Defining Evolutionary Trajectories: Molecular adaptation to antibiotic resistance

细菌病原体之间的抗生素耐药性仍然是公众面临的巨大挑战之一 当今世界的健康问题。抗生素的广泛使用促进了多重耐药的增加 有可能破坏现代医学非凡成就的病原体。城市发展中心 疾控中心已将多重耐药肠球菌定为“严重威胁” 需要迅速和持续的活动来限制扩散。达托霉素是一种一线抗生素， 对革兰氏阳性菌有效，对多种药物的使用频率越来越高 耐药肠球菌，如耐万古霉素肠球菌(VRES)。这项提议的目标是 综合绘制导致粪肠球菌对DAP耐药性的进化轨迹 并阐明了已识别的蛋白质结构-功能变化如何建立物理化学 根据观察到的耐药表型。我们在一部小说中使用了定量的实验进化， 连续培养生物反应器系统对最重要的进化轨迹进行识别和排序 导致抵抗。基于这些结果，我们然后描述了最相关的蛋白质和 使用生化和结构方法相结合的方式产生达托霉素耐药性的途径 将生物物理性质的变化与抗药性联系起来。技术包括X射线结晶学， 酶活性、配基亲和力、蛋白质稳定性研究、RNAseq、qPCR等。这种方法 寻求确定，不仅是抗性的生化基础，还包括那些候选蛋白质 以及非常适合开发新一类辅助药物的途径，这种药物将 靶向并延缓抗药性的发展。我们的研究也可以提供有价值的分子 出现阻力的指标。利用我们在实验进化方面的专业知识，我们还 开发了一种新的方法来利用进化的力量和大部分未被探索的 自然界最好的抗生素产生体之一的生物化学多样性和杀灭策略： 链霉菌。我们使用微乳液滴中的实验进化来产生选择 识别玫瑰孢链霉菌(“捕食者”)提高杀灭能力的变种的条件 一种VRE菌株(猎物)。在每个微滴中，我们诱捕两个种群(捕食者和猎物)。 如果捕食者能够适应杀死猎物，适应的捕食者就拥有显著的资源优势， 与未适应的捕食者相比，繁殖成功率更高。总而言之，这个项目需要 多管齐下揭示进化的机制和物理化学基础 并将实验进化扩展到包括一种新的发现方法 新的抗菌剂。

Antibiotic resistance among bacterial pathogens remains one of the great challenges confronting public health in the world today. The widespread use of antibiotics has facilitated the rise of multi-drug resistant pathogens that threaten to undermine the remarkable success of modern medicine. The Centers for Disease Control and Prevention have identified multi-drug resistant enterococci as a “Serious Threat” requiring prompt and sustained activity to limit proliferation. Daptomycin is a frontline antibiotic with efficacy against Gram positive organisms and is used with increasing frequency against multi-drug resistant enterococci such as vancomycin-resistant enterococci (VREs). The goal of this proposal is to comprehensively map the evolutionary trajectories leading to DAP resistance in Enterococcus faecium and elucidate how the identified changes in protein structure-function establish the physicochemical basis for the observed resistance phenotypes. We use quantitative experimental evolution in a novel, continuous culture bioreactor system to identify and rank the most important evolutionary trajectories leading to resistance. Based upon these results, we then characterize the most relevant proteins and pathways to daptomycin resistance using a combination of biochemical and structural approaches that link the change in biophysical properties to resistance. Techniques include X-ray crystallography, enzyme activity, ligand affinity, protein stability studies, RNAseq, qPCR, and others. This approach seeks to determine, not only the biochemical basis for resistance, but also those candidate proteins and pathways that would be well suited for the development of a new class of co-drugs that would target and delay the development of resistance. Our studies can also provide valuable molecular indicators of emerging resistance. Using our expertise in experimental evolution, we have also developed a new approach to harness both the power of evolution and the largely unexplored biochemical diversity and killing strategies of one of Nature's best antibiotic producing organisms: Streptomyces. We use experimental evolution within micro-emulsion droplets to produce selection conditions to identify variants of S. roseosporus (the “Predator”) that have improved their ability to kill a VRE strain (the “Prey”). Within each micro-droplet, we trap the two populations (Predator and a Prey). If the Predator can adapt to kill the Prey, the adapted Predator has a significant resource advantage, and increased reproductive success, over the un-adapted Predator. Taken together, this project takes a multi-pronged approach to uncovering the mechanisms and physicochemical basis for the evolution of antibiotic resistance and extends experimental evolution to include a novel method for discovering new antimicrobials.