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耐药性的细菌病原体的流行率日益增加，这是一个令人担忧的情况， 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来自微生物产生的天然产物，现在人们对使用新方法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被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的事实所混淆。我们已经鉴定了从新墨西哥州四个不同洞穴深处获得的独特微生物集合。由于这些细菌是从遥远的、未被充分探索的地方分离出来的，这些地方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的平台来识别和纯化分子，从这个多样化的洞穴细菌集合中对抗多药耐药（MDR）细菌。首先，我们将使用我们最近开发的细菌细胞学分析（BCP）的方法，以确定天然产品的抗菌活性的粗有机提取物或直接在平板上。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。靶向不同细胞途径和途径内不同步骤的抗生素产生独特的细胞学特征，允许在几小时内鉴定新分离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，使其能够用于指导天然产品的纯化。我们将对产生抗生素的菌株进行测序，然后使用靶向基因组挖掘（TDGM）和异源生物合成基因簇（BGC）过量生产来鉴定新的产生抗菌素的BGC。通常沉默的BGC的杂合子过表达将使我们能够识别传统筛选遗漏的分子。

 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.