Structural studies of bacterial quorum sensing regulator

描述（由申请人提供）：持续性细菌感染是囊性纤维化患者和免疫功能低下患者死亡的主要原因 个人。许多革兰氏阴性细菌，包括假单胞菌 铜绿假单胞菌是囊性纤维化的主要病原体，可引起以下感染： 难以治疗，因为细菌形成“生物膜群落”，导致 他们对传统抗生素不太敏感。群体感应，介导 酰基高丝氨酸内酯 (AHL) 信号分子，调节发病机制和 铜绿假单胞菌中生物膜的形成。因此，了解分子 群体感应的基础是新型药物开发的重中之重 抗菌剂。该项目的长期目标是延长 对原子水平的群体感应系统的理解，以开发 控制细菌发病机制的详细描述。 该提案的主要焦点是产生 AHL 的酶类 信号，AHL 合成酶，因为缺乏 AHL 信号的细菌无法变成 致病性或形成稳定的生物膜。虽然有机制模型 AHL 合成酶的作用，目前没有任何 AHL 的结构 合酶。高分辨率结构信息对于 充分认识AHL合成机理，为AHL合成提供基础 用于未来基于结构的抑制剂设计，用于开发新型 疗法。 本项目的具体目标是：（一）确定高分辨率 Pantoea Steartii 亚种的晶体结构。紫茎泽兰 AHL 合酶 (EsaI) 了解其功能、机制以及与其他酶的关系 使用类似的基材。进行诱变、结合和动力学 使用 EsaI 进行实验以更好地了解催化机制 底物特异性。 (II) 研究铜绿假单胞菌 AHL 合酶 LasI，使用 结构和生化技术来了解 AHL 的特异性 产量确定。 (III) 确定 AHL 合酶同源物是否 在不同的生物体中，利用质量产生高丝氨酸内酯信号 光谱测定和活性测定。研究结构和机制 确定与其他 AHL 合酶的相似性。

DESCRIPTION (provided by applicant): Persistent bacterial infections are a major cause of death in cystic fibrosis patients and immune-compromised individuals. A number of gram-negative bacteria including Pseudomonas aeruginosa, a major pathogen in cystic fibrosis, cause infections that are difficult to treat because the bacteria form a "biofilm community" that renders them less sensitive to traditional antibiotics. Quorum sensing, mediated by acylhomoserine lactone (AHL) signaling molecules, regulates pathogenesis and biofilm formation in P. aeruginosa. Therefore, understanding the molecular basis of quorum sensing is a high priority in the development of novel anti-bacterial agents. The long term goal of this project is to extend the understanding of the quorum-sensing system to the atomic level to develop a detailed description of the mechanisms that control bacterial pathogenesis. The main focus of this proposal is the class of enzymes that produce the AHL signal, AHL-synthases, because bacteria lacking the AHL signal fail to become pathogenic or form stable biofilms. Although there are models of the mechanism of action of the AHL-synthases, there are currently no structures of any AHL synthase. High resolution structural information is absolutely essential for fully understanding the mechanism of AHL synthesis and will provide the basis for future structure-based inhibitor design for development of novel therapeutics. The specific aims for this project are: (I) determine the high resolution crystal structure of the Pantoea stewartii subsp. Stewartii AHL-synthase (EsaI) to understand its function, mechanism, and relationship to other enzymes that utilize similar substrates. Perform mutagenesis, binding and kinetics experiments with EsaI to better understand the catalytic mechanism and substrate specificity. (II) Study the P. aeruginosa AHL-synthase, LasI, using structural and biochemical techniques to understand how specificity of AHL production is determined. (III) Establish whether the AHL-synthase homologues in divergent organisms produce a homoserine lactone signal using mass spectrometry and activity assays. Study the structures and mechanisms to determine similarities to other AHL synthases.