Defining Host-Microbial Interactions Using Functional Metagenomics

使用功能宏基因组学定义宿主-微生物相互作用

• 批准号: 10386268
• 负责人: Louis Jared Cohen
• 金额: $ 3.41万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-01 至 2021-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10386268
• 关键词: Bacteria; Bacterial Model; Cell Line; Cell physiology; Cells; Chemicals; Collection; DNA; DNA Library; Foundations; Genes; Glycine; Goals; Health; Human; Human Biology; Human Microbiome; Image; Immune; Individual; Libraries; Manuscripts; Mentors; Metagenomics; Methods; Microbe; Modality; Pathway interactions; Patients; Phenotype; Physiology; Problem Solving; Reporter; Research; Research Methodology; Sampling; Signaling Molecule; Structure; Techniques; Universities; commensal bacteria; host-microbe interactions; interest; novel therapeutics; screening; skills; small molecule; stool sample; therapeutic development

PROJECT SUMMARY The trillions of bacteria that make up the human microbiome are believed to encode functions that are important to human health; however, little is known about the specific effectors that commensal bacteria use to interact with the human host. The inability to culture many commensal bacteria renders these microbes incompatible with the most heavily relied upon techniques for characterizing effector molecules. To solve this problem requires the application of methods where bacterial effectors can be isolated and observed to interact with human biology. In functional metagenomic studies fragments of DNA extracted from an environmental sample are cloned into a model bacterial host, and the resulting metagenomic clones are examined for phenotypes of interest. This approach circumvents the culture barrier allowing for the simultaneous identification of effectors from both cultured and uncultured microbes. In a recently submitted manuscript we demonstrated the use of functional metagenomic techniques to isolate commensal effector molecules. In this study we created three metagenomic libraries from DNA isolated from phenotypically diverse patient stool samples. High content imaging of a human reporter cell line was used to identify effector molecules produced by metagenomic clones that activate human cellular NFκB pathways. This study led to the discovery of 26 biosynthetic commensal effector genes and a small molecule, N-acyl-3OH- palmitoyl-glycine which structurally mimics endogenous signaling molecules in humans and modulates immune cell functions. The central hypothesis is that our functional metagenomic screening method can be broadly applied to the human microbiome to discover effector molecules by expanding our metagenomic library collection and repertoire of human cellular reporters (Aim 1). Once metagenomic clones are isolated that interact with human cells it is straightforward to identify each effector gene and molecule (Aim 2) and explore the chemical diversity of similar molecules produced by other commensals (Aim 3). The rationale that underlies this proposal is that the isolation of bioactive molecules creates a strong foundation for future research to understand how commensal effector functions dictate host physiology and to target these host-microbial interactions for therapeutic development. To achieve these aims I will be supported by a primary mentor who is a scientific leader in the application of functional metagenomic research methods, Dr. Sean Brady (Rockefeller University). The mentor will help me complete the individual aims of this project and develop the skills required to pursue my long term goal to understand how commensal effector molecules define host-microbial interactions.

项目摘要 据信构成人类微生物组的数万亿细菌编码的功能 重要的是人类健康;然而，很少有人知道的具体效应，肠道细菌使用， 与人类宿主互动由于无法培养许多肠道细菌， 与最依赖的表征效应分子的技术不相容。解决这个 这一问题需要应用一些方法，其中可以分离细菌效应物并观察其相互作用 人类生物学。在功能宏基因组研究中，从环境中提取的DNA片段 将样品克隆到模型细菌宿主中，并检查所得宏基因组克隆的 感兴趣的表型。这种方法绕过了文化障碍，允许同时识别 来自培养和未培养微生物的效应子。 在最近提交的手稿中，我们展示了功能宏基因组技术的使用， 分离细胞效应分子。在这项研究中，我们从分离的DNA中创建了三个宏基因组文库， 从表型多样的患者粪便样品中分离。使用人报告细胞系的高含量成像 鉴定由宏基因组克隆产生的激活人细胞NFκB通路的效应分子。 这项研究发现了26个生物合成的细胞效应基因和一个小分子，N-酰基-3OH-。 棕榈酰-甘氨酸，其在结构上模拟人的内源性信号分子并调节免疫 细胞功能。核心假设是我们的功能宏基因组筛选方法可以广泛地应用于 应用于人类微生物组，通过扩大我们的宏基因组文库来发现效应分子。 和人类细胞报告基因库（Aim 1）。一旦宏基因组克隆被分离， 在人类细胞中，识别每个效应基因和分子（目标2）并探索化学作用是很简单的。 由其他生物产生的类似分子的多样性（目标3）。这一建议的基本原理是 生物活性分子的分离为未来的研究奠定了坚实的基础， 植物效应器功能决定宿主生理学，并靶向这些宿主-微生物相互作用， 治疗发展 为了实现这些目标，我将得到一位主要导师的支持，他是该应用程序的科学领导者 功能宏基因组学研究方法，肖恩布雷迪博士（洛克菲勒大学）。导师会帮我的 完成这个项目的个人目标，并发展追求我的长期目标所需的技能， 了解微生物效应分子如何定义宿主-微生物相互作用。