Membrane protein effectors of pathogen interactions with host

 描述（由申请人提供）：本项目的总体目标是开发一个框架，用于了解病原体细胞膜上表达的膜蛋白介导病原体与其人类宿主相互作用的方式。我们专注于Ail（附着入侵位点），一种在鼠疫耶尔森氏菌（Yersinia pestis）外细胞膜中表达的蛋白质，它是鼠疫的病原体。鼠疫耶尔森鼠疫菌致病性强，传播迅速，人类死亡率极高。虽然它对有限的抗生素敏感，但具有生物工程抗生素抗性的气溶胶细菌的潜在武器化以及缺乏有效的疫苗或疗法是主要问题，有助于将其归类为1级生物威胁剂。Ail是一种关键的细菌毒力因子，由于其介导细菌粘附宿主细胞和促进细菌对人类免疫的抗性的功能，是治疗开发的主要候选者。本项目的重点是阐明这些关键功能的Ail的分子机制，通过确定其三维结构和结构特征的Ail与其人配体的相互作用。这些研究将集中在AIL及其配合物的样品中纳入无洗涤剂的脂质双层膜，因为AIL的生物活性显着改变胶束洗涤剂的存在下。此外，这些研究将利用NMR光谱，一种擅长表征结构和动力学的方法，因为介导与宿主相互作用的Ail的细胞外环是高度动态的。最后，这些研究将补充AIL在脂质双层膜中产生的实验数据，通过开发专门针对其磷脂双层膜的功能环境内的膜蛋白的整体NMR结构分析的计算方法。这些方法将被设计为便于结构测定和提高结构质量，并且它们将与广泛使用的NMR结构计算程序相结合，以扩大我们工作的影响。

 DESCRIPTION (provided by applicant): The overall goal of this project is to develop a framework for understanding the way in which membrane proteins expressed on pathogen cell membranes mediate the interactions of pathogens with their human host. We focus on Ail (attachment invasion locus), a protein expressed in the outer cell membrane of the bacterium Yersinia pestis, which is the causative agent of plague. The Y. pestis bacterium is highly pathogenic, spreads rapidly and causes extremely high human mortality. Although it is sensitive to a restricted panel of antibiotics, the potential weaponization of aerolized bacteria with bio-engineered antibiotic resistance and the lack of an effective vaccine or therapy are major concerns, contributing to its classification as a Tier 1 Biothreat Agent. Ail is a key bacterial virulence factor and a prime candidate for therapeutic development due to its functions in mediating bacterial adhesion to host cells, and promoting bacterial resistance to human immunity. This project focuses on elucidating the molecular mechanisms of these key functions of Ail by determining its three-dimensional structure and structurally characterizing the interactions of Ail with its human ligands. These studies will focus on samples of Ail and its complexes incorporated in detergent-free lipid bilayer membranes because the biological activity of Ail is dramatically altered in the presence of micellar detergents. Furthermore, these studies will utilize NMR spectroscopy, a method adept at characterizing both structure and dynamics, because the extracellular loops of Ail that mediate the interactions with host are highly dynamic. Finally, these studies will complement the experimental data generated for Ail in lipid bilayer membranes, by developing computational methods tailored specifically for NMR structural analysis of integral membrane proteins within their functional environment of the phospholipid bilayer membrane. The methods will be designed to facilitate structure determination and increase structural quality, and they will be integrated with widely used programs for programs for NMR structure calculations so as to broaden the impact of our work.