Structure-Function Analysis of AI-2 Quorum Sensing

AI-2群体感应的结构功能分析

• 批准号: 8112157
• 负责人: FREDERICK M HUGHSON
• 金额: $ 11.82万
• 依托单位: PRINCETON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-15 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8112157
• 关键词: Agonist; Anti-Bacterial Agents; Bacteria; Behavior Control; Biochemical; Biochemistry; Biological; Cell Communication; Cell physiology; Cells; Chemical Structure; Chemicals; Chemotaxis; Communities; Coupled; Crystallography; Development; Drug Design; Enzymes; Funding; Goals; Human; Lead; Microbial Biofilms; Modeling; Molecular; Molecular Genetics; Molecular Probes; Names; Organic Chemistry; Organic Synthesis; Pathway interactions; Phosphotransferases; Principal Investigator; Process; Pyridoxal Phosphate; Research; Resource Sharing; Sensory; Signal Transduction; Structure; Testing; Vibrio; Vibrio cholerae; Virulence; Virulence Factors; bacterial genetics; base; chemical genetics; extracellular; high throughput screening; novel; pathogen; programs; public health relevance; quorum sensing; receptor; sensor

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.

描述（由申请人提供）：本研究的长期目标是探索细菌用于细胞间通讯的分子机制。在这里，我们提出了一个综合的结构，化学和生物学研究最近确定的群体感应电路在两个相关的细菌，哈维氏弧菌和霍乱弧菌。为了从分子上了解群体感应信号（称为自诱导物）是如何被检测到的，以及感觉信息是如何被转导到社区范围内控制行为的，我们将结合联合收割机合成有机化学、细菌遗传学、生物化学和X射线晶体学进行深入研究。我们将鉴定信号激动剂和拮抗剂，为开发旨在调节群体感应的抗菌药物提供先导化合物。更一般地说，细菌信号领域的一个长期问题是了解细胞外信息是如何被转导到细胞中的。拟议的研究将进一步我们的跨膜信号转导机制的理解，通过双组分传感器激酶，其中这些群体感应受体代表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，其中广泛的结构/功能研究导致了一个特定的机制模型的信号转导的群体感应受体LuxPQ。这种机制与基于趋化性受体研究的经典机制有根本不同。在第一个目标中，我们将使用分子遗传学方法结合X射线晶体学来测试和扩展我们的模型。第二个目标是使用有机合成和高通量筛选，以确定新的LuxPQ激动剂和拮抗剂。生物化学和结构研究将用于表征其作用模式。目的3和4代表了一个新的努力，以表征在人类病原体霍乱弧菌占主导地位的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自诱导剂CAI-1的化学结构。我们已经纯化和结晶的CAI-1合酶CqsA，磷酸吡哆醛酶，并在第三个目标，我们建议确定其结构，确定其底物，并表征其酶促机制。在第四个目标中，我们将结合联合收割机的遗传和化学筛选与X射线晶体学，探索CAI-1和其细胞受体之间相互作用的分子细节。 公共卫生相关性：群体感应是一种细胞间通讯的过程，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，并确定靶向群体感应以抑制毒力的分子。