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

高度的分类学和化学冗余是微生物菌株库来源的主要限制 用于药物发现。目前，这些图书馆的创建依赖于过时和昂贵的方法，即视觉 琼脂平板菌落形态差异检查，或核糖体RNA基因测序 不能指示微生物产生特定代谢物（SM）的能力的方法。尽管 微生物产生SM的惊人潜力，这种冗余仍然是药物治疗的主要障碍。 探索的努力。为了克服这一点，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 公众我们的平台将采用基质辅助激光解吸/电离飞行时间MS（MALDI-TOF MS）， 解决药物发现过程中的一些主要障碍。首先，我们将开发高通量MALDI- TOF MS方法能够从基于琼脂的单菌落细菌收集两个不同的数据集 多样性板：a）核糖体蛋白指纹，其用于纯化鉴定植物的属和种; 菌落，和B）每个菌落的SM指纹，以阐明SM产生的种内差异（目的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种菌株。 这是一个显着的进步，当与其他质谱或基因组学为基础的 剖析方法。当应用于可培养环境中的数千个细菌菌落时 微生物组，该平台将有助于最大限度地提高进入图书馆的分类和化学多样性， 最小化实现这一点所需的菌株数量（例如，添加300个菌株而不是3，000个菌株）。 第二，我们将开发一个简单的荧光/MS检测平台， 现有细菌菌株文库的活性化学空间（目标2）。使用现有的放线菌文库作为 为了验证概念，我们将在48孔琼脂板中在八种不同的培养条件下培养每种菌株。我们 然后将开发和实施一系列ESKAPE荧光报告菌株的抗生素测定 病原体，并使用MALDI-TOF MS检测存在于抑制区内的生物活性SM， 每种都产生放线菌。这种方法使研究人员能够同时观察生长抑制， 荧光成像和鉴定在单一/独特培养下产生生物活性SM的菌株 条件这避免了无活性细菌的费力的液体培养和层析步骤（目前的方法）。 实践）。将通过开发一个基于网络的半自动可视化工具，促进数据分析 科学界将可免费使用这一管道（目标3）。成功实现这些目标将 导致更有针对性的，成本效益高的和可获得的方法来发现微生物药物，并代表了一个 前端微生物文库生成的重大创新，可以说几十年来没有取得进展。

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 Currently, the creation of these libraries relies on outdated and costly methods, namely visual inspection of morphological differences of colonies from agar plates, or ribosomal RNA gene sequencing methods that are not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.