N-terminal acylation of Lipoproteins in Firmicutes

厚壁菌门中脂蛋白的 N 末端酰化

• 批准号: 10187587
• 负责人: Timothy C. Meredith
• 金额: $ 31.41万
• 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/10187587
• 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.

项目概要 该应用的长期目标是了解厚壁菌门中的脂蛋白为何以及如何产生 脂蛋白是锚定在细菌上的膜相关球蛋白。 膜表面通过脂化的N-末端半胱氨酸残基它们是普遍存在的细胞膜。 在革兰氏阳性菌和阴性菌中均发现的结构，占典型细菌中所有基因的 1-5% 细菌基因组在细菌细胞生理学的几乎各个方面都发挥着作用。 营养物质的获取对介导细胞接触也很重要。 由于其多种重要的细胞作用，增强初始免疫反应以清除细菌感染。 丰度、普遍分布及其独特且高度保守的结构，先天免疫检测到 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。 根据 N-酰化状态，使用 TLR-1 或 TLR-6 通过形成异二聚体来形成脂蛋白。 因此，了解某些细菌如何以及为何利用脂蛋白结构变异与以下方面相关： 基础细菌生理学和感染生物学。 特别是，该应用研究了粪肠球菌中N-酰化的作用。 有证据表明，厚壁菌门 A 类细菌中脂蛋白的 N 酰化程度存在差异。 在粪肠球菌中鉴定出名为 Lit 的整合膜 N-酰化蛋白，该蛋白可产生溶菌形式 该生物体中的脂蛋白的溶酶体具有独特的酰基链分布模式。 虽然二酰基甘油形式在甘油基残基上具有两条酰基链，而三酰基形式则具有这些 两条酰基链加上第三条 N 端酰基链，通过酰胺键与 α-氨基半胱氨酸连接 基团，溶血形式在 N 末端和甘油基单元上都有一个酰基链。 表明酰基链从二酰基甘油基单元中移除并转移至 N 末端以形成 溶血结构或有脂蛋白酯酶与 Lit 协同工作。 通过重组表达重建 Lit 活性，以便使用以下方法测试所提出的分子内转移酶 专门标记的二酰基甘油脂蛋白底物将通过 MALDI 质量进行表征。 光谱测定法确定酰基链供体来源的第二个目的是测试环境压力条件。 为了使用粪肠球菌/Δlit 同基因配对菌株组深入了解 N-酰化的生物学作用。 第三个目标是探究 Lit 如何影响 TLR-2 信号传导，因为初步数据表明溶血形式 脂蛋白与 TLR2 的结合方式比其三酰基甘油和二酰基甘油同源物更复杂。 的目标是开发功能测定法来发现其他脂蛋白 N 末端修饰酶。