Diagnosing reactive oxygen species in bacteria

细菌学家提出，大量的压力对细菌是有毒的，因为它们 刺激细胞内产生超氧化物和过氧化氢。在大多数情况下，没有 分子机制已经确定，没有一个是不言而喻的。这些建议中有许多是 基于氧化还原活性荧光染料的数据，这些染料被认为可以检测细胞内的反应 氧物种(ROS)。然而，这些结果并不总是得到其他更直接的支持 方法：研究方法。这项提案包括初步证据，突显了对这些染料的两个担忧： 它们可以被ROS以外的细胞内物种氧化，而且负载的染料的量 会受到压力的影响。目的1将系统地测试荧光素染料的用途，其中 被羟基自由基和乙基染料氧化，与超氧化物发生反应。信号将是 在生物相关性范围内调节ROS的大肠杆菌菌株中进行量化。染料 使用一种新技术，信号将被归一化为其细胞内浓度。结果将是 将是对它们作为ROS传感器的有效性的严格测试。 AIM 2将开发一种氧化应激的替代标记，预计该标记将是可靠的， 相关的，非专家可以访问的。丝氨酸脱水酶是一种分布广泛的酶，其 铁-硫团簇被氧化剂转化为独特的[3Fe-4S]形式，可以通过以下方法进行诊断 简单的体外实验方法。这一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将调查两种抗生素，它们的毒性作用被认为依赖于 细胞内ROS。链球菌素被认为通过氧化还原产生有毒数量的过氧化氢- 循环，与过氧化氢，然后与药物结合的铁(II)反应，产生致命的羟基自由基。然而， 这里考虑的另一个模型表明，氧化还原循环可能是最小的，而游离的过氧化氢可能 而不是参与。实验将区分这两种假设。甲氧苄啶是一种 胸腺嘧啶合成的抑制剂，公布的数据表明，过氧化氢可导致其致命性。 作者推测，胸腺嘧啶饥饿的细胞会产生有毒的过氧化氢。另一种可能性是 外源性过氧化氢造成DNA损伤，无胸腺细胞难以修复。这两个分部- AIMS介绍了ROS可能驱动抗生素作用的几种不同方式。这些概念 实验方法可以指导对先导化合物的分析，这些化合物的抗生素作用是 涉嫌牵涉到Ros。

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.