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: 10429910
• 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/10429910
• 关键词: Address; Adoption; Anaerobic Bacteria; Antibiotics; Archives; Area; Attention; Back; Bacteria; Bacterial Physiology; Bacteriology; Bar Codes; Biological Process; Cells; 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; Outcome; Pharmaceutical Preparations; Phenotype; Phylogenetic Analysis; Physiological; Physiology; Population; Protocols documentation; Research; Research Personnel; Resolution; Resources; Role; Sorting - Cell Movement; Source; Stress; Study models; System; Technology; Testing; Time; Volatile Fatty Acids; Work; antimicrobial peptide; base; 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; transposon sequencing

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.

项目摘要 我们胃肠道中的微生物社区，统称为肠道微生物群， 构成已知的最密集，最多样化的细菌生态系统之一。而亲密关系 在人类和他们的菌群之间代表了工程人类健康的巨大潜力，我们目前是 限制揭示内在复杂性所需的工具。我们可以预测利用微生物群的能力 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工具进行研究，并且与 精心研究的模型细菌以通过同源性准确地传递基因注释。我们的这个主要差距 必须通过系统的努力来解决对人肠道细菌中基因功能的功能理解， 这将需要多个互补的专业知识。 高通量遗传学是一种表征基因生物学功能的有吸引力的方法 在人类微生物群中。扰动应用于大量基因修饰的大群 细菌允许对几乎所有基因的平行评估。类似的高通量策略可能可能 应用于人类的肠道微生物组，但我们旨在解决这个问题的多个主要障碍 项目：（1）非模型细菌的转化仍然具有挑战性，并且是一项逐个试验的努力，（2） 非模型细菌的新遗传系统的开发是耗时的，（3）采用 多种技术和实验室工作流使整个团队的数据比较复杂化，（4）体内 理想情况下，应在通过感兴趣的突变体定植的无前小鼠中进行小鼠实验。 为此赠款组装的团队包括在新颖耕种方法的最前沿的领导者， 用于遗传转化的电穿孔，以及用于在体外和体内评估基因功能的工具。在AIM 1中， 我们将迅速为大量人类肠道菌株开发遗传工具，并具有最终目标 生成全基因组随机条形码转座子突变库以进行测序。我们将利用 这些文库测试了所有非必需基因在许多体外的表型重要性（AIM 2） 体内（AIM 3）在全球范围内发现新基因功能。通过我们的联合专业知识 细菌学，微流体，高通量筛查，宿主 - 微叶相互作用和成像，我们将产生 庞大的微生物群研究人员的遗传工具和健身数据以前所未有的规模和 深入了解人类肠道菌群的生理学。