Antibiotics induce redox-related physiological alterations as part of their lethality

• Published in: Proceedings of the National Academy of Sciences - PNAS 2014-05, Vol.111 (20), p.E2100-E2109
• Main Authors: Dwyer, Daniel J, Belenky, Peter A, Yang, Jason H, MacDonald, I Cody, Martell, Jeffrey D, Takahashi, Noriko, Chan, Clement T Y, Lobritz, Michael A, Braff, Dana, Schwarz, Eric G, Ye, Jonathan D, Pati, Mekhala, Vercruysse, Maarten, Ralifo, Paul S, Allison, Kyle R, Khalil, Ahmad S, Ting, Alice Y, Walker, Graham C, Collins, James J
• Format: Article
• Language: English
• Subjects: Anti-Bacterial Agents - pharmacology; Antibiotics; Antioxidants - chemistry; Bacteria; Biological Sciences; Catalase - metabolism; Cell Respiration; DNA Repair; Escherichia coli - drug effects; Escherichia coli Proteins - metabolism; Experiments; Fluorescent Dyes; Gene Expression Regulation, Bacterial - drug effects; Genetics; Genotype & phenotype; Green Fluorescent Proteins - metabolism; Hydrogen Peroxide - chemistry; Mutagenesis; MutS DNA Mismatch-Binding Protein - metabolism; Oligonucleotide Array Sequence Analysis; Oxidation-Reduction; Oxidative Stress; Oxygen - metabolism; Physiology; Plasmids - metabolism; PNAS Plus; Reactive Oxygen Species
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1401876111

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 has been challenged recently. Here we robustly test the hypothesis using biochemical, enzymatic, and biophysical assays along with genetic and phenotypic experiments. We first used a novel intracellular H ₂O ₂ 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 supraphysiological levels of H ₂O ₂. We next developed a method to quantify cellular respiration dynamically and found that bactericidal antibiotics elevate oxygen consumption, indicating significant alterations to bacterial redox physiology. We further show that overexpression of catalase or DNA mismatch repair enzyme, MutS, and antioxidant pretreatment 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 by the addition of alternative electron acceptors, indicating that environmental factors play a role in killing cells physiologically primed for death. This work provides direct evidence that, downstream of their target-specific interactions, bactericidal antibiotics induce complex redox alterations that contribute to cellular damage and death, thus supporting an evolving, expanded model of antibiotic lethality.