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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.

项目总结细菌的生存需要一个几乎恒定和特定的全球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 DNA压缩DNA旋转酶。我们提出了第一个无偏高吞吐量探索控制这些酶的丰度和活性的基因，这是拥有巨大的未开发的治疗操作的前景。病原体是如何变化的感染过程中的DNA超级卷曲隐藏了对克服当前DNA旋转酶抑制剂的毒性效应和耐药细菌的出现这些特工。我们将检测病原体肠炎沙门氏菌血清型的突变小鼠在感染相关条件下通过部署用于改变的DNA超螺旋一种新的高通量DNA超螺旋或FEDS的荧光评价我们实验室开发的报告全球DNA超螺旋状态的方法活细菌。为了揭示改变的DNA超螺旋的遗传基础，我们将识别并探索突变基因之间的遗传交互作用，揭示突变合成致命性的新实例，从而揭示了两种新的遗传途径以及操纵DNA超级卷曲的新方法。我们将解开DNA的碱基通过测定DNA含量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，二磷酸腺苷和特定的多胺--DNA旋转酶的关键调节因子活动。拟议的研究计划开创了对心脏的生理学理解。在广泛的细菌物种中可利用的基本细胞特性来识别抗菌药物的新靶点。