Targeted discovery of antibiotics from cave bacteria

 描述（由申请人提供）：对大多数临床批准的抗生素具有抗药性的细菌病原体的日益流行是一个令人震惊的情况， 激发了人们对抗生素发现计划的新兴趣。由于大多数抗生素都源自微生物产生的天然产物，因此现在人们对使用新方法来筛选遗传和化学多样性的细菌集合产生了浓厚的兴趣。然而，从细菌提取物中鉴定新分子会因先前鉴定的分子的压倒性存在以及生物体的大部分生物合成潜力通常在实验室生长条件下不表达这一事实而受到困扰。我们对从新墨西哥州四个不同洞穴深处获得的一组独特的微生物进行了表征。由于这些细菌是从偏远、未充分开发的地方分离出来的，而这些地方才刚刚开始开采抗生素，因此识别具有独特化学结构和新作用模式的分子的可能性增加。该项目的目标是使用两个新的强大平台从不同的洞穴细菌中识别和纯化对多重耐药（MDR）细菌具有活性的分子。首先，我们将使用我们最近开发的细菌细胞学分析 (BCP) 方法来鉴定粗有机提取物中或直接在平板上具有抗菌活性的天然产物。 BCP 使用定量荧光显微镜来测量抗生素治疗对单个细胞的影响。针对不同细胞途径和途径内不同步骤的抗生素会产生独特的细胞学特征，从而可以在几个小时内鉴定出新分离的化合物的可能细胞靶点。 BCP 适用于复杂的粗提物和后续馏分，使其可用于指导天然产物的纯化。我们将对产生抗生素的菌株进行测序，然后使用目标定向基因组挖掘（TDGM）和异源生物合成基因簇（BGC）过量生产来识别新型抗生素产生的BGC。通常沉默的 BGC 的异源过度表达将使我们能够识别传统筛选中遗漏的分子。

 DESCRIPTION (provided by applicant): The increasing prevalence of bacterial pathogens that are resistant to most of the clinically approved antibiotics is an alarming situation that has spurred renewed interest in antibiotic discovery programs. Since most antibiotics are derived from natural products produced by microorganisms, there is now intense interest in using new methods to screen genetically and chemically diverse collections of bacteria. However, identifying new molecules from bacterial extracts is confounded by the overwhelming presence of previously identified molecules as well as the fact that most of the biosynthetic potential of a organism is typically not expressed under laboratory growth conditions. We have characterized a unique collection of microbes obtained from deep within four different caves of New Mexico. Since these bacteria were isolated from remote, underexplored locations that are only just beginning to be mined for antibiotics, there is an increased probability of identifying molecules with unique chemical structures and new modes of action. The goal of this project is to use two new powerful platforms to identify and purify molecules active against multidrug resistant (MDR) bacteria from this diverse collection of cave bacteria. First, we will use our recently developed bacterial cytological profiling (BCP) approach to identify natural products with antibacterial activities in crude organic extracts or directly on plates. BCP uses quantitative fluorescence microscopy to measure the effects of antibiotic treatment on individual cells. Antibiotics that target different cellular pathways and different steps within a pathway generate unique cytological profiles, allowing identification of the likely cellular target of newly isolated compounds in a few hours. BCP works in complex crude extracts and subsequent fractions, allowing it to be used to guide natural product purification. We will sequence strains producing antibiotics and then use target directed genome mining (TDGM) and heterologous biosynthetic gene cluster (BGC) overproduction to identify novel antibacterial producing BGCs. Heterologous overexpression of normally silent BGCs will allow us to identify molecules missed by traditional screening.