Membrane protein effectors of pathogen interactions with host

 描述(由申请人提供)：这个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如何介导病原体与其人类宿主的相互作用。我们关注于鼠疫的病原体--鼠疫耶尔森氏菌的外细胞膜上表达的一种蛋白AIL(Attach Inventory Locus)。鼠疫耶尔森氏菌具有高致病性，传播迅速，导致极高的人类死亡率。尽管它对有限的抗生素小组敏感，但具有生物工程抗生素耐药性的雾化细菌的潜在武器化以及缺乏有效的疫苗或治疗方法是主要问题，导致其被归类为一级生物制剂。AIL是一种关键的细菌毒力因子，由于其介导细菌与宿主细胞的黏附，并促进细菌对人类免疫的抵抗力，因此是治疗开发的首选候选药物。本项目致力于通过确定AIL的三维结构和结构表征AIL与其人类配体的相互作用来阐明AIL这些关键功能的分子机制。这些研究将集中在AIL及其络合物的样品上，因为AIL的生物活性在胶束洗涤剂的存在下发生了显着的变化。此外，这些研究将利用核磁共振波谱，这是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胞外环路是高度动态的。最后，这些研究将通过开发专门为磷脂双层膜功能环境中的完整膜蛋白的核磁共振结构分析而定制的计算方法，来补充在脂质双层膜中产生的AIL的实验数据。这些方法的设计将有助于结构确定和提高结构质量，并将与广泛使用的核磁共振结构计算程序集成在一起，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.