N-terminal acylation of Lipoproteins in Firmicutes

厚壁菌门中脂蛋白的 N 末端酰化

• 批准号: 9923708
• 负责人: Timothy C. Meredith
• 金额: $ 31.42万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-05-01 至 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9923708
• 关键词: Accounting; Acylation; Affect; Affinity; Amides; Bacteria; Bacterial Genome; Bacterial Infections; Bacterial Physiology; Bacterial Proteins; Binding; Biological; Biological Assay; Biology; Cell Adhesion; Cell membrane; Complex; Copper; Cysteine; Data; Databases; Detection; Deuterium; Diglycerides; Elements; Enterococcus faecalis; Environment; Environmental Monitoring; Enzymes; Firmicutes; Genes; Genetic Transcription; Glycerol; Goals; Gram-Negative Bacteria; Gram-Positive Bacteria; Immune response; Immune system; In Vitro; Infection; Integral Membrane Protein; Ions; Label; Lipoprotein Binding; Lipoproteins; Mass Spectrum Analysis; Measures; Mediating; Membrane; Methods; Modification; Monitor; Multiprotein Complexes; Mutase; N acylation; N-terminal; Names; Natural Immunity; Nutrient; Operon; Organism; Pathway interactions; Pattern; Phenotype; Phospholipids; Physiology; Plasmids; Play; Process; Proteins; Receptor Signaling; Recombinants; Report (document); Reporter; Research; Resistance; Role; Signal Transduction; Source; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Staphylococcus aureus; Stress; Structure; Surface; System; TLR1 gene; TLR2 gene; TLR6 gene; Testing; Time; Toll-like receptors; Variant; Virulence; Virulence Factors; amino group; antimicrobial drug; bacterial fitness; beta-Galactosidase; cell envelope; esterase; fluorophore; genome-wide; globular protein; insight; novel; paralogous gene; pressure; prevent; promoter; reconstitution; response; theories; uptake; whole genome

Project Summary The long term goal of the application is to understand why and how lipoproteins from Firmicutes undergo modifications. Lipoproteins are membrane associated globular proteins anchored to the bacterial membrane surface through a lipidated N-terminal cysteine residue. They are ubiquitous cell envelope structures found in both gram positive and negative bacteria, accounting for 1-5% of all genes in a typical bacterial genome. Lipoproteins play roles in nearly every aspect of bacterial cell envelope physiology, from nutrient acquisition to mediating cellular contacts. Lipoproteins are also important during the detection and mounting of initial immune responses to clear bacteria infections. Due to their multiple essential cellular roles, abundance, universal distribution, and their unique and highly conserved structure, innate immunity detects the presence of bacteria through binding lipoproteins using Toll-like receptor 2 (TLR-2) complexes. TLR2 binds lipoproteins through forming heterodimers, using either TLR-1 or TLR-6 depending on the state of N-acylation. As such, understanding how and why certain bacteria utilize lipoprotein structural variations is pertinent to both fundamental bacterial physiology and infection biology. In particular, this application studies the role of N-acylation in Enterococcus faecalis. There is emerging evidence that lipoproteins are differentially N-acylated amongst bacteria in the Firmicutes phylum. A class of integral membrane N-acylating proteins named Lit was identified in E. faecalis that makes lyso- form lipoproteins in this organism. The lyso- form of lipoprotein has a unique acyl chain distribution pattern. Whereas the diacylglyceryl form has both acyl chains on the glyceryl residue, and the triacyl form has these two acyl chains plus a third N-terminal acyl chain connected through an amide bond to the α-amino cysteine group, the lyso-form has a single acyl chain on both the N-terminus and glyceryl unit. The lyso- structure suggests an acyl chain is removed from the diacylglyceryl unit and transferred to the N-terminus to form the lyso- structure or that there are lipoprotein esterases working concert with Lit. The application aims to reconstitute Lit activity by recombinant expression so as to test the proposed intramolecular transferase using specifically labeled diacylglyceryl lipoprotein substrates. The products will be characterized by MALDI mass spectrometry to determine acyl chain donor origin. A second aim is to test environmental stress condition in order to gain insight into the biological role for N-acylation using the E. faecalis/Δlit isogenic paired strain set. The third aim is to probe how Lit affects TLR-2 signaling, as preliminary data has suggested lyso-form lipoproteins bind in more complex ways to TLR2 than their triacyl- and diacyl-glycerol congeners. The final aim is to develop functional assays to uncover other lipoprotein N-terminal modifying enzymes.

项目摘要 该应用程序的长期目标是了解厚壁菌门的脂蛋白为什么以及如何 进行修改。脂蛋白是一种膜相关的球状蛋白质， 膜表面通过脂化的N-末端半胱氨酸残基。它们是无处不在的细胞被膜 在革兰氏阳性和阴性细菌中都发现了这种结构，占典型细菌中所有基因的1-5%。 细菌基因组脂蛋白几乎在细菌细胞包膜生理学的各个方面都发挥作用， 营养获取到介导细胞接触。脂蛋白在检测过程中也很重要， 对清除细菌感染的初始免疫反应的增加。由于其多种重要的细胞作用， 丰富，普遍分布，以及它们独特和高度保守的结构，先天免疫检测到的免疫系统。 通过使用Toll样受体2（TLR-2）复合物结合脂蛋白来检测细菌的存在。TLR 2结合 根据N-酰化状态，使用TLR-1或TLR-6通过形成异源二聚体与脂蛋白结合。 因此，了解某些细菌如何以及为什么利用脂蛋白结构变异与这两个方面有关。 基础细菌生理学和感染生物学。 特别地，本申请研究了N-酰化在粪肠球菌中的作用。正在形成 这表明在厚壁菌门的细菌中脂蛋白是不同的N-酰化。一类 在E.使之形成溶菌的粪便 脂蛋白。脂蛋白的溶血形式具有独特的酰基链分布模式。 而二酰基甘油形式在甘油基残基上具有两个酰基链，并且三酰基形式具有这些酰基链。 两条酰基链加上第三条N-末端酰基链，通过酰胺键与α-氨基半胱氨酸连接 溶血形式在N-末端和甘油基单元上都具有单个酰基链。溶酶体结构 表明酰基链从二酰基甘油基单元中除去并转移到N-末端以形成 溶血结构或有脂蛋白酯酶与Lit协同工作。该申请旨在 通过重组表达重建Lit活性，以便使用 专门标记的二酰基甘油脂蛋白底物。产品将通过MALDI质量进行表征 光谱法以确定酰基链供体来源。第二个目的是测试环境压力条件， 为了深入了解N-酰化的生物学作用，使用E. faecalis/Δlit等基因配对菌株集。 第三个目的是探索Lit如何影响TLR-2信号传导，因为初步数据表明Lit是溶血形式的。 脂蛋白以比它们的三酰基-和二酰基-甘油同源物更复杂的方式与TLR 2结合。最终目的 是开发功能性检测，以发现其他脂蛋白N-末端修饰酶。