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.

项目摘要 应用程序的长期目标是了解医学中细菌脂蛋白的原因和细菌脂蛋白 重要的公司门对经历结构修饰。脂蛋白是膜相关蛋白 通过酰化的N末端半胱氨酸锚将细菌表面拴在细菌表面上。它们是无处不在的细胞 革兰氏阳性和革兰氏阴性细菌中的信封结构，几乎在各个方面发挥关键作用 细菌细胞包膜生理。由于其功能重要性，丰富性，普遍分布以及 结构独特的酰化N末端半胱氨酸，先天免疫系统通过结合检测细菌 使用Toll样受体2（TLR2）家族的脂蛋白的N末端。 TLR2激活触发了 炎性细胞因子/趋化因子对清除细菌的反应以及调节体液免疫。 但是，有新的证据表明，来自Firmicutes门的脂蛋白中的酰化模式不是 典型的TLR2配体也不是静态的结构。 Firmicutes合成脂蛋白化学型的变化 使用一系列辅助脂蛋白生物合成基因，酰基链数，长度和固定位置。 基因分布在属和物种水平上均有所不同，甚至可能在应变水平上有所不同 循环质粒/转座子编码的脂蛋白重塑基因。脂蛋白组成也是 由生长环境动态调节，包括已显示诱导表达的铜 某些N末端修饰基因。该项目旨在揭示生理需求 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末端的TLR2逃避能力 在环境谱系中也发现了酰化/乙酰化，这表明宽阔的选择压力。给出 铜共诱导脂蛋白N末端修饰和铜耐药基因，已提出 该铜直接与游离的N末端结合并抑制细胞生长，除非N模化。这个项目将 研究N末端脂蛋白修饰对铜结合的影响，通过测量 铜挑战条件需要特定脂蛋白功能，使用TN-Seq比较化学型特异性 铜敏化基因网络，并分析了从两者中分离出的模型脂肽的氧化损伤 积极呼吸细胞和体外反应条件。在第二个目标中，最近发现的 将重构来自金黄色葡萄球菌的脂蛋白N-酰化酶LNSAB。目标突变， 酰基链供体以及可能的蛋白质 - 蛋白质复合物将使用遗传和生化检查 方法。该项目的最后一部分将检查如何使用遗传来制作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，该项目将阐明脂蛋白之间的复杂相互作用 化学型，铜暴露，TLR2识别和临界轴的临界轴，控制宿主 - 固定的相互作用。

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