Metabolic carbohydrate cell wall probes for bacterial structure and immune recognition studies

用于细菌结构和免疫识别研究的代谢碳水化合物细胞壁探针

• 批准号: 9750646
• 负责人: Catherine Leimkuhler Grimes
• 金额: $ 33.18万
• 依托单位: UNIVERSITY OF DELAWARE
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-11 至 2021-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9750646
• 关键词: Adjuvant; Alkynes; Amino Sugars; Anabolism; Antibiotic Therapy; Antibiotic susceptibility; Antibiotics; Architecture; Azides; Bacillus subtilis; Bacteria; Binding; Biological; Carbohydrates; Cell Shape; Cell Wall; Cells; Cellular biology; Charge; Chemistry; Complement; Data; Discipline; Disease; Dose; Elements; Engineering; Enzymes; Escherichia coli; G Cells; Generations; Genetic Engineering; Genome; Glucosamine; Goals; Grant; Growth; Health; Helicobacter pylori; Heterogeneity; Human; Human body; Immune; Immune response; Immunologist; Inflammatory; Label; Lactobacillus; Light; Link; Medical; Metabolic; Methodology; Methods; Microbe; Muramic Acid; Mycobacterium tuberculosis; Natural Immunity; Nature; Organism; Osmotic Pressure; Pathogenesis; Pathway interactions; Peptides; Peptidoglycan; Permeability; Phosphotransferases; Polymers; Polysaccharides; Problem Solving; Production; Property; Proteins; Recycling; Reporter; Research Personnel; Shapes; Stomach; Structure; Substrate Specificity; Techniques; Transferase; United States National Institutes of Health; Uridine Diphosphate Sugars; Vertebral column; biochemical tools; body sense; carbohydrate structure; chemical synthesis; commensal bacteria; commensal microbes; crosslink; design; drug development; effective therapy; immune activation; innovation; large scale production; mycobacterial; novel; off-label use; pathogen; pathogenic bacteria; pathogenic microbe; pressure; small molecule; sugar; tool; uptake

PROJECT SUMMARY Bacterial cells surround themselves with a peptidoglycan (PG) cell wall, an essential structure that resists changes in osmotic pressure and other environmental insults. To a certain degree, PG is also essential to humans as antibiotics target its destruction and fragments activate immune responses. The basic building blocks of PG have been known for over fifty years; however, the higher architectural features of this polymer and complete set of immunostimulating fragments remain unknown. We hypothesize that differences in overall PG structure and fragment generation are important for sensing pathogenic bacteria. The glycan of the PG is essential for immune recognition; study of this important structure has been hampered by a lack of tools to label and track the fate of PG carbohydrate precursors and the resultant polymer in living cells. Currently, researchers are limited to few carbohydrate probes and even fewer larger fragments. Chemical synthesis is laborious and challenging to even expert carbohydrate chemists. The goal of this U01 proposal is to develop a method to label the glycan of the PG in a range of microbes to facilitate identification, tracking, manipulation and analysis of the glycans derived from PG with their biological binding partners and determine their functions. We propose to utilize a metabolic labeling approach in which the necessary functionalized PG biosynthetic building blocks are synthesized, provided to the microbe and incorporated in the backbone of the polymer. This has not been done before as the synthesis of the UDP-sugar building blocks is challenging and the uptake and processing pathways for the free sugars are not widely distributed. To overcome this challenge we propose parallel approaches which utilize either chemoenzymatic synthesis or genetic engineering. The bacterial PG recycling enzymes, AmgK and MurU have relaxed substrate specificity for N-acetyl-muramic acid lactols, allowing the production of labeled UDP-PG precursors. In Aim One, a large-scale chemoenzymatic synthesis of a variety of UDP-PG derivatives will be optimized and these molecules will be provided to a variety of pathogenic and commensal microbes for subsequent PG incorporation. Kits will be developed to distribute these essential carbohydrates. For Aim Two tagged lactol substrates will be provided to cells whose genomes have been engineered to encode for AmgK and MurU. As Escherichia coli and Bacillus subtilis are amendable to this approach, this methodology will be extended to pathogens such as Helicobacter pylori (Hp) and Mycobacterium tuberculosis (Mtb) as well as commensal bacteria. Aim Three will showcase the utility of this method for immunologists and microbiologists: (1) glycan-containing immunostimulatory molecules from Mtb, and Hp will be tracked, sorted and identified; (2) Hp and Mtb's PG structural features related to pathogenesis and antibiotic susceptibility will be interrogated. This innovative carbohydrate metabolic labeling method for peptidoglycan will be an approachable yet powerful technique for biomedical researchers and a valuable addition to the Glycoscience Consortium.

项目概要 细菌细胞周围有肽聚糖 (PG) 细胞壁，这是抵抗细菌侵害的重要结构。 渗透压的变化和其他环境损害。从某种程度上来说，PG也是必不可少的 人类作为抗生素的目标是破坏它，碎片会激活免疫反应。基本建筑 PG 块已为人所知已有五十多年；然而，这种聚合物具有更高的结构特征 完整的免疫刺激片段仍然未知。我们假设总体差异 PG结构和片段生成对于检测病原菌非常重要。 PG的聚糖是 对于免疫识别至关重要；由于缺乏工具，对这一重要结构的研究受到了阻碍 标记并追踪 PG 碳水化合物前体和所得聚合物在活细胞中的命运。现在， 研究人员仅限于少数碳水化合物探针，甚至更少的较大片段。化学合成是 即使对于专业的碳水化合物化学家来说，这也是费力且具有挑战性的。该 U01 提案的目标是开发一个 标记一系列微生物中 PG 聚糖的方法，以方便识别、跟踪和操作 分析 PG 衍生的聚糖及其生物结合伴侣并确定其功能。 我们建议利用代谢标记方法，其中必要的功能化 PG 生物合成 合成结构单元，提供给微生物并结合到聚合物的主链中。这 以前从未有人做过，因为 UDP-糖构建块的合成具有挑战性，并且吸收和 游离糖的加工途径分布并不广泛。为了克服这一挑战，我们建议 利用化学酶合成或基因工程的并行方法。细菌PG 回收酶、AmgK 和 MurU 放宽了对 N-乙酰胞壁酸乳醇的底物特异性， 允许生产标记的 UDP-PG 前体。在“目标一”中，大规模化学酶合成 多种UDP-PG衍生物将被优化，这些分子将提供给多种 用于随后 PG 掺入的病原微生物和共生微生物。将开发套件来分发 这些必需的碳水化合物。为了目标，将向其基因组的细胞提供两种标记的乳醇底物 已被设计为编码 AmgK 和 MurU。由于大肠杆菌和枯草芽孢杆菌是可修正的 对于这种方法，该方法将扩展到病原体，例如幽门螺杆菌（Hp）和 结核分枝杆菌 (Mtb) 以及共生细菌。目标三将展示此功能的实用性 免疫学家和微生物学家的方法：（1）来自结核分枝杆菌的含聚糖免疫刺激分子， Hp将被跟踪、分类和识别； (2) Hp和Mtb与发病机制相关的PG结构特征 并将询问抗生素敏感性。这种创新的碳水化合物代谢标记方法 对于生物医学研究人员来说，肽聚糖将是一种平易近人但功能强大的技术，也是一种有价值的技术。 除了糖科学联盟之外。