Document text
Principal Investigator: JAMES A. IMLAY
Organization: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
Fiscal Year: 2024
Award: $305,434
Funding agency: National Institute of General Medical Sciences
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.
Terms: <1,1'-dimethyl-4,4'-bipyridinium><Active Oxygen><Affect><Anaerobic Bacteria><Anti-Bacterial Agents><Antibiotic Agents><Antibiotic Drugs><Antibiotics><Antioxidants><Artifacts><Attention><Awareness><Bacteria><Binding><Biologic Phenomena><Biological><Biological Phenomena><Cell Body><Cell Communication and Signaling><Cell Protection><Cell Signaling><Cells><Cellular injury><Chemical Actions><Clinical><Coloring Agents><Communities><Cytoprotection><DNA><DNA Damage><DNA Injury><DNA Repair Inhibition><Data><Deaminase><Defect><Dehydratases><Deoxyribonucleic Acid><Diagnosis><Dose><Drugs><Dyes><E coli><E. coli><Enzyme Gene><Enzymes><Equipment><Escherichia coli><Fe element><Fluorescein><Fluorescence><Fluorescence Agents><Fluorescent Agents><Fluorescent Dyes><H2O2><Herbicides><Hydrases><Hydro-Lyases><Hydrogen Peroxide><Hydroperoxide><Hydroxyl><Hydroxyl Radical><Immune system><In Vitro><In vivo analysis><Injury><Intracellular Communication and Signaling><Iron><L-Serine><Lead><Literature><Measurement><Medication><Metabolic stress><Metals><Methodology><Methods><Methyl Viologen><Microbiology><Miscellaneous Antibiotic><Modeling><Molecular><Molecular Interaction><Morphologic artifacts><NIH><National Institutes of Health><Natural Products><Nutrient><O element><O2 element><Organism><Oxidants><Oxidation-Reduction><Oxidative Stress><Oxidizing Agents><Oxygen><Oxygen Radicals><Paper><Paraquat><Pb element><Pharmaceutical Preparations><Phenazines><Play><Poison><Position><Positioning Attribute><Pro-Oxidants><Production><Proloprim><Protocol><Protocols documentation><Publishing><Quinone Compound><Quinones><Reaction><Reactive Oxygen Species><Redox><Role><S element><Serine><Side><Signal Transduction><Signal Transduction Systems><Signaling><Site><Starvation><Stress><Structure><Suggestion><Sulfur><Superoxide Anion><Superoxide Radical><Superoxides><System><Techniques><Testing><Thymine><Toxic Actions><Toxic Chemical><Toxic Substance><Toxic effect><Toxicities><Trimethoprim><Trimpex><United States National Institutes of Health><Variant><Variation><Voice><Work><anaerobe><anti-bacterial><anti-tumor agent><assault><biologic><biological signal transduction><cell damage><cell injury><cellular damage><chemical library><cytoprotective><damage to cells><detection method><detection procedure><detection technique><drug action><drug/agent><electron acceptor><experience><experiment><experimental research><experimental study><experiments><fluorescent dye/probe><heavy metal Pb><heavy metal lead><improved><in vivo><in vivo evaluation><in vivo testing><inhibitor><injuries><injury to cells><intervention design><living system><metalloenzyme><naturally occurring product><novel><oxidation><oxidation reduction reaction><oxidative damage><oxidative injury><repair><repaired><sensor><side effect><skills><small molecule libraries><social role><therapy design><toxic compound><treatment design>