A universal pipeline for functional characterization of the human microbiota at a massive scale

大规模人类微生物群功能表征的通用管道

• 批准号: 10626097
• 负责人: Cullen Richard Buie
• 金额: $ 147.59万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10626097
• 关键词: Address; Adoption; Antibiotics; Archives; Area; Assessment tool; Attention; Back; Bacteria; Bacterial Physiology; Bacteriology; Bar Codes; Biological Process; Cells; Chromosome Mapping; Collection; Communities; Complement; Consumption; Data; Data Set; Development; Diet; Disease; Distant; Ecosystem; Electroporation; Engineering; Environment; Feeds; Fiber; Gastrointestinal tract structure; Gene Transfer; Generations; Genes; Genetic; Genetic Transformation; Genotype; Germ-Free; Gnotobiotic; Goals; Grant; Growth; Health; Heterogeneity; Human; Human Engineering; Image; In Vitro; Individual; Intestines; Inulin; Laboratories; Libraries; Life Style; Magic; Maps; Measurement; Metagenomics; Methods; Microbe; Microfluidics; Molecular Genetics; Mucins; Mus; Nutrient; Organism; Osmosis; Outcome; Pharmaceutical Preparations; Phenotype; Phylogenetic Analysis; Physiological; Physiology; Population; Protocols documentation; Research; Research Personnel; Resolution; Resources; Role; Shapes; Sorting; Source; Stress; Study models; System; Technology; Testing; Time; Volatile Fatty Acids; Work; antimicrobial peptide; commensal bacteria; commensal microbes; cross immunity; experimental study; extreme temperature; feeding; fitness; gene function; genome-wide; gut bacteria; gut microbiome; gut microbiota; high throughput screening; host-microbe interactions; human microbiota; imaging modality; in vivo; innovation; innovative technologies; insight; interest; microbiome; microbiota; microorganism; mutant; novel; personalized medicine; tool

Project Summary The community of microorganisms within our gastrointestinal tract, collectively known as the gut microbiota, constitutes one of the densest and most diverse bacterial ecosystems known. While the close relationship between humans and their microbiota represents vast potential for engineering human health, we are currently limited in tools required to unravel the intrinsic complexity. Our ability to predictably harness the microbiota for beneficial health outcomes requires a fundamental understanding of the physiology of these bacteria, yet most human gut bacteria have never been studied using molecular genetic tools and are too distantly related from well-studied model bacteria to accurately transfer gene annotations by homology. This major gap in our functional understanding of gene functions in human gut bacteria must be addressed with systematic efforts, which will require multiple complementary expertise. High-throughput genetics is an attractive approach for characterizing the biological functions of genes within the human microbiota. Application of perturbations en masse to large populations of genetically modified bacteria permits the parallel assessment of nearly all genes. A similar high-throughput strategy can potentially be applied to the human gut microbiome, but there are multiple major obstacles that we aim to resolve in this project: (1) transformation of non-model bacteria remains challenging and is a largely trial-by-error effort, (2) the development of a new genetic system for a non-model bacterium is time-consuming, (3) the adoption of multiple technologies and laboratory workflows complicates the comparison of data across teams, (4) in vivo mouse experiments should ideally be carried out in ex-germ-free mice colonized by mutants of interest. The team assembled for this grant includes leaders at the forefront of novel cultivation methods, electroporation for genetic transformation, and tools for assessing gene function in vitro and in vivo. In Aim 1, we will rapidly develop genetic tools for a large number of human gut commensal strains, with the ultimate goal of generating genome-wide randomly barcoded transposon mutant libraries for sequencing. We will utilize these libraries to test the phenotypic importance of all non-essential genes across a multitude of in vitro (Aim 2) and in vivo (Aim 3) conditions to globally discover new gene functions. Through our combined expertise in bacteriology, microfluidics, high-throughput screening, host-microbe interactions, and imaging, we will produce genetic tools and fitness data for the vast community of microbiota researchers at unprecedented scale, and deliver deep insight into the physiology of the human gut microbiota.

项目概要 我们胃肠道内的微生物群落，统称为肠道微生物群， 构成了已知最密集、最多样化的细菌生态系统之一。虽然关系密切 人类与其微生物群之间的关系代表着工程人类健康的巨大潜力，我们目前正在 解决内在复杂性所需的工具有限。我们有能力以可预测的方式利用微生物群 有益的健康结果需要对这些细菌的生理学有基本的了解，但大多数 人类肠道细菌从未使用分子遗传学工具进行过研究，并且与人类肠道细菌的亲缘关系太远。 经过充分研究的模型细菌，可通过同源性准确转移基因注释。我们的这一重大差距 对人类肠道细菌基因功能的功能性理解必须通过系统的努力来解决， 这将需要多种互补的专业知识。 高通量遗传学是表征基因生物学功能的一种有吸引力的方法 人类微生物群内。将扰动集中应用于大量转基因群体 细菌允许对几乎所有基因进行并行评估。类似的高通量策略有可能 应用于人类肠道微生物组，但我们希望在此解决多个主要障碍 项目：（1）非模型细菌的转化仍然具有挑战性，并且很大程度上是一项反复试验的工作，（2） 为非模式细菌开发新的遗传系统非常耗时，（3）采用 多种技术和实验室工作流程使团队之间的数据比较变得复杂，(4) 体内 理想情况下，小鼠实验应该在被目标突变体定植的无菌小鼠中进行。 为这项资助组建的团队包括处于新颖栽培方法前沿的领导者， 用于遗传转化的电穿孔，以及体外和体内评估基因功能的工具。在目标 1 中， 我们将快速开发针对大量人类肠道共生菌株的遗传工具，最终目标 生成用于测序的全基因组随机条形码转座子突变体文库。我们将利用 这些文库可在多种体外测试中测试所有非必需基因的表型重要性（目标 2） 和体内（目标 3）条件，以在全球范围内发现新的基因功能。通过我们综合的专业知识 细菌学、微流体、高通量筛选、宿主-微生物相互作用和成像，我们将生产 以前所未有的规模为广大微生物群研究人员提供遗传工具和健康数据，以及 深入了解人类肠道微生物群的生理学。