Structural studies of bacterial quorum sensing regulator

描述(申请人提供)：持续性细菌感染是囊性纤维化患者和免疫受损患者死亡的主要原因。 个人。包括假单胞菌在内的一些革兰氏阴性细菌 铜绿假单胞菌是囊性纤维化的主要病原体，可引起 很难治疗，因为细菌形成了一个“生物膜群落”，使 他们对传统抗生素不太敏感。仲裁感应，由 酰基高丝氨酸内酯(AHL)信号分子，调节发病和 铜绿假单胞菌生物膜的形成。因此，理解分子 群体感应的基础是小说发展的重中之重 抗菌剂。这个项目的长期目标是延长 了解原子级的群体感应系统，以开发一种 详细描述控制细菌致病的机制。 这项建议的主要焦点是产生AHL的酶的类别。 信号，AHL-合成酶，因为缺乏AHL信号的细菌无法成为 致病或形成稳定的生物膜。尽管有一些机制的模型 AHL-合成酶的作用，目前还没有任何AHL的结构 合成酶。高分辨率的构造信息对于 充分理解AHL的合成机理，为下一步研究提供依据 为今后基于结构的缓蚀剂设计开发提供新的思路 治疗学。 本项目的具体目标是：(一)确定高分辨率 斯氏泛藻亚种的晶体结构。Stewartii AHL合酶(ESAI) 以了解其功能、机制以及与其他酶的关系 利用类似的底物。进行诱变、结合和动力学 用ESAI进行实验，以更好地了解催化机理和 底物专一性。(Ii)研究铜绿假单胞菌AHL-合成酶，LASI，使用 用结构和生化技术了解急性淋巴细胞性白血病的特异性 产量是确定的。(Iii)确定AHL-合成酶同源物是否 在不同的生物体中使用质量产生高丝氨酸内酯信号 光谱分析和活性分析。研究结构和机制，以 确定与其他AHL合成酶的相似性。

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.