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

绘制新型四环素抗性酶家族的进化图谱

• 批准号: 10686080
• 负责人: Luke Evan Diorio-Toth
• 金额: $ 5.27万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10686080
• 关键词: Acinetobacter; Amino Acids; Antibiotic Resistance; Antibiotics; Base Sequence; Binding Sites; Biochemical; Characteristics; Chemicals; Clinical; Codon Nucleotides; Computer Analysis; Detection; Development; Diagnostic; Directed Molecular Evolution; Distal; Environment; Enzymes; Evolution; Family; Farm; Flavins; Generations; Genes; Genomics; Goals; Health; Healthcare; Hospitals; Human; Individual; Knowledge; Libraries; Maps; Measures; Medical; Mixed Function Oxygenases; Modern Medicine; Molecular Evolution; Morbidity - disease rate; Mutagenesis; Mutate; Mutation; Natural Resistance; Pathway interactions; Phylogenetic Analysis; Plasmids; Point Mutation; Proteins; Public Health; Randomized; Research; Resistance; Resolution; Resources; Ribosomes; Sampling; Site; Structure; Testing; Tetanus Helper Peptide; Tetracycline Resistance; Tetracyclines; Therapeutic; Work; anthropogenesis; antibiotic efflux; antineoplastic antibiotics; clinically relevant; cofactor; deep sequencing; disability; drug resistant pathogen; efflux pump; emerging antibiotic resistance; enzyme mechanism; experimental study; fitness; improved; in vivo; inhibitor; interest; mortality; multi-drug resistant pathogen; multidrug-resistant Pseudomonas aeruginosa; mutant; next generation; novel; pathogen; predictive modeling; preservation; pressure; rational design; resistance gene; resistance mechanism; screening; synthetic biology; tigecycline; water sampling

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.

项目概要/摘要 四环素是一类重要的抗生素，但耐药性的增加威胁着它们的疗效。四环素再 据认为，耐药性主要通过抗生素外流和核糖体保护发生。阻力越来越大 新一代四环素，包括最后手段的抗生素，通过酶灭活正在被取消 在环境和临床样本中进行检测。这些酶被称为四环素破坏酶，现在 被广泛认为是一种临床相关的耐药机制。尽管最近人们对这些酶产生了兴趣， 精确的序列要求将它们与其他黄素酶区分开来，并使其具有活性 四环素类尚不清楚。为了填补这些知识空白，我将结合使用细菌基因组学、同步技术 主题生物学和分子进化。该提案的长期目标是更好地理解进化论 四环素破坏酶的起源和结构特征，以便合理设计更好的诊断 抽动症和抑制剂，以在这一类重要抗生素成为广泛的病因之前恢复其功效 发病率和死亡率。我提出了两个独立但互补的具体目标：（1）描述这些特征 四环素破坏酶的序列-结构-功能空间，以及（2）确定相关fla-的能力 voenzymes 具有四环素灭活活性。第一个目标将检验序列的假设 FMO 向四环素失活进化的决定因素包括结构上远离的区域 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 选择不同的四环素破坏酶来抵抗不同代的四环素抗生素 耳药和化学抑制剂。对所选文库的计算分析将揭示酶上的位点 严格选择底物特异性活性。第二个目标将检验四环素去 结构酶在四环素生物合成途径中具有进化起源。我将进行定向进化 使用四环素和四环素的诱变和选择的迭代循环，系统发育相关的黄素酶 直到这些酶具有与四环素破坏酶相当的抗性水平。硒- 克隆的测序将揭示针对四环素活性优化这些酶的残基或结构域 活动性。这项拟议的研究意义重大，因为抗生素耐药性是一场公共卫生危机，而四环素- 能够降解所有已知四环素的克莱破坏酶已经广泛分布在环境中。这 拟议的研究具有影响力，因为它提供了对 这些酶的序列-结构-功能空间，这将有助于联合治疗的主动开发 有潜力减轻这一新兴威胁的治疗剂和诊断剂。