Diagnosing reactive oxygen species in bacteria

诊断细菌中的活性氧

基本信息

批准号：
10181793
负责人：
JAMES A. IMLAY
金额：
$ 30.54万
依托单位：
UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-04-01 至 2025-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10181793
关键词：
Affect Anaerobic Bacteria Anti-Bacterial Agents Antibiotics Antioxidants Attention Awareness Bacteria Biological Biological Phenomena Cells Chemicals Clinical Communities DNA DNA Damage DNA Repair Data Deaminase Defect Diagnosis Dose Dyes Enzymes Equipment Escherichia coli Fluorescein Fluorescence Fluorescent Dyes Herbicides Hydro-Lyases Hydrogen Peroxide Hydroxyl Radical Immune system In Vitro Iron Lead Literature Measurement Metabolic stress Metals Methodology Methods Microbiology Modeling Molecular Morphologic artifacts Natural Products Nutrient Organism Oxidants Oxidation-Reduction Oxidative Stress Oxides Oxygen Paper Paraquat Pharmaceutical Preparations Phenazines Play Poison Positioning Attribute Production Protocols documentation Publishing Quinones Reaction Reactive Oxygen Species Role Serine Side Signal Transduction Site Stress Structure Suggestion Sulfur Superoxides System Techniques Testing Thymine Toxic Actions Toxic effect Trimethoprim United States National Institutes of Health Variant Voice Work antitumor agent assault base cell injury detection method drug action experience experimental study improved in vivo inhibitor/antagonist metalloenzyme novel oxidation oxidative damage repaired sensor side effect skills small molecule libraries therapy design

项目摘要

Bacteriologists have proposed that numerous stresses are toxic to bacteria because they stimulate the intracellular production of superoxide and hydrogen peroxide. In most cases, no molecular mechanism has been determined, and none is self-evident. Many of these proposals are based upon data from redox-active fluorescent dyes, which are believed to detect intracellular reactive oxygen species (ROS). However, those results have not always been supported by other more-direct methods. This proposal includes preliminary evidence that highlights two concerns about these dyes: that they can be oxidized by intracellular species other than ROS, and that the amount of dye that loads into cells can be affected by stress. Aim 1 will systematically test the utility of fluorescein dyes, which are oxidized by hydroxyl radicals, and ethidine dyes, which react with superoxide. Signals will be quantified in E. coli strains in which ROS are adjusted over the range of biological relevance. Dye signals will be normalized to their intracellular concentrations, using a novel technique. The result will be a rigorous test of their validity as ROS sensors. Aim 2 will develop an alternative marker of oxidative stress that is expected to be reliable, relevant, and accessible to non-experts. Serine dehydratase is a widely distributed enzyme whose iron-sulfur cluster is converted by oxidants to a unique [3Fe-4S] form that can be diagnosed through simple in vitro methods. This Aim will optimize these measurements and test whether the enzyme responds to the levels of ROS that are pertinent in vivo. Aim 3 will then investigate two antibiotics whose toxic actions have been proposed to depend upon intracellular ROS. Streptonigrin is thought to generate toxic amounts of H2O2 through redox- cycling, with the H2O2 then reacting with drug-bound Fe(II) to produce fatal hydroxyl radicals. However, an alternative model considered here suggests that redox-cycling may be minimal and free H2O2 may not participate. Experiments will distinguish between these two hypotheses. Trimethoprim is an inhibitor of thymine synthesis, and published data indicates that H2O2 can contribute to its lethality. Authors conjectured that thymine-starved cells produce toxic amounts of H2O2. A different possibility is that exogenous H2O2 creates DNA damage that thymineless cells struggle to repair. These two sub- aims introduce several distinct ways in which ROS may drive the action of antibiotics. These concepts and experimental approaches can guide the analysis of lead compounds whose antibiotic actions are suspected of involving ROS.

细菌学家提出，许多压力对细菌来说是有毒的，因为它们刺激细胞内产生超氧化物和过氧化氢。大多数情况下，没有分子机制已被确定，但没有一个是不言而喻的。其中许多建议是基于氧化还原活性荧光染料的数据，该染料被认为可以检测细胞内反应性氧物质（ROS）。然而，这些结果并不总是得到其他更直接的支持方法。该提案包括初步证据，强调了对这些染料的两个担忧：它们可以被 ROS 以外的细胞内物质氧化，并且负载的染料量进入细胞可能会受到压力的影响。目标 1 将系统地测试荧光素染料的效用，被羟基自由基和乙啶染料氧化，与超氧化物发生反应。信号将是在大肠杆菌菌株中进行定量，其中 ROS 在生物相关性范围内进行调整。染料使用新技术将信号标准化为其细胞内浓度。结果将对其作为 ROS 传感器的有效性进行严格的测试。目标 2 将开发一种氧化应激的替代标记，预计是可靠的，相关且非专家也可以理解。丝氨酸脱水酶是一种分布广泛的酶，其铁硫簇被氧化剂转化为独特的[3Fe-4S]形式，可以通过以下方式诊断简单的体外方法。该目标将优化这些测量并测试酶是否对体内相关的 ROS 水平做出反应。然后目标 3 将研究两种抗生素，其毒性作用被认为取决于依赖于细胞内的 ROS。链黑素被认为会通过氧化还原作用产生有毒量的 H2O2 循环，然后 H2O2 与药物结合的 Fe(II) 反应，产生致命的羟基自由基。然而，这里考虑的另一种模型表明，氧化还原循环可能是最小的，并且游离的 H2O2 可能是不参加。实验将区分这两种假设。甲氧苄啶是一种胸腺嘧啶合成抑制剂，已发表的数据表明 H2O2 有助于其致死性。作者推测，缺乏胸腺嘧啶的细胞会产生有毒量的 H2O2。另一种可能性是外源性 H2O2 会造成 DNA 损伤，而无胸腺细胞很难修复这种损伤。这两个子目标介绍了ROS驱动抗生素作用的几种不同方式。这些概念实验方法可以指导其抗生素作用的先导化合物的分析怀疑涉及ROS。