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

项目总结 细菌细胞被肽聚糖(PG)细胞壁包围，这是一种抵抗 渗透压的变化和其他环境侮辱。在某种程度上，PG也是 人类作为抗生素的靶标破坏它，碎片激活免疫反应。基础性建筑 PG块已经有50多年的历史了；然而，这种聚合物的更高的建筑特征 完整的免疫刺激片段仍然未知。我们假设总体上的差异 PG结构和片段的产生对检测病原菌具有重要意义。PG的葡聚糖是 对于免疫识别是必不可少的；对这一重要结构的研究由于缺乏工具而受到阻碍 标记和跟踪PG碳水化合物前体和生成的聚合物在活细胞中的命运。目前， 研究人员被限制在很少的碳水化合物探针，甚至更少的较大片段。化学合成是 即使对于碳水化合物化学家专家来说，这也是一项艰巨而具有挑战性的工作。这份U01提案的目标是开发一种 一种标记PG在多种微生物中的葡聚糖以便于识别、跟踪、操作的方法 以及分析来自PG与其生物结合伙伴的多糖并确定其功能。 我们建议利用一种代谢标记方法，在这种方法中，必要的功能化PG生物合成 合成构建块，提供给微生物，并结合到聚合物的主干中。这 以前从未做过，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不广。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。细菌型PG 循环酶AmgK和Muru放宽了对N-乙酰-胞壁酸乳糖醇的底物专一性， 允许生产标记的UDP-PG前体。在目标一中，大规模的化学酶合成 将对各种UDP-PG衍生品进行优化，这些分子将提供给各种 致病微生物和共生微生物用于随后的PG掺入。将开发套件以分发 这些必需的碳水化合物。为此，将向其基因组的细胞提供两种标记的乳糖醇底物 都被设计成对AmgK和Muru进行编码。由于大肠杆菌和枯草芽孢杆菌是可修饰的 对于这种方法，这种方法将扩展到病原体，如幽门螺杆菌(Hp)和 结核分枝杆菌(Mtb)以及共生菌。目标三将展示这一功能的实用性。 免疫学家和微生物学家的方法：(1)来自结核分枝杆菌的含有多糖的免疫刺激分子， 并对Hp进行跟踪、分类和鉴定；(2)Hp和Mtb与发病机制相关的PG结构特征 并将对抗生素敏感性进行讯问。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，肽聚糖将是一种可接近但功能强大的技术，也是一种有价值的 加入糖科学联盟。

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.