Defining Host-Microbial Interactions Using Functional Metagenomics

 描述（由申请人提供）：组成人类微生物组的数万亿细菌被认为编码对人类健康至关重要的功能;然而，对肠道细菌用于与人类宿主相互作用的特定效应物知之甚少。不能培养许多肠道细菌使得这些微生物与最严重依赖的用于表征效应分子的技术不相容。为了解决这个问题，需要应用可以分离和观察细菌效应物与人类生物学相互作用的方法。在功能宏基因组研究中，将从环境样品中提取的DNA片段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，允许同时识别来自两种细胞的效应子。 培养和未培养的微生物。在最近提交的手稿中，我们展示了使用功能性宏基因组技术来分离细胞效应分子。在这项研究中，我们创建了三个宏基因组文库从DNA分离的表型不同的患者粪便样本。使用人报告细胞系的高含量成像来鉴定由宏基因组克隆产生的激活人细胞NFκB途径的效应分子。这项研究发现了26个生物合成的唾液酸效应基因和一种小分子，N-酰基-3OH-棕榈酰-甘氨酸，它在结构上模拟了人类的内源性信号分子并调节免疫细胞功能。中心假设是，我们的功能性宏基因组筛选方法可以广泛应用于人类微生物组，通过扩大我们的宏基因组库和人类细胞报告基因库来发现效应分子（目标1）。一旦分离出与人类细胞相互作用的宏基因组克隆，就可以直接鉴定每个效应基因和分子（Aim 2），并探索其他生物产生的类似分子的化学多样性（Aim 3）。该提议的基本原理是，生物活性分子的分离为未来的研究创造了坚实的基础，以了解植物效应子功能如何决定宿主生理学，并靶向这些宿主-微生物相互作用以用于治疗开发。为了实现这些目标，我将得到主要导师Sean布雷迪博士（洛克菲勒大学）和共同导师Sergio里拉博士（西奈山）的支持，前者是功能性宏基因组研究方法应用的科学领导者，后者具有宿主-微生物相互作用和粘膜免疫学方面的专业知识。每个导师将帮助我完成这个项目的个人目标，并发展所需的技能，以追求我的长期目标，了解植物效应分子如何定义宿主-微生物相互作用。

 DESCRIPTION (provided by applicant): 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) and a co- mentor with expertise in host-microbial interactions and mucosal immunology, Dr. Sergio Lira (Mount Sinai). Each 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.