N-terminal acylation of Lipoproteins in Firmicutes

厚壁菌门中脂蛋白的 N 末端酰化

• 批准号: 10394728
• 负责人: Timothy C. Meredith
• 金额: $ 31.4万
• 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/10394728
• 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 这项应用的长期目标是了解为什么以及如何从菲米库特中提取脂蛋白 进行修改。脂蛋白是固定在细菌上的膜相关球状蛋白。 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包膜 在革兰氏阳性和阴性细菌中都发现了结构，占典型肺炎中所有基因的1-5% 细菌基因组。脂蛋白在细菌细胞包膜生理的几乎每个方面都发挥着作用，从 从营养获取到调节细胞接触。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。由于它们具有多种基本的细胞功能， 丰度，普遍分布，及其独特和高度保守的结构，先天免疫检测 通过Toll样受体2(TLR-2)复合体结合脂蛋白，细菌的存在。TLR2绑定 脂蛋白通过形成异二聚体，根据N-酰化状态使用TLR-1或TLR-6。 因此，了解某些细菌如何以及为什么利用脂蛋白结构变异对两者都有关系。 基础细菌生理学和感染生物学。 特别是，这项应用研究了N-酰化在粪肠球菌中的作用。有一个正在出现的 脂蛋白在细菌中N-酰化程度不同的证据。一类人 在粪肠球菌中鉴定出一种完整的膜N-酰化蛋白LIT，该蛋白可产生溶菌体 这种有机体中的脂蛋白。脂蛋白的溶血型具有独特的酰基链分布模式。 而二酰甘油形式在甘油残基上具有两个酰基链，而三酰基形式具有这些 两个酰基链加上第三个N-末端的酰基链，通过酰胺键连接到α-氨基半胱氨酸 裂解形式在N-末端和甘油基团上都有单一的酰基链。里索结构 暗示从二酰基甘油基团上去掉一条酰基链，并转移到N-末端形成 溶质结构或有脂蛋白酯酶与LIT协同工作。该应用程序旨在 通过重组表达来重建LIT活性，以检测所提出的分子内转移酶 特别标记的二酰甘油基脂蛋白底物。产品将通过MALDI质量进行表征 光谱分析以确定酰链给体来源。第二个目标是测试环境压力条件 为了利用粪肠球菌/ΔLIT等基因配对菌株组深入了解N-酰化的生物学作用。 第三个目标是探索LIT如何影响TLR-2信号，因为初步数据表明它是Lyso形式的 脂蛋白与TLR2结合的方式比它们的三酰基和二酰甘油同系物复杂得多。最终目标 是开发功能分析来发现其他脂蛋白N末端修饰酶。