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: 10481828
• 负责人: Luke Evan Diorio-Toth
• 金额: $ 3.27万
• 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/10481828
• 关键词: Acinetobacter; Amino Acids; Antibiotic Resistance; Antibiotics; Base Sequence; Binding Sites; Biochemical; Centers for Disease Control and Prevention (U.S.); 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; Measures; Medical; Mixed Function Oxygenases; Modern Medicine; Molecular Evolution; Morbidity - disease rate; Mutagenesis; Mutate; Mutation; Natural Resistance; Oxides; Pathway interactions; Phylogenetic Analysis; Plasmids; Point Mutation; Proteins; Public Health; Randomized; Research; Resistance; Resolution; Resort; Resources; Ribosomes; Sampling; Site; Structure; Testing; Tetanus Helper Peptide; Tetracycline Resistance; Tetracyclines; Work; 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; Acinetobacter; Amino Acids; Antibiotic Resistance; Antibiotics; Base Sequence; Binding Sites; Biochemical; Centers for Disease Control and Prevention (U.S.); 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; Measures; Medical; Mixed Function Oxygenases; Modern Medicine; Molecular Evolution; Morbidity - disease rate; Mutagenesis; Mutate; Mutation; Natural Resistance; Oxides; Pathway interactions; Phylogenetic Analysis; Plasmids; Point Mutation; Proteins; Public Health; Randomized; Research; Resistance; Resolution; Resort; Resources; Ribosomes; Sampling; Site; Structure; Testing; Tetanus Helper Peptide; Tetracycline Resistance; Tetracyclines; Work; 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.

项目摘要/摘要 四环素是至关重要的抗生素类别，但增加的抗药性威胁到它们的功效。四环素重新 人们认为齐平主要通过抗生素外排和核糖体保护发生。越来越多的阻力 通过酶促失活到包括最后一度的抗生素在内的晚期四环素 在环境和临床样品中调查。这些酶，称为四环素破坏酶，现在 被广泛认为是临床上与临床相关的电阻机制。尽管最近对这些酶有兴趣，但 精确的序列要求，将它们与其他黄酮酶区分开，并使活动能够 四环素尚不清楚。为了填补这些知识的空白，我将使用细菌基因组学的组合，合成 Thetic Biology和分子进化。该提议的长期目标是更好地了解Evolu- 四环素破坏酶的含量起源和结构特征，以便在理性上设计更好的诊断 - 在这种重要类别的抗生素成为广泛的原因之前 发病率和死亡率。我提出了两个独立但互补的特定目的：（1）表征se- 四环素破坏酶的验证结构功能空间，（2）确定相关FLA-的能力 Voenzymes进化四环素灭活活性。第一个目的将检验序列的假设 FMO进化向四环素灭活的决定因素包括在结构远端的区域 底物和辅因子结合位点。我将对随机单二元库进行深入测序 选择不同世代四环素抗抗抗性的不同四环素破坏性酶 口语和化学抑制剂。选定库的计算分析将揭示酶上的地点 大量选择用于底物特异性活性。第二个目的将检验四环素解除的假设 结构在四环素生物合成途径中具有进化起源。我将执行定向演变 使用诱变的迭代循环和四环素和四环素的迭代周期 杀菌剂直到这些酶赋予与四环素破坏酶相当的抗性水平。 se 克隆的示例将揭示残基或域，这些残基或域将这些酶优化为四环素ac- 暴力。拟议的研究很重要，因为抗生素耐药性是一种公共卫生危机，而四范围 - 降解所有已知四环素的降解酶已经在环境中广泛分布。这 拟议的研究具有影响力，因为它对对 这些酶的序列结构功能空间，这将有助于主动发展共同发展 具有减轻这种新兴威胁的潜力的诊断和诊断剂。