Diagnosing reactive oxygen species in bacteria

点击翻译按钮获取中文摘要

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.