N-terminal acylation of Lipoproteins in Firmicutes

厚壁菌门中脂蛋白的 N 末端酰化

• 批准号: 10737277
• 负责人: Timothy C. Meredith
• 金额: $ 31.89万
• 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 2027-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10737277
• 关键词: Acetates; Acetyl Coenzyme A; Acetylation; Acylation; Address; Adhesions; Amino Acids; Attenuated; Bacillus cereus; Bacillus subtilis; Bacteria; Bacterial Proteins; Binding; Biochemical; Biochemistry; Biological Assay; Carbohydrates; Cell surface; Cells; Chromosomes; Complex; Copper; Cysteine; Cytosol; Drug Metabolic Detoxication; Enterococcus faecalis; Environment; Enzymes; Event; Family; Fatty Acids; Firmicutes; Gene Transfer; Genes; Genetic; Genome; Genus staphylococcus; Germination; Goals; Gram-Negative Bacteria; Growth; Humoral Immunities; Immune system; In Vitro; Infection; Inflammatory; Innate Immune System; Ions; Knock-out; Label; Length; Ligands; Lipase; Lipoprotein (a); Lipoprotein Binding; Lipoproteins; Maps; Measures; Medical; Membrane; Membrane Proteins; Modeling; Modification; Mutation; N acylation; N-terminal; Names; Natural Immunity; Nutrient; Orthologous Gene; Pathogenicity; Pathway interactions; Pattern; Phenotype; Physiological; Physiology; Plasmids; Play; Polyunsaturated Fatty Acids; Positioning Attribute; Process; Protein Secretion; Proteins; Reaction; Recombinants; Reproduction spores; Role; Signal Transduction; Source; Staphylococcus aureus; Structure; Surface; System; TLR2 gene; Testing; Transacylase; Transferase; Variant; Virulence Factors; attenuation; bacterial fitness; cell envelope; cell growth; cell type; chemokine; cytokine; detection platform; extracellular; fitness; gene network; member; microbial; oxidation; oxidative damage; pressure; prevent; protein complex; protein protein interaction; receptor; reconstitution; resistance gene; response; transposon sequencing

Project Summary The long term goal of the application is to understand why and how bacterial lipoproteins in the medically important Firmicutes phylum undergo structural modifications. Lipoproteins are membrane associated proteins tethered to the bacterial surface through an acylated N-terminal cysteine anchor. They are ubiquitous cell envelope structures in both gram-positive and gram-negative bacteria, playing key roles in nearly every aspect of bacterial cell envelope physiology. Due to their functional importance, abundance, universal distribution, and the structurally unique acylated N-terminal cysteine, the innate immune system detects bacteria by binding the N-terminus of lipoproteins using the Toll-like receptor 2 (TLR2) family. TLR2 activation triggers a pro- inflammatory cytokine/chemokine response to clear bacteria as well as to orchestrate humoral immunity. However, there is emerging evidence that acylation patterns in lipoproteins from the Firmicutes phylum are not canonical TLR2 ligands nor are they static in structure. Firmicutes synthesize lipoprotein chemotypes varying in acyl chain number, length, and attachment position using an array of accessory lipoprotein biosynthetic genes. Gene distribution varies at both the genera and species level, and can even differ at the strain level due to circulating plasmid/transposon encoded lipoprotein remodeling genes. Lipoprotein composition is also dynamically regulated by the growth environment, including copper which has been shown to induce expression of certain N-terminal modifying genes. This project aims to uncover the physiological need(s) for distinct lipoprotein N-terminal modification systems and to characterize the enzymes involved. While select lipoprotein structural modifications confer TLR2 evading capabilities in host associated bacteria, lipoprotein N-terminal acylation/acetylation is also found in environmental lineages which suggests a broad selective pressure. Given copper co-induces both lipoprotein N-terminal modification and copper resistance genes, it has been proposed that copper directly binds to the free N-terminus and inhibits cell growth unless N-modified. This project will investigate the impact of N-terminal lipoprotein modifications on copper binding by measuring growth under copper challenge conditions requiring specific lipoprotein functions, using Tn-seq to compare chemotype-specific copper sensitization gene networks, and assaying oxidative damage to model lipopeptides isolated from both actively respiring cells and under in vitro reaction conditions. In the second aim, the recently discovered lipoprotein N-acylating enzymes LnsAB from Staphylococcus aureus will be reconstituted. Targeted mutations, acyl chain donor, and possible protein-protein complex formation will be examined using genetic and biochemical approaches. The final part of the project will examine how N-acetylated lipoproteins are made using genetic transposon screens and biochemical assays, and what impact lipoprotein acetylation has on key phenotypes. By understanding lipoprotein synthesis, the project will shed light on the complex interplay between lipoprotein chemotype, copper exposure, TLR2 recognition, and in turn a critical axis governing host-Firmicutes interactions.

