Structural and Functional Studies of Bacterial Adhesins and Protein Nanotubes

细菌粘附素和蛋白质纳米管的结构和功能研究

• 批准号: RGPIN-2014-06218
• 负责人: Audette, Gerald
• 金额: $ 2.55万
• 依托单位: York University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2015
• 资助国家: 加拿大
• 起止时间: 2015-01-01 至 2016-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=588876
• 关键词: Structural; Functional; Studies; Bacterial; Adhesins

The ability to stick to surfaces is an essential capability of bacteria that enables growth and adaptation in various environments. Bacteria routinely produce complex nanoscale functional structures such as flagella and pili (fibre-like protein polymers) for use in motility, as well as surface and cellular adherence and to transfer DNA and other molecules across membranes. These protein-based adhesion systems are very robust and are assembled and disassembled rapidly by the cell through the use of various secretion systems, which are themselves highly specialized nanomachines. Our research program aims to understand at a structural level how these systems assemble, and how their multiple interactions affect infection and virulence, increase genetic diversity and propagate resistance strategies. One main focus of our research program is the type II secretion system (T2SS) that assembles the type IV pilus (T4P) in several organisms, including Pseudomonas aeruginosa. We are building upon our work characterizing the assembly of protein nanotubes (PNTs) from an engineered form of the P. aeruginosa type IV pilin. Specifically, our research is focusing on: (a) a structural characterization the PNT assembly process, both in solution and at surfaces; (b) exploring the integration of the PNT architecture into functional biosensors and nanodevices; and (c) characterizing the sensing and signal transduction properties of the PNTs within the context of a functional biosensor targeting specific biochemical signals/triggers. A second focus of our research program studies the T4P and T2SS assembly machinery used by the gram-negative bacteria Francisella tularensis and Coxiella burnetii. Both organisms have recently been shown to assemble T4P through associated T2SSs and that the pilus mediates adherence to cells during infection. In addition, methods for testing for Coxiella and Francisella infections are limited. Our research focuses on the structural characterization of the type IV pilins and T2SS component proteins of both F. tularensis and C. burnetii, primarily through X-ray crystallography. We will also explore the possibility of adapting these type IV pilins for biosensor applications using analogous strategies to our studies of the P. aeruginosa PNTs. Our research will provide structural and functional insights into the roles that these T4P play in surface adherence, their mechanisms of assembly, and the adaptability of these proteins for biosensor applications. We are also exploring the type IV secretion system (T4SS) of the F plasmid from E. coli. This system is a main route for the transfer of DNA among a bacterial population, a process known as conjugation, which leads to greater diversity, adaptability and resistance to challenging environments. We are interested in understanding how the proteins of the F-T4SS interact structurally to assemble the conjugative pilus. These studies will provide a clearer picture into how these systems are assembled, leading to the design of new strategies to combat the spread of DNA through T4SS-mediated conjugation. Our research program encompasses a range of biochemical, physical and analytical methods to study several bacterial adhesins and their associated assembly systems. It will lead to a greater understanding of how these structures are assembled, the specific interactions that occur at the binding interface during surface/cellular adherence, and will further advance pilin-derived PNTs for applications in biosensors.

粘附在表面上的能力是细菌的一种基本能力，它使细菌能够在各种环境中生长和适应。细菌通常产生复杂的纳米级功能结构，如鞭毛和皮利（纤维样蛋白聚合物），用于运动，以及表面和细胞粘附，并转移DNA和其他分子穿过膜。这些基于蛋白质的粘附系统非常强大，并且通过使用各种分泌系统由细胞快速组装和分解，这些分泌系统本身是高度专业化的纳米机器。我们的研究计划旨在了解这些系统如何在结构层面组装，以及它们的多重相互作用如何影响感染和毒力，增加遗传多样性和传播抗性策略。 我们的研究计划的一个主要重点是II型分泌系统（T2 SS），组装IV型菌毛（T4 P）在几种生物体，包括铜绿假单胞菌。我们正在建立我们的工作特征的蛋白质纳米管（PNT）的组装从铜绿假单胞菌IV型菌毛蛋白的工程形式。具体而言，我们的研究集中在：（a）PNT组装过程的结构表征，无论是在溶液中还是在表面上;（B）探索PNT架构整合到功能性生物传感器和纳米器件中;以及（c）在功能性生物传感器的背景下表征PNT的传感和信号转导特性，以特定的生化信号/触发器为目标。 我们研究计划的第二个重点是研究革兰氏阴性菌土拉弗朗西斯菌和贝氏柯克斯体所使用的T4 P和T2 SS组装机制。这两种生物体最近已被证明通过相关的T2 SS组装T4 P，并且在感染期间菌毛介导粘附细胞。此外，用于检测Coxiella和Francisella感染的方法有限。我们的研究集中在F. tularensis和C. burnetii，主要是通过X射线晶体学。我们还将探索使用与我们对铜绿假单胞菌PNT的研究类似的策略使这些IV型菌毛蛋白适应生物传感器应用的可能性。我们的研究将提供结构和功能的见解，这些T4 P在表面粘附中发挥的作用，它们的组装机制，以及这些蛋白质对生物传感器应用的适应性。 我们还对E.杆菌该系统是细菌群体之间DNA转移的主要途径，这一过程称为接合，导致更大的多样性，适应性和对挑战性环境的抵抗力。我们有兴趣了解F-T4 SS的蛋白质如何在结构上相互作用以组装接合菌毛。这些研究将为这些系统如何组装提供更清晰的画面，从而设计新的策略来对抗通过T4 SS介导的结合传播的DNA。 我们的研究计划包括一系列的生物化学，物理和分析方法来研究几种细菌粘附素及其相关的组装系统。这将导致更好地了解这些结构是如何组装的，在表面/细胞粘附过程中在结合界面发生的特定相互作用，并将进一步推进菌毛蛋白衍生的PNT在生物传感器中的应用。