Bacterial outer membrane biogenesis through a novel periplasmic protein that recognizes and traffics phospholipids

通过识别和运输磷脂的新型周质蛋白进行细菌外膜生物发生

• 批准号: RGPIN-2018-04994
• 负责人: Overduin, Michael
• 金额: $ 7.29万
• 依托单位: University of Alberta
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=744094
• 关键词: Bacterial; outer; membrane; biogenesis; through

The overall aim of this project is to determine how bacteria recognize and organize lipids in their outer membrane. The challenge we are tackling here is best described by Bruce Alberts, Editor of Science, who stated "I am painfully aware of the huge gap that remains in our understanding of even the simplest cells. Consider, for example, the common bacterium E. coli, which served as a predominant model organism in the early years of molecular biology. It is very sobering to report that more than 50 years later, nearly a quarter of the more than 4000 proteins encoded by its genome have functions that remain unknown. Might some new functional classes of biological molecules, common to all cells, be discovered by a focus on such proteins?"We will address this gap by elucidating the mechanism of an E. coli protein called YraP. There are at least 7000 proteins with similarity to YraP which have been found in a range of bacteria including various pathogens, and hence there are implications for infectious diseases. Moreover, as a unique gene product that is expressed in response to stress, YraP could offer opportunities for the design of antimicrobial agents once its structure, function and screening assays are available. The evolutionary relatives of YraP includes hemolysins, mechanosensitive channels, the membrane-pore forming protein Secretin, and a variety of eukaryotic proteins. We propose that they share a common function based on lipid recognition. Our focus is on understanding the cellular function, molecular interactions and 3D structures of YraP. We will reveal how this lipoprotein engages the phospholipids found in the membrane that surrounds all Gram negative bacteria. The outer membrane's role is to form a semi-permeable layer that controls the entry and exit of nutrients and other materials including drug molecules. The proteins inserted into this membrane include pores, channels and antigens that act as targets of immune responses. We propose that they all depend on YraP to deliver a specific lipid to the outer membrane. We will investigate how YraP recognizes and organizes this lipid, and how it traffics lipid from inside the cell. We will use methods including magnetic resonance spectroscopy to visualize the atoms of the structures and binding interfaces to expose the specific roles of its amino acid residues and lipids they contact. The resulting principles of ligand recognition and membrane biogenesis will inform the discovery of inhibitors, vaccines and antimicrobial agents. This project is collaborative, involving detailed structure-function analysis of a novel target by researchers at Canada's national NMR facility and the UK-based Institute of Microbiology and Infection. We will capitalize on our joint discovery of the role of YraP in lipid trafficking, along with a new NMR system geared to elucidate how this superfamily of proteins recognizes lipids, shapes membranes and protect cells.

该项目的总体目标是确定细菌如何识别和组织其外膜中的脂质。我们在这里解决的挑战用《科学》杂志的编辑Bruce Alberts描述得最好，他说：“我痛苦地意识到我们对最简单的细胞的理解仍然存在着巨大的差距。例如，考虑常见的细菌大肠杆菌，它在分子生物学的早期被用作主要的模式生物。令人警醒的是，50多年后，它的基因组编码的4000多种蛋白质中，有近四分之一的功能仍然未知。通过对这些蛋白质的关注，是否会发现一些所有细胞都共有的新的生物分子功能类别？“我们将通过阐明一种名为YraP的大肠杆菌蛋白质的机制来解决这一差距。在包括各种病原体在内的一系列细菌中发现了至少7000种与Yrap相似的蛋白质，因此，这与传染病有关。此外，作为一种独特的应激反应基因产物，YraP一旦其结构、功能和筛选方法可用，就可以为抗菌剂的设计提供机会。YraP的进化近缘包括溶血素、机械敏感通道、膜孔形成蛋白分泌素和多种真核蛋白。我们认为它们共享一个共同的基于脂质识别的功能。我们的重点是了解YraP的细胞功能、分子相互作用和3D结构。我们将揭示这种脂蛋白如何与所有革兰氏阴性细菌周围的膜中发现的磷脂结合。外膜的作用是形成一个半透层，控制营养物质和包括药物分子在内的其他材料的进出。插入到膜中的蛋白质包括作为免疫反应目标的毛孔、通道和抗原。我们认为它们都依赖于YraP将特定的脂质运送到外膜。我们将研究YraP是如何识别和组织这种脂质的，以及它是如何从细胞内运输脂质的。我们将使用包括磁共振光谱在内的方法来可视化结构和结合界面的原子，以揭示其氨基酸残基和它们接触的脂类的具体作用。由此产生的配体识别和膜生物发生原理将为发现抑制剂、疫苗和抗菌剂提供信息。这个项目是合作的，加拿大国家核磁共振设施和英国微生物和感染研究所的研究人员对一个新靶点进行了详细的结构-功能分析。我们将利用我们共同发现的YraP在脂质运输中的作用，以及一个新的核磁共振系统，旨在阐明这个蛋白质超家族如何识别脂质、塑造膜和保护细胞。