Defining Evolutionary Trajectories: Molecular adaptation to antibiotic resistance

细菌病原体的耐药性仍然是公众面临的最大挑战之一 当今世界的健康。抗生素的广泛使用促进了多重耐药菌的增加， 这些病原体可能会破坏现代医学的巨大成就。中心的 疾病控制和预防已将耐多药肠球菌确定为“严重威胁” 需要迅速和持续的活动来限制扩散。达托霉素是一线抗生素， 对革兰氏阳性微生物的有效性，并且越来越频繁地用于对抗多种药物 耐万古霉素肠球菌（VRE）。本提案的目的是 全面绘制屎肠球菌中导致DAP耐药性的进化轨迹 并阐明蛋白质结构-功能的确定变化如何建立物理化学 观察到的抗性表型的基础。我们在小说中使用定量实验进化， 连续培养生物反应器系统，以识别和排名最重要的进化轨迹 导致抵抗。基于这些结果，我们然后表征最相关的蛋白质， 使用生物化学和结构方法的组合， 将生物物理特性的变化与抗性联系起来。技术包括X射线晶体学， 酶活性、配体亲和力、蛋白质稳定性研究、RNAseq、qPCR等。这种方法 不仅试图确定抗性的生化基础，而且还试图确定那些候选蛋白质， 以及非常适合开发一类新的联合药物的途径， 瞄准并延缓耐药性的发展。我们的研究还可以提供有价值的分子生物学信息。 新出现的阻力指标。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，既利用进化的力量， 自然界最好的抗生素生产生物之一的生物化学多样性和杀死策略： 链霉菌我们使用微乳液液滴内的实验进化来产生选择 条件来鉴定S. roseosporus（“捕食者”），提高了他们的能力，杀死 VRE菌株（“猎物”）。在每个微滴中，我们捕获两个种群（捕食者和猎物）。 如果捕食者能适应杀死猎物，适应捕食者有一个显着的资源优势， 和繁殖成功率的提高。总的来说，这个项目需要 一个多管齐下的方法来揭示机制和物理化学基础的演变 抗生素耐药性，并扩展实验进化，包括一种新的方法，发现 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.