Real-Time Single-Cell Imaging Reveals Accelerating Lipid Peroxyl Radical Formation in Escherichia coli Triggered by a Fluoroquinolone Antibiotic (original) (raw)
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Antibiotics induce redox-related physiological alterations as part of their lethality
Proceedings of the National Academy of Sciences, 2014
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 2 O 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 2 O 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. reactive oxygen species | DNA repair | mutagenesis T he increasing incidence of antibiotic-resistant infections coupled with a declining antibiotic pipeline has created a global public health threat (1-6). Therefore there is a pressing need to expand our conceptual understanding of how antibiotics act and to use insights gained from such efforts to enhance our antibiotic arsenal. It has been proposed that different classes of bactericidal antibiotics, regardless of their drug-target interactions, generate varying levels of deleterious reactive oxygen species (ROS) that contribute to cell killing . This unanticipated notion, built upon important prior work (9-11), has been extended and supported by multiple laboratories investigating wide-ranging drug classes (e.g., β-lactams, aminoglycosides, and fluoroquinolones) and bacterial species (e.g., Escherichia coli, Pseudomonas aeruginosa, Salmonella enterica, Mycobacterium tuberculosis, Bacillus subtilis, Staphylococcus aureus, Acinetobacter baumannii, Burkholderia cepecia, Streptococcus pneumonia, Enterococcus faecalis) using independent lines of evidence (12-39). Importantly, these ongoing efforts have served to refine aspects of the initial model and show that antibioticinduced ROS generation is a more complex process than originally suggested, likely involving additional means of production.
Contribution of Oxidative Damage to Antimicrobial Lethality
Antimicrobial Agents and Chemotherapy, 2009
A potential pathway linking hydroxyl radicals to antimicrobial lethality was examined by using mutational and chemical perturbations of Escherichia coli. Deficiencies of sodA or sodB had no effect on norfloxacin lethality; however, the absence of both genes together reduced lethal activity, consistent with rapid conversion of excessive superoxide to hydrogen peroxide contributing to quinolone lethality. Norfloxacin was more lethal with a mutant deficient in katG than with its isogenic parent, suggesting that detoxification of peroxide to water normally reduces quinolone lethality. An iron chelator (bipyridyl) and a hydroxyl radical scavenger (thiourea) reduced the lethal activity of norfloxacin, indicating that norfloxacin-stimulated accumulation of peroxide affects lethal activity via hydroxyl radicals generated through the Fenton reaction. Ampicillin and kanamycin, antibacterials unrelated to fluoroquinolones, displayed behavior similar to that of norfloxacin except that these two agents showed hyperlethality with an ahpC (alkyl hydroperoxide reductase) mutant rather than with a katG mutant. Collectively, these data are consistent with antimicrobial stress increasing the production of superoxide, which then undergoes dismutation to peroxide, from which a highly toxic hydroxyl radical is generated. Hydroxyl radicals then enhance antimicrobial lethality, as suggested by earlier work. Such findings indicate that oxidative stress networks may provide targets for antimicrobial potentiation.
Killing by Bactericidal Antibiotics Does Not Depend on Reactive Oxygen Species
Science, 2013
Antibiotic Mechanisms Revisited Several recent studies have suggested that bactericidal antibiotics kill cells by a common mechanism involving reactive oxygen species (ROS). Two groups tested this hypothesis using diverse experiments, with both finding that quinolone, lactam, and aminoglycoside antibiotics had similar efficacy for killing in the presence or absence of oxygen (or nitrate). Liu et al. (p. 1210 ) saw no increase in hydrogen peroxide production in antibiotic-exposed cells and found no association between antibiotic exposure and the expected symptoms of oxidative damage, such as the breakdown of iron-sulfur clusters in enzymes or of hydroxyl radical injuries to DNA. Similarly, Keren et al. (p. 1213 ) found no correlation between the production of ROS