Structural and Functional Analysis of a Quorum-Sensing Inhibition Mechanism

细菌可以表现出社会行为，其中之一就是所谓的群体感应。群体感应调节一系列功能，通常涉及宿主相互作用，如发病机制和共生关系，通过信号分子响应细菌种群密度。一旦信号分子（例如，变形杆菌的酰化高丝氨酸内酯或ahl）达到阈值浓度，它就会有效地结合并激活一个转录因子，该转录因子调节负责细菌集体活动的基因的表达。尽管AHL群体感应在微生物系统中的作用已被广泛接受，但对AHL调控回路的机制研究，包括AHL的产生和扩散、AHL应答转录因子的激活和抑制以及基因表达的控制，仍处于早期阶段。本项目的目的是研究农杆菌群体感应的结构和生化基础，农杆菌是一种模式植物病原体，也是群体感应机制研究的首要系统，使用一系列遗传、生化和结构方法，特别是蛋白质x射线晶体学。在a . tummefaciens中，群体感应需要ahl响应的TraR转录因子，在非诱导条件下，该蛋白通过形成高度稳定的杂合体而被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活性的生化和功能复杂性。其次，将通过x射线晶体学确定抗活化剂杂络合物（TraM- trar - ahl）的结构，并辅以生物物理和动力学技术对该复合物进行生化分析，研究TraM的抑制机制。第三，将进行功能研究，以验证通过共晶结构揭示的TraM抑制TraR功能的预测。这些研究将首次对群体感应途径中转录因子抑制的机制方面进行结构研究，并首次对TraR上的TraM拮抗分子事件进行动力学表征。这项工作的结果将为TraM-TraR相互作用提供一个全面的结构-功能理解。这些发现可能会导致通过故意干预其群体感应来限制农业环境中传染性肿瘤杆菌传播的新方法。这些研究也将在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，如设计干扰细菌通讯的化合物，以控制和对抗微生物入侵。这项工作还将为本科生的参与和研究生的培养提供丰富的机会，与印第安纳大学丰富的教育环境相结合。

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.