of undergraduates and the training of graduate students, integrating with the rich educational environment at Indiana University.

细菌可以表现出社会行为，其中之一是被称为群体感应的过程。 群体感应调节一系列功能，通常涉及宿主相互作用，如致病和共生，通过信号分子响应细菌种群密度。 一旦信号分子（例如，用于变形菌的酰化高丝氨酸内酯或AHL）达到阈值浓度，其有效地结合并激活转录因子，所述转录因子调节负责集体细菌活性的基因的表达。 虽然阿勒群体感应在微生物系统中的作用是相对较好地接受，机制的研究，包括生产和扩散的AHL，激活和抑制的AHL响应转录因子，和控制基因表达的调节电路，仍然处于早期阶段。 本项目的目的是利用一系列遗传、生物化学和结构方法，特别是蛋白质X射线晶体学方法，研究根癌农杆菌群体感应的结构和生物化学基础。 以.根癌农杆菌群体感应需要AHL响应性TraR转录因子，并且在非诱导条件下，该蛋白质通过形成高度稳定的异源复合物而被TraM抗激活剂抑制。 本研究的目的如下。 首先，基于PI最近解决的TraM晶体结构，将继续进行TraM的结构和功能研究。 这些努力将剖析TraM活动的生物化学和功能复杂性。 其次，TraM抑制机制将通过使用X射线晶体学确定抗激活剂杂合物（TraM-TraR-阿勒）的结构来研究，并使用生物物理和动力学技术对该复合物进行生化分析。 第三，将进行功能研究以测试通过共晶体结构揭示的关于TraM抑制TraR功能的预测。 这些研究将代表第一个结构的研究机制方面的转录因子抑制在群体感应途径，和第一个动力学特性的分子事件的TraM拮抗TraR。 这项工作的结果将提供一个全面的结构-功能的理解TraM-TraR相互作用。 这些发现可能会导致新的方法来限制传染性A。在农业环境中，通过故意干预它们的群体感应来控制根瘤菌。 这些研究也将对微生物细胞间通讯领域产生重大影响，并可能有助于开发新的方法，例如设计干扰细菌通讯的化合物，以控制和对抗微生物入侵。这项工作也将为本科生的参与和研究生的培养提供丰富的机会，与印第安纳州大学丰富的教育环境相结合。