Harnessing the gut microbiome to detect and quantify glycans

利用肠道微生物组来检测和量化聚糖

• 批准号: 10798384
• 负责人: Guy Edmund Townsend
• 金额: $ 12.93万
• 依托单位: PENNSYLVANIA STATE UNIV HERSHEY MED CTR
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10798384
• 关键词: Affinity; Affinity Chromatography; Animals; Automobile Driving; Bacteroides; Bacteroides thetaiotaomicron; Binding Proteins; Biological; Biological Process; Carbohydrates; Complement; Complex; Complex Mixtures; Detection; Disease; Dose; Engineering; Genes; Goals; Harvest; Human; In Vitro; Individual; Intestinal Content; Intestines; Libraries; Life; Mass Spectrum Analysis; Measurement; Measures; Metabolic; Metabolism; Methods; Microbe; Modeling; Modification; Natural regeneration; Nuclear Magnetic Resonance; Nucleic Acids; Nutrient; Oligonucleotides; Organism; Output; Plants; Polymers; Polysaccharides; Proteins; Recombinants; Reporter; Reporting; Resources; Sampling; Sensitivity and Specificity; Source; Specificity; Standardization; Structure; Surveys; System; Therapeutic; biological preparation; clinically relevant; cost; detection method; detection platform; gut bacteria; gut microbes; gut microbiome; gut microbiota; health determinants; interest; member; microbial; microbiome composition; novel; predictive tools; response; therapeutic target; tool; tool development

Project Summary Detecting various compositionally similar but structurally distinct glycans present in heterogenous mixtures has traditionally been challenging due to the limitations facing current glycomics approaches. These limitations have hindered our understanding of the availability and abundance of individual glycans simultaneously present in the mammalian intestine, which contains complex mixtures of microbial, mammalian, and plant-derived oligo- and polysaccharides. Moreover, intestinal glycans are a primary driver of gut microbiome composition and metabolism, which represents an increasingly important human health determinant, and necessitates a deep understanding of the glycomic-microbial interface to identify important biological interactions and putatively develop glycan-derived therapeutics to target specific microbial activities. Therefore, new tools are necessary to detect and measure the relative abundance of individual glycan substrates present in the heterogeneous mixtures prepared from biological sources such as mammalian intestinal contents. We have harnessed the glycan detection machinery employed by dominant members of the gut microbiota to detect, measure and isolate individual glycan substrates present in heterogenous mixtures extracted from the mammalian intestine. Herein, we demonstrate robust, specific, and scalable approaches by which engineered microbes report the presence of individual glycan substrates with incredible sensitivity. Furthermore, we demonstrate that this approach can accurately measure the abundance of individual glycans present in mixtures across wide linear ranges and that the specificity and sensitivity of these measurements can be tuned by modifying particular microbial glycan utilization genes. Finally, we demonstrate that microbially-encoded glycan-binding proteins can be used to isolate individual target glycans from mixtures for downstream compositional and structural determination. We propose to 1.) develop arrayed libraries of distinct gut microbial species, each engineered to report the presence of unique target glycans, 2.) develop a rapid glycan isolation pipeline to purify individual substrates of interest for downstream structural and functional characterization, and 3.) develop genetically modified microbial strains with enhanced sensitivity or target specificity. In addition to offering a rapid and inexpensive alternative to quantifying known glycans, we believe that further development of these tools will reveal the presence and abundance of previously undetectable glycans and dramatically enhance our understanding of the interactions between gut microbes and their mammalian hosts.

项目概要 检测异质混合物中存在的各种组成相似但结构不同的聚糖 由于当前糖组学方法面临的局限性，传统上一直具有挑战性。这些限制有 阻碍了我们对同时存在于细胞中的单个聚糖的可用性和丰度的理解 哺乳动物肠道，含有微生物、哺乳动物和植物来源的寡聚和 多糖。此外，肠聚糖是肠道微生物组组成的主要驱动因素， 新陈代谢是人类健康日益重要的决定因素，需要深入研究 了解糖组-微生物界面以确定重要的生物相互作用并推定 开发针对特定微生物活性的聚糖衍生疗法。因此，需要新的工具 检测并测量异源中存在的单个聚糖底物的相对丰度 由生物来源（例如哺乳动物肠道内容物）制备的混合物。我们已经利用了 肠道微生物群的主要成员使用聚糖检测机器来检测、测量和分离 存在于从哺乳动物肠道中提取的异质混合物中的单个聚糖底物。在此处， 我们展示了强大的、特定的和可扩展的方法，通过这些方法工程微生物可以报告存在 具有令人难以置信的灵敏度的单个聚糖底物。此外，我们证明这种方法可以 准确测量混合物中存在的单个聚糖在宽线性范围内的丰度 这些测量的特异性和灵敏度可以通过修改特定的微生物聚糖来调整 利用基因。最后，我们证明微生物编码的聚糖结合蛋白可用于分离 从混合物中分离出单个目标聚糖，用于下游成分和结构测定。我们建议 1.) 开发不同肠道微生物物种的阵列文库，每个文库都经过改造以报告独特的存在 目标聚糖，2.) 开发快速聚糖分离管道来纯化各个感兴趣的底物 下游结构和功能表征，以及 3.) 开发转基因微生物菌株 增强的敏感性或目标特异性。除了提供快速且廉价的量化替代方案之外 已知聚糖，我们相信这些工具的进一步开发将揭示已知聚糖的存在和丰度 以前无法检测到的聚糖，极大地增强了我们对肠道之间相互作用的理解 微生物及其哺乳动物宿主。