项目概要 该应用程序的长期目标是了解细菌脂蛋白在医学上的原因和方式 重要的厚壁菌门经历结构修饰。脂蛋白是膜相关蛋白 通过酰化的 N 末端半胱氨酸锚定物连接到细菌表面。它们是无处不在的细胞 革兰氏阳性菌和革兰氏阴性菌的包膜结构，几乎在每个方面都发挥着关键作用 细菌细胞包膜生理学。由于它们的功能重要性、丰富性、普遍分布和 结构独特的酰化 N 末端半胱氨酸，先天免疫系统通过结合 使用 Toll 样受体 2 (TLR2) 家族的脂蛋白 N 末端。 TLR2 激活触发亲 炎症细胞因子/趋化因子对清除细菌的反应以及协调体液免疫。 然而，有新的证据表明，厚壁菌门脂蛋白的酰化模式并非如此。 典型的 TLR2 配体在结构上也不是静态的。厚壁菌门合成脂蛋白化学型不同 使用一系列辅助脂蛋白生物合成基因来确定酰基链的数量、长度和附着位置。 基因分布在属和种水平上都有所不同，甚至在品系水平上也会有所不同，因为 循环质粒/转座子编码脂蛋白重塑基因。脂蛋白的组成也 受生长环境动态调节，包括已被证明可诱导表达的铜 某些 N 末端修饰基因。该项目旨在揭示不同的生理需求 脂蛋白 N 末端修饰系统并表征所涉及的酶。同时选择脂蛋白 结构修饰赋予宿主相关细菌、脂蛋白 N 末端 TLR2 逃避能力 酰化/乙酰化也存在于环境谱系中，这表明存在广泛的选择压力。给定 铜共同诱导脂蛋白 N 末端修饰和铜抗性基因，有人提出 铜直接结合到游离的 N 末端并抑制细胞生长，除非 N 进行修饰。该项目将 通过测量生长条件下的生长来研究 N 末端脂蛋白修饰对铜结合的影响 需要特定脂蛋白功能的铜挑战条件，使用 Tn-seq 比较化学型特异性 铜敏化基因网络，并测定从两者分离的模型脂肽的氧化损伤 活跃呼吸的细胞和体外反应条件下。在第二个目标中，最近发现 来自金黄色葡萄球菌的脂蛋白 N-酰化酶 LnsAB 将被重构。靶向突变， 酰基链供体，以及可能的蛋白质-蛋白质复合物的形成将使用遗传和生化方法进行检查 接近。该项目的最后部分将研究如何利用基因技术制造 N-乙酰化脂蛋白 转座子筛选和生化测定，以及脂蛋白乙酰化对关键表型的影响。 通过了解脂蛋白合成，该项目将揭示脂蛋白之间复杂的相互作用 化学型、铜暴露、TLR2 识别，以及控制宿主与厚壁菌相互作用的关键轴。

项目成果

Spatial regulation of protein A in Staphylococcus aureus.

• DOI: 10.1111/mmi.14734
• 发表时间: 2021-08
• 期刊: Molecular microbiology
• 影响因子: 3.6
• 作者: Zhang R;Shebes MA;Kho K;Scaffidi SJ;Meredith TC;Yu W
• 通讯作者: Yu W