Connecting structure and fitness landscapes to overcome antibiotic resistance

连接结构和健身景观以克服抗生素耐药性

• 批准号: 10679332
• 负责人: Christian Bernard Macdonald
• 金额: $ 6.95万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-13 至 2025-09-12
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10679332
• 关键词: Acetylation; Amino Acids; Antibiotic Resistance; Antibiotics; Area; Binding; Biochemistry; Biological Assay; Biophysics; Chemosensitization; Clinical; Collaborations; Complex; Coupled; Coyotes; Crystallography; Development; Drug Design; Drug resistance; Enterococcus faecium; Environment; Enzymatic Biochemistry; Escherichia coli; Event; Evolution; Failure; Family; Future; Genes; Genetic Recombination; Goals; Grant; Heterogeneity; Homologous Gene; In Vitro; Laboratories; Libraries; Link; Longevity; Maps; Measurement; Measures; Mediating; Mentorship; Microbe; Modeling; Molecular; Molecular Conformation; Mutation; Nature; Outcome; Pathway interactions; Phenotype; Phylogenetic Analysis; Play; Positioning Attribute; Process; Property; Proteins; Research; Resistance; Resources; Ribosomes; Role; Sampling; Scanning; Sequence Analysis; Streptogramins; Streptomyces; Structural Biochemistry; Structure; Substrate Specificity; Techniques; Temperature; Universities; Variant; Work; analog; antimicrobial; biophysical model; biophysical properties; combinatorial; enzyme activity; fitness; gain of function; global health; improved; insertion/deletion mutation; insight; microbial; mutant; mutation screening; novel; novel strategies; pathogen; permissiveness; predictive modeling; preempt; protein function; protein structure; resistance gene; resistance mechanism; response; screening; training opportunity

Project summary - Connecting structure and fitness landscapes to overcome antibiotic resistance Antibiotic resistance is a pressing, multifaceted challenge. Pathogen evolution is outstripping the supply of new compounds and analogues, threatening a global health crisis. New approaches to understand adaptation are clearly necessary, but this is a difficult problem. The mechanisms of resistance are often unknown, as well as the overall combination of changes that produce overall microbial fitness changes. Technical developments in high-throughput biochemistry have allowed massive variant libraries to be assayed, which opens the door to constructing predictive models of resistance, but these have until our recent work ignored complex mutations, specifically insertions and deletions, which play a massive role by producing major changes to underlying fitness landscapes with small mutations. To study how, we will combine experimental evolution, deep mutational scanning, and multitemperature crystallography to produce an integrated model for how insertions and deletions permit rapid changes to protein function. We will use the Streptogramin A family as our model antibiotic. These are ribosome-targeting compounds produced by Streptomyces. Resistance occurs through Vat proteins, which specifically inactivate Streptogramin A (SA) via acetylation. A collaboration with the Seiple lab at UCSF has led to a modular synthesis of SA that allows variants to be simply produced, as well as several novel compounds with demonstrated reduced acetylation in vitro. We will determine how adaptation to these novel compounds proceeds, and how indels within a key variable substrate-binding loop modulate it. In my first aim, I will use experimental evolution to uncover how Vat proteins adapt to streptogramins, and how the addition of insertions and deletions within this loop change the adaptive potential. In my second aim, I will conduct high-throughput stability measurements to determine the mechanistic basis for resistance changes, and then use deep mutational scanning to measure the mutational accessibility and biophysical basis for an evolved adaptive trajectory. In my third aim, I will use cryo- and multitemperature crystallography to determine the static and dynamic basis for indel-potentiated changes to substrate specificity towards new SAs. The completion of these aims will produce an integrated picture of microbial adaptation and essential new insight into how complex but understudied mutations radically shift the adaptive potential of genes. The cross- disciplinary nature of the project will expose me to a number of techniques and approaches that will provide me with excellent training opportunities under the mentorship of field-defining experts. The new frameworks and techniques I will develop will help establish my scientific maturity as I pursue my goal of an independent research position studying the mechanistic basis of molecular protein evolution at a university.

项目摘要 - 连接结构和健身景观以克服抗生素耐药性 抗生素耐药性是一个紧迫的、多方面的挑战。病原体的进化超过了新病原体的供应 化合物和类似物，威胁着全球健康危机。理解适应的新方法是 显然是必要的，但这是一个难题。耐药机制通常是未知的，而且 产生总体微生物适应性变化的变化的总体组合。技术发展 高通量生物化学允许分析大量变异文库，这打开了大门 构建耐药性预测模型，但直到我们最近的工作之前，这些模型都忽略了复杂的突变， 特别是插入和删除，它们通过对底层产生重大变化而发挥着巨大作用 具有小突变的健身景观。为了研究如何，我们将结合实验进化、深度 突变扫描和多温度晶体学可生成插入如何进行的集成模型 和缺失使得蛋白质功能快速改变。 我们将使用链阳菌素 A 家族作为我们的模型抗生素。这些是核糖体靶向化合物 由链霉菌产生。耐药性是通过 Vat 蛋白产生的，Vat 蛋白会特异性地灭活 链阳菌素 A (SA) 通过乙酰化作用。与加州大学旧金山分校 Seiple 实验室的合作开发了一个模块化的 SA 的合成，可以简单地生产变体，以及几种具有 体外证明乙酰化作用降低。我们将确定如何适应这些新化合物 的进展，以及关键可变底物结合环中的插入缺失如何调节它。 在我的第一个目标中，我将利用实验进化来揭示 Vat 蛋白如何适应链阳菌素，以及如何 在这个循环中添加插入和删除会改变适应性潜力。在我的第二个目标中，我会 进行高通量稳定性测量以确定电阻变化的机械基础， 然后使用深度突变扫描来测量突变的可及性和生物物理基础 进化出的自适应轨迹。在我的第三个目标中，我将使用低温和多温晶体学来确定 新 SA 底物特异性的插入缺失增强变化的静态和动态基础。 这些目标的完成将产生微生物适应和重要新功能的综合图景。 深入了解复杂但尚未充分研究的突变如何从根本上改变基因的适应潜力。交叉 该项目的学科性质将使我接触到许多技术和方法，这些技术和方法将为我提供帮助 在领域定义专家的指导下提供良好的培训机会。新的框架和 当我追求独立的目标时，我将开发的技术将有助于建立我的科学成熟度 在大学研究分子蛋白质进化的机制基础的研究职位。