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

高度的分类和化学冗余是寻找微生物菌株文库的主要限制 用于药物研发。目前，这些库的创建依赖于过时和昂贵的方法，即可视化 琼脂平板上菌落形态差异的检验，或核糖体RNA基因测序 不能表明微生物产生特定代谢物(SM)的能力的方法。尽管 微生物产生短信的惊人潜力，这种冗余仍然是药物的主要障碍 探索的努力。为了克服这一点，我们将开发一种快速、易用的质谱仪(MS)。 这项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、基于Web的生物信息学管道相结合，该管道将提供给 公开的。我们的平台将采用基质辅助激光解吸/电离飞行时间质谱仪(MALDI-TOF MS)来 解决药物发现过程中的几个主要障碍。首先，我们将开发一种高通量的MALDI- 能够从琼脂基单菌落中收集两个不同数据集的TOF MS方法 多样性图板：a)核糖体蛋白指纹，用于推测鉴定该属和种 B)每个群体的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 与其他基于质谱学或基因组学的方法相比，这是一个显著的进步 侧写接近了。当应用于可培养环境中的数千个细菌菌落时 微生物组，这个平台将用于最大限度地增加进入图书馆的分类和化学多样性，同时 将实现这一目标所需的菌株数量降至最低(例如，增加300个菌株，而不是3,000个)。 其次，我们将开发一个简便的荧光/MS检测平台，用于对未开采的生物进行讯问 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 概念验证，我们将每个菌株在8个不同的培养条件下在48孔琼脂平板中生长。我们 然后将开发和实施一系列ESKAPE荧光报告菌株的抗生素检测 病原体，并使用MALDI-TOF MS检测存在于抑制区内的生物活性SMS 每一种都产生放线菌。这种方法允许研究人员同时观察生长抑制通过 荧光成像和鉴定在单一/单一培养条件下产生生物活性短信的菌株 条件。这就放弃了费力的液体培养和非活性细菌的层析步骤(当前 练习)。将通过开发基于网络的半自动可视化来促进数据分析 将向科学界免费提供的管道(目标3)。这些目标的成功实现将 带来更有针对性、成本效益和可获得性的微生物药物发现方法，并代表着一种 在前端微生物文库生成方面的重大创新，可以说几十年来没有看到任何进展。

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 Currently, the creation of these libraries relies on outdated and costly methods, namely visual inspection of morphological differences of colonies from agar plates, or ribosomal RNA gene sequencing methods that are not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