Peptidoglycan Fragment Library to Investigate Innate Immune Responses

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。这项提议的目标是开发一个PG 捕获自然多样性以促进适当的先天免疫和获得性免疫的片段文库 生物学研究。PG片段被先天免疫系统用来正确识别和响应 细菌的存在。我们假设Pg的多样性自然存在于众多细菌中 物种对于为宿主防御产生正确的免疫反应是必不可少的。对这些重要问题的研究 PG片段由于缺乏可重现的高纯度化合物而受到阻碍。目前，研究人员 仅限于少数碳水化合物探针，如MDP，甚至更少的较大片段。我们建议 功能化三个碳水化合物核心：单糖、双糖和多肽二聚体，带有精选的氨基 在各种细菌物种中高度存在的酸。合成的工作流程是模块化的， 在生产高纯度PG碎片方面建立在我们的专业知识基础上，并允许使用化学品进行修改 针对图书馆成员的多个精确点的生物探针。此类型的大型PG片段库尚未 以前也曾制作过。为了克服这一挑战，我们建议对这三种产品都使用通用中间体 碳水化合物核心。我们的目标是使用这个文库来询问在 巨噬细胞。在目标一中，将进行大规模、模块化的合成努力，以生产&gt；400 会员库。出于第二个目的，二糖和多肽连接的二聚体家族成员将用于 一种无偏见的全基因组转录组分析，以揭开定义 骨髓来源的巨噬细胞对PG类的反应。我们将合成化学生物学 用于研究受体和信号伙伴的探针。目的三是探讨人的先天免疫反应。 通过生产PG-微阵列来获得PG。该阵列将PG衍生物固定在多个方向 允许潜在的受体横跨化学空间。这些数组将用于评估 PG识别中多种先天免疫受体的底物结合偏好。这个图书馆 将准确捕捉PG片段的多样性，为所提出的免疫学 微生物和免疫学领域的化验和新工具。

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. The goal of this proposal is to develop a PG fragment library that captures the natural diversity to facilitate the proper innate and adaptive immunity biological studies. PG fragments are used by the innate immune system to correctly recognize and respond to the presence of bacteria. We hypothesize that PG diversity naturally present across the multitude of bacterial species is essential for generating the correct immune response for host defense. Study of these important PG fragments has been hampered by the lack of reproducible, high purity compounds. Currently, researchers are limited to few carbohydrate probes, such as MDP, and even fewer larger fragments. We propose to functionalize three carbohydrate cores: monosaccharide, disaccharide and peptide dimers, with select amino acids which are highly represented across a variety of bacterial species. The synthetic workflow is modular, builds off our expertise in producing highly pure PG fragments and permits for modification with chemical biology probes at multiple pinpoints on the library members. A large PG fragment library of this type has not been produced before. To overcome this challenge we propose to use common intermediates for all three carbohydrate cores. We aim to use this library to interrogate innate immune responses elicited in macrophages. In Aim One, a large-scale, modular synthetic effort will be undertaken to produce the >400 member library. For Aim Two the disaccharide and peptide linked dimer family members will be employed in an unbiased genome wide transcriptome analysis to unravel gene expression signatures that define the responses to PG classes in bone marrow derived macrophages. We will synthesize the chemical biology probes to investigate receptors and signaling partners. Aim Three will probe the innate immune response of the PG through the production of a PG-microarray. This array will fix the PG derivatives in multiple orientations permitting the sweeping of potential receptors across chemical space. The arrays will be used to assess the substrate binding preferences of a variety of innate immune receptors indicated in PG recognition. This library will accurately capture the PG fragment diversity, providing powerful probes for the proposed immunological assays and new tools for the microbial and immunological communities.