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.

项目摘要 细菌细胞用肽聚糖（PG）细胞壁包围自己，这是一种抵抗细菌生长的基本结构。 渗透压和其他环境损害的变化。在某种程度上，PG也是必不可少的， 人类作为抗生素的目标，它的破坏和片段激活免疫反应。的基本构建 PG块已经知道了五十多年;然而，这种聚合物的更高结构特征 并且完整的免疫刺激片段集仍然未知。我们假设， PG结构和片段产生对于传感病原菌是重要的。PG的聚糖是 对于免疫识别至关重要;由于缺乏工具， 标记和跟踪PG碳水化合物前体和活细胞中所得聚合物的命运。目前， 研究人员限于很少的碳水化合物探针和更少的较大片段。化学合成 甚至对专家碳水化合物化学家来说也是费力和具有挑战性的。本U 01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，以便于识别、跟踪、操作 分析PG衍生的聚糖及其生物结合配偶体，并确定其功能。 我们建议利用代谢标记方法，其中必要的官能化PG生物合成 合成结构单元，提供给微生物并掺入聚合物的主链中。这 以前没有做过，因为UDP-糖结构单元的合成具有挑战性， 游离糖的加工途径分布不广。为了克服这一挑战，我们建议 利用化学酶促合成或基因工程的平行方法。细菌PG 再循环酶，AmgK和MurU对N-乙酰基-胞壁酸乳糖醇具有松弛的底物特异性， 允许产生标记的UDP-PG前体。目标一，大规模化学酶促合成 将优化各种UDP-PG衍生物的分子，并将这些分子提供给各种实验室。 病原微生物和真菌用于随后的PG掺入。将开发工具包， 这些必需的碳水化合物。为了达到目的，将两种标记的乳醇底物提供给基因组 已经被设计成编码AmgK和MurU。由于大肠杆菌和枯草芽孢杆菌是可降解的， 为了这种方法，这种方法将扩展到病原体，如幽门螺杆菌（Hp）， 结核分枝杆菌（Mtb）以及细菌。目标三将展示这一工具的实用性 免疫学家和微生物学家的方法：（1）来自Mtb的含聚糖的免疫刺激分子， （2）Hp和Mtb与致病相关的PG结构特征 和抗生素敏感性进行调查。这种创新的碳水化合物代谢标记方法， 肽聚糖对于生物医学研究人员来说将是一种平易近人但功能强大的技术，也是一种有价值的 加入糖科学联盟。