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Application of the redox metabolite detection method for profiling redox state following pharmacologic perturbations of redox balance in cells (part III)

ABSTRACT: This study aimed to test our optimized method for detection of redox metabolites from mammalian cells upon redox stress and metabolism perturbations. Redox balance was perturbed using H2O2 and diamide, metabolism was perturbed by methotrexate or oligomycin. This experiment is an independent repeat.

ORGANISM(S): Human Homo Sapiens

TISSUE(S): Cultured Cells

SUBMITTER: Boryana Petrova

PROVIDER: ST001771 | MetabolomicsWorkbench | Thu Apr 22 00:00:00 BST 2021

REPOSITORIES: MetabolomicsWorkbench

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Project description:Deeper understanding of antibiotic-induced physiological responses is critical to identifying means for enhancing our current antibiotic arsenal. Bactericidal antibiotics with diverse targets have been hypothesized to kill bacteria, in part, by inducing production of damaging reactive species. This notion has been supported by many groups, but recently challenged. Here we robustly test the hypothesis using biochemical, enzymatic and biophysical assays along with genetic and phenotypic experiments. We first used a novel intracellular hydrogen peroxide (H2O2) sensor, together with a chemically diverse panel of fluorescent dyes sensitive to an array of reactive species, to demonstrate that antibiotics broadly induce redox stress. Subsequent gene expression analyses reveal that complex antibiotic-induced oxidative stress responses are distinct from canonical responses generated by supra-physiological levels of H2O2. We next developed a method to dynamically quantify cellular respiration and found that bactericidal antibiotics elevate oxygen consumption, indicating significant alterations to bacterial redox physiology. We further show that catalase or DNA mismatch repair enzyme overexpression, as well as antioxidant pre-treatment limit antibiotic lethality, indicating that reactive oxygen species causatively contribute to antibiotic killing. Critically, the killing efficacy of antibiotics was diminished under strict anaerobic conditions, but could be enhanced by exposure to molecular oxygen or addition of alternative electron acceptors, suggesting that environmental factors play a role in killing cells physiologically primed for death. This work provides direct evidence that bactericidal antibiotics, downstream of their target-specific interactions, induce complex redox alterations that contribute to cellular damage and death, thus supporting an evolving, expanded model of antibiotic lethality. Here, we used microarrays to analyze oxidative stress responses to bactericidal antibiotic treatment in wildtype and mutant E coli WT or mutant E coli cells were grown to OD ~0.3. Untreated cells were harvested at time 0 as controls. Treated cells given the appropriate chemical perturbation and harvested 1 hour post-treatment. All experiments were performed in technical triplicate.

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Application of the redox metabolite detection method for profiling redox state following pharmacologic perturbations of redox balance in cells (part III)

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