A Novel High-Throughput Exploration of DNA Supercoiling toward the Therapeutic Manipulation of Bacteria

DNA 超螺旋的新型高通量探索对细菌的治疗性操作

基本信息

批准号：
10741397
负责人：
Eduardo Groisman
金额：
$ 25.13万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-05-17 至 2025-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10741397
关键词：
Acceleration Adenosine Diphosphate Adenosine Triphosphate Animal Model Anti-Bacterial Agents Anti-Infective Agents Antibiotics Antimicrobial Resistance Bacteria Bacterial DNA Bacterial Physiology Behavior Biological Cells Chloroquine DNA DNA Gyrase Development Enzymes Evaluation Exposure to Face Fluorescence Fluoroquinolones Genes Genetic Growth Human Infection Infectious Agent Laboratories Link Magnesium Methods Modeling Mutate Mutation Mycobacterium tuberculosis Osmolar Concentration Output Pathway interactions Pharmaceutical Preparations Phenotype Physiological Polyamines Predisposition Process Property Proteins Putrescine RNA Relaxation Reporting Research Salmonella typhimurium Sepharose Site Spermidine Stress Superhelical DNA Technology Temperature Therapeutic Time Toxic effect Type I DNA Topoisomerases antimicrobial bacterial resistance base enzyme activity experience exposure pathway gene discovery high throughput screening human pathogen inhibitor mutant novel novel strategies pathogen plasmid DNA programs response targeted agent technological innovation

项目摘要

PROJECT SUMMARY Bacterial survival requires a nearly constant and specific global DNA supercoiling status. This is achieved by the opposing functions of the DNA-relaxing topoisomerase I and DNA-compacting DNA gyrase. We propose the first unbiased high-throughput exploration of genes governing the abundance and activity of these enzymes, which hold great untapped promise for therapeutic manipulation. How pathogens change DNA supercoiling during infection conceals vulnerabilities critical to overcoming the toxic effects of current DNA gyrase inhibitors and emergence of bacteria resistant to these agents. We will examine mutants of the pathogen Salmonella enterica serovar Typhimurium for altered DNA supercoiling in infection-relevant conditions by deploying Fluorescence Evaluation of DNA Supercoiling – or FEDS – a new high-throughput method developed in our laboratory that reports the global DNA supercoiling status of living bacteria. To expose the genetic basis for altered DNA supercoiling, we will identify the mutated genes and explore genetic interactions among the mutations to reveal novel instances of synthetic lethality, thereby uncovering both novel genetic pathways and novel ways to manipulate DNA supercoiling. We will solve the bases for the DNA supercoiling behavior of the identified mutants by determining the amounts of DNA gyrase and topoisomerase I as well as the abundance of adenosine triphosphate, adenosine diphosphate, and specific polyamines – all critical regulators of DNA gyrase activity. The proposed research program pioneers the physiological understanding of an essential cellular property exploitable in a wide range of bacterial species to identify novel targets for antibacterial agents.

项目概要细菌的生存需要几乎恒定且特定的整体 DNA 超螺旋状态。这是通过 DNA 松弛拓扑异构酶 I 和拓扑异构酶 I 的相反功能实现的。我们提出了第一个无偏高通量的 DNA 压缩 DNA 旋转酶。探索控制这些酶的丰度和活性的基因，病原体如何变化具有巨大的未开发前景。感染期间的 DNA 超螺旋隐藏了对于克服病毒至关重要的漏洞当前 DNA 旋转酶抑制剂的毒性作用以及耐药细菌的出现我们将检查病原体肠道沙门氏菌血清型的突变体。鼠伤寒杆菌通过部署改变感染相关条件下的 DNA 超螺旋 DNA 超螺旋（或 FEDS）的荧光评估——一种新的高通量我们实验室开发的方法可报告全球 DNA 超螺旋状态为了揭示 DNA 超螺旋的遗传基础，我们将鉴定活细菌。突变基因并探索突变之间的遗传相互作用以揭示从而揭示了合成致死性的新实例，揭示了两种新的遗传途径以及操纵 DNA 超螺旋的新方法我们将解决 DNA 的碱基问题。通过测定 DNA 量来确定突变体的超螺旋行为旋转酶和拓扑异构酶 I 以及三磷酸腺苷的丰度，二磷酸腺苷和特定多胺——DNA 旋转酶的所有关键调节因子拟议的研究计划开创了对生理学的理解。可在多种细菌物种中利用的基本细胞特性来识别抗菌剂的新靶点。