Structure-Function Analysis of AI-2 Quorum Sensing

描述(申请人提供)：这项研究的长期目标是探索细菌用于细胞间交流的分子机制。在这里，我们建议对最近在两个相关细菌--哈维氏弧菌和霍乱弧菌--中发现的群体感应电路进行结构、化学和生物学方面的综合研究。为了从分子上了解群体感应信号(称为自动诱导物)是如何被检测到的，以及如何在社区范围内传递感觉信息来控制行为，我们将进行结合合成有机化学、细菌遗传学、生物化学和X射线结晶学的深入研究。我们将确定信号激动剂和拮抗剂，为设计用于调节群体感应的抗菌药物的开发提供先导化合物。更广泛地说，细菌信号领域的一个长期问题是了解细胞外信息是如何传递到细胞内的。这些拟议的研究将进一步加深我们对通过双组分传感器激酶进行跨膜信号转导的机制的理解，其中这些群体感应受体是特别容易处理的例子。拟议的目标建立在第一个资助期的重大进展的基础上，其中广泛的结构/功能研究导致了通过群体感应受体LuxPQ进行信号转导的特定机制模型。这种机制与基于趋化受体研究的典型机制有根本的不同。在第一个目标中，我们将使用分子遗传学方法结合X射线结晶学来测试和扩展我们的模型。第二个目标是利用有机合成和高通量筛选来鉴定新的LuxPQ激动剂和拮抗剂。将使用生物化学和结构研究来描述它们的作用模式。AIMS 3和4代表了一项新的努力，以表征人类病原体霍乱弧菌中主导群体感应途径的分子机制。在初步研究中，我们已经确定了相关的自动诱导剂CAI-1的化学结构。我们已经提纯并结晶了一种名为CqsA的磷酸吡哆醛合成酶，第三个目的是确定它的结构，鉴定它的底物，并研究它的作用机制。在第四个目标中，我们将结合遗传和化学筛选与X射线结晶学来探索CAI-1与其细胞受体相互作用的分子细节。与公共卫生相关：群体感应是一种细胞间的交流过程，允许细菌共同控制包括生物膜形成和毒力因子分泌在内的过程。我们建议研究人类主要病原体霍乱弧菌的群体感应，并识别针对群体感应的分子以抑制毒力。

DESCRIPTION (provided by applicant): The long-term goal of this research is to explore the molecular mechanisms that bacteria use for cell-cell communication. Here we propose an integrated structural, chemical, and biological study of recently identified quorum sensing circuits in two related bacteria, Vibrio harveyi and Vibrio cholerae. To develop a molecular understanding of how quorum sensing signals (called autoinducers) are detected, and how sensory information is transduced to control behavior on a community-wide scale, we will carry out in-depth studies that combine synthetic organic chemistry, bacterial genetics, biochemistry, and x-ray crystallography. We will identify signaling agonists and antagonists to provide lead compounds for the development of antibacterial drugs designed to modulate quorum sensing. More generally, a longstanding problem in the bacterial signaling field is to understand how extracellular information is transduced into cells. The proposed studies will further our mechanistic understanding of transmembrane signal transduction via two-component sensor kinases, of which these quorum sensing receptors represent particularly tractable examples. The proposed aims build on significant progress during the first funding period, in which extensive structure/function studies led to a specific mechanistic model for signal transduction by the quorum sensing receptor LuxPQ. This mechanism differs fundamentally from the canonical mechanism based on studies of chemotaxis receptors. In the first aim, we will use molecular genetic approaches coupled with x-ray crystallography to test and extend our model. The second aim is to use organic synthesis and high-throughput screening to identify novel LuxPQ agonists and antagonists. Biochemical and structural studies will be used to characterize their mode of action. Aims 3 and 4 represent a new effort to characterize the molecular mechanisms underlying the dominant quorum sensing pathway in the human pathogen V. cholerae. In preliminary studies, we have determined the chemical structure of the relevant autoinducer, CAI-1. We have purified and crystallized the CAI-1 synthase CqsA, a pyridoxal phosphate enzyme, and in the third aim, we propose to determine its structure, identify its substrates, and characterize its enzymatic mechanism. In the fourth aim, we will combine genetic and chemical screens with x-ray crystallography to probe the molecular details of the interaction between CAI-1 and its cellular receptor. PUBLIC HEALTH RELEVANCE: Quorum sensing is a process of cell-cell communication that allows bacteria to collectively control processes including biofilm formation and the secretion of virulence factors. We propose to study quorum sensing in the major human pathogen, Vibrio cholerae, and to identify molecules that target quorum sensing to inhibit virulence.