Mapping the evolutionary landscape of a novel family of tetracycline resistance enzymes

项目总结/摘要 四环素类是一类重要的抗生素，但日益增加的耐药性威胁到它们的功效。四环素Re- 耐药性主要通过抗生素外排和核糖体保护作用产生。越来越多的抵抗 到新一代四环素，包括抗生素的最后手段，通过酶失活，正在去- 检测环境和临床样本。这些酶被称为四环素破坏酶， 被广泛认为是临床相关的耐药机制。尽管最近对这些酶感兴趣， 精确的序列要求，使它们与其他黄素酶区分开来，并使活性， 四环素是不清楚的。为了填补这些知识空白，我将使用细菌基因组学，同步- 心理生物学和分子进化。这项提案的长期目标是更好地了解演变- 四环素破坏酶的来源和结构特点，以便合理设计更好的诊断方法， 抽搐和抑制剂，以恢复这类重要的抗生素的功效，然后才成为一个广泛的原因， 发病率和死亡率。我提出了两个独立的，但互补的具体目标：（1）表征本身- 四环素破坏酶的序列-结构-功能空间;（2）确定相关弗拉- voenzymes发展四环素失活活性。第一个目标是检验一个假设， FMO向四环素失活进化的决定因素包括结构上远离四环素的区域， 底物和辅因子结合位点。我将对随机单密码子文库进行深度测序， 选择对不同世代的四环素抗体具有抗性的不同四环素破坏酶， 药物和化学抑制剂。对所选文库的计算分析将揭示酶上的位点， 被大量选择用于底物特异性活性。第二个目的是检验四环素脱- 结构酶在四环素生物合成途径中具有进化起源。我将对 使用四环素和四环素选择的诱变和选择的迭代循环， 抗生素，直到这些酶赋予与四环素破坏酶相当的抗性水平。塞- 克隆的测序将揭示使这些酶对四环素AC最优化的残基或结构域。 活力这项拟议中的研究意义重大，因为抗生素耐药性是一场公共卫生危机，而四环素- 降解所有已知四环素类的渐变群破坏酶已经广泛分布于环境中。的 拟议的研究是有影响力的，因为它提供了一个全面和高分辨率的理解， 这些酶的序列-结构-功能空间，这将有助于协同疗法的积极发展， 有可能减轻这一新出现的威胁的治疗剂和诊断剂。

PROJECT SUMMARY/ABSTRACT Tetracyclines are a vital class of antibiotics, but increasing resistance threatens their efficacy. Tetracycline re- sistance is thought to mainly occur through antibiotic efflux and ribosomal protection. Increasingly, resistance to late-generation tetracyclines, including antibiotics of last-resort, through enzymatic inactivation is being de- tected in environmental and clinical samples. These enzymes, known as tetracycline destructases, are now widely recognized as a clinically-relevant resistance mechanism. Despite recent interest in these enzymes, the precise sequence requirements that distinguish them from other flavoenzymes, and enable activity towards tetracyclines is unclear. To fill these gaps in knowledge, I will use a combination of bacterial genomics, syn- thetic biology, and molecular evolution. The long-term goal for this proposal is to better understand the evolu- tionary origins and structural features of tetracycline destructases in order to rationally design better diagnos- tics and inhibitors to re-store efficacy of this vital class of antibiotics before they become a widespread cause of morbidity and mortality. I propose two independent, yet complementary specific aims: (1) Characterize the se- quence-structure-function space of tetracycline destructases, and (2) Determine the capacity of related fla- voenzymes to evolve tetracycline inactivation activity. The first aim will test the hypothesis that the sequence determinants of FMO evolution toward tetracycline inactivation include regions that are structurally distal to substrate and cofactor binding sites. I will perform deep sequencing of randomized single-codon libraries of different tetracycline destructases that are selected for resistance to different generations of tetracycline antibi- otics and chemical inhibitors. Computational analysis of selected libraries will reveal sites on the enzymes that are heavily selected for substrate-specific activity. The second aim will test the hypothesis that tetracycline de- structases have evolutionary origins in tetracycline biosynthetic pathways. I will perform directed evolution on phylogenetically-related flavoenzymes using iterative cycles of mutagenesis and selection with tetracycline an- tibiotics until these enzymes confer levels of resistance that are comparable to tetracycline destructases. Se- quencing of clones will reveal the residues or domains which optimize these enzymes towards tetracycline ac- tivity. The proposed research is significant because antibiotic resistance is a public health crisis, and tetracy- cline destructases that degrade all known tetracyclines are already widely distributed in the environment. The proposed research is impactful because it provides a comprehensive and high-resolution understanding of the sequence-structure-function space of these enzymes, which will aid in the proactive development of co-thera- peutic and diagnostic agents that have the potential to mitigate this emerging threat.