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。病原体进化超过了新病原体的供应 化合物和类似物，威胁全球健康危机。理解适应的新方法是 这显然是必要的，但这是一个难题。耐药性的机制往往是未知的，以及 产生整体微生物适合度变化的变化的整体组合。的技术发展 高通量生物化学已经允许测定大量的变体文库，这为以下打开了大门： 构建耐药性的预测模型，但直到我们最近的工作才忽略了复杂的突变， 特别是插入和缺失，它们通过产生基础基因的重大变化而发挥着巨大的作用。 适应性景观的小突变。为了研究如何，我们将联合收割机结合实验进化， 突变扫描和多温晶体学，以产生一个综合模型， 并且缺失允许蛋白质功能的快速变化。 我们将使用链阳性菌素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Streptomyces生产。抗性通过Vat蛋白发生， 链阳性菌素A（SA）通过乙酰化。与加州大学旧金山分校的Seiple实验室合作， SA的合成，其允许简单地产生变体，以及几种新化合物， 在体外证明乙酰化降低。我们将确定如何适应这些新化合物 以及关键变量底物结合环内的插入缺失如何调节它。 在我的第一个目标中，我将使用实验进化来揭示Vat蛋白如何适应链阳性菌素，以及如何 在该环内插入和缺失的添加改变了适应性潜力。在我的第二个目标中，我将 进行高通量稳定性测量以确定电阻变化的机理基础， 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， 进化的适应性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 对新SA的底物特异性的indel增强变化的静态和动态基础。 这些目标的完成将产生一个微生物适应和必要的新的综合图片。 深入了解复杂但研究不足的突变如何从根本上改变基因的适应潜力。十字架- 该项目的学科性质将使我接触到一些技术和方法，这将为我提供 在领域界定专家的指导下，有极好的培训机会。新框架和 我将发展的技术将有助于建立我的科学成熟，因为我追求我的目标是一个独立的 在一所大学从事研究蛋白质分子进化的机械基础的研究工作。