A new paradigm for the creation and mining of microbial libraries for drug discovery (Equipment)

用于药物发现的微生物库的创建和挖掘的新范例（设备）

• 批准号: 9895367
• 负责人: Isabel Cruz
• 金额: $ 18.24万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-10 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9895367
• 关键词: Actinobacteria class; Address; Agar; Antibiotics; Automatic Data Processing; Bacteria; Bioinformatics; Biological; Biological Assay; Chemicals; Chemistry; Chromatography; Collection; Communities; Coupled; Data; Data Analyses; Data Set; Detection; Development; Environment; Equipment; Fermentation; Fingerprint; Fluorescence; Foundations; Generations; Genomics; Growth; Hour; Imagery; Knowledge; Laboratories; Lead; Libraries; Liquid substance; MALDI-TOF Mass Spectrometry; Mass Spectrum Analysis; Methods; Microbe; Mining; Morphology; Nutrient; Online Systems; Process; Production; Protein Fingerprints; Proteins; Reporter; Research Personnel; Ribosomal Proteins; Sampling; Series; Source; Subgroup; Taxonomy; Techniques; Visual; base; bioinformatics tool; chemical fingerprinting; cost; cost effective; cost efficient; data visualization; digital; drug discovery; fluorescence imaging; graphical user interface; innovation; microbial; microbiome; microorganism; pathogen; rRNA Genes; small molecule; species difference; web interface; web portal

A high degree of taxonomic and chemical redundancy is a major limitation in sourcing microbial strain libraries for drug discovery. Currently, the creation of these libraries relies on outdated and costly methods, namely visual inspection of morphological differences of colonies from agar plates, or ribosomal RNA gene sequencing methods that are not indicative of a microbe’s capacity to produce specialized metabolites (SM). Despite the incredible potential of microorganisms to produce SMs, this redundancy remains a primary barrier to drug discovery efforts. In order to overcome this, we will develop a rapid, easy to use mass spectrometry (MS) technique that will maximize both the taxonomic and chemical diversity entering into microbial strain libraries. This will be coupled to our semi-automated, web-based bioinformatics pipeline that will be made available to the public. Our platform will employ matrix-assisted laser desorption/ionization time of flight MS (MALDI-TOF MS) to address a few major obstacles in the drug discovery process. First, we will develop a high-throughput MALDI- TOF MS method capable of gathering two distinct datasets from single colonies of bacteria from agar-based diversity plates: a) ribosomal protein fingerprints that are used to putatively identify the genus and species of the colony, and b) SM fingerprints of each colony to elucidate intra-species differences in SM production (Aim 1). Importantly, our MALDI-TOF MS platform is capable of processing and analyzing 384 strains in a 4-hour period, which is a significant advance when compared to other mass spectrometry or genomics-based profiling approaches. When applied to thousands of bacterial colonies of a cultivatable environmental microbiome, this platform will serve to maximize the taxonomic and chemical diversity entering a library, while minimizing the number of strains required to achieve this (e.g. addition of 300 strains as opposed to 3,000). Second, we will develop a facile fluorescence/MS-detection platform to interrogate the unmined biologically active chemical space of existing bacterial strain libraries (Aim 2). Using an existing Actinobacteria library as proof of concept, we will grow each strain under eight different cultivation conditions in 48-well agar plates. We will then develop and implement a series of antibiotic assays with fluorescent reporter strains of ESKAPE pathogens, and use MALDI-TOF MS to detect biologically active SMs that exist within zones of inhibition from each producing actinomycete. This method allows researchers to simultaneously observe growth inhibition via fluorescence imaging and to identify strains that produce bioactive SMs under single/unique cultivation conditions. This foregoes laborious liquid cultivation and chromatography steps of inactive bacteria (current practice). Data analysis will be facilitated through development of a web-based, semi-automated visualization pipeline that will be freely available to the scientific community (Aim 3). Successful completion of these aims will result in more a targeted, cost efficient, and accessible approach to microbial drug discovery, and represents a major innovation to front end microbial library generation that has arguably not seen an advance in decades.

高度的分类和化学冗余是采购微生物菌株库的主要限制 目前，这些库的创建依赖于过时且昂贵的方法，即视觉方法。 检查琼脂平板菌落的形态差异，或核糖体 RNA 基因测序 尽管有这些方法，但并不能表明微生物产生特殊代谢物（SM）的能力。 微生物生产 SM 的潜力令人难以置信，但这种冗余仍然是药物的主要障碍 为了克服这一困难，我们将开发一种快速、易于使用的质谱（MS）。 该技术将最大限度地提高进入微生物菌株库的分类学和化学多样性。 这将与我们的半自动化、基于网络的生物信息学管道相结合，该管道将提供给 我们的平台将采用基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF MS）来 首先，我们将开发高通量 MALDI- 解决药物发现过程中的几个主要障碍。 TOF MS 方法能够从基于琼脂的细菌单菌落中收集两个不同的数据集 多样性板：a) 核糖体蛋白指纹，用于推定鉴定生物的属和种 b) 每个菌落的 SM 指纹，以阐明 SM 生产的种内差异（目标 1）。 重要的是，我们的 MALDI-TOF MS 平台能够在 4 小时内处理和分析 384 个菌株 与其他基于质谱或基因组学的方法相比，这是一个显着的进步 当应用于可培养环境的数千个细菌菌落时。 微生物组，该平台将最大限度地提高进入图书馆的分类学和化学多样性，同时 最大限度地减少实现这一目标所需的菌株数量（例如添加 300 株而不是 3,000 株）。 其次，我们将开发一个简便的荧光/MS检测平台来对未开采的生物进行询问 现有细菌菌株库的活性化学空间（目标 2）。 为了验证概念，我们将在 48 孔琼脂平板中的八种不同培养条件下培养每种菌株。 然后将开发并实施一系列使用 ESKAPE 荧光报告菌株的抗生素检测 病原体，并使用 MALDI-TOF MS 检测存在于病原体抑制区内的生物活性 SM 每个产生放线菌的方法允许研究人员通过同时观察生长抑制。 荧光成像并鉴定在单一/独特培养下产生生物活性 SM 的菌株 这放弃了非活性细菌的费力液体培养和色谱步骤（当前）。 数据分析将通过开发基于网络的半自动可视化来促进。 将向科学界免费提供管道（目标 3）。 为微生物药物发现提供了一种更有针对性、成本低廉且易于使用的方法，并代表了一种 前端微生物文库生成的重大创新，几十年来一直没有取得进展，颇有争议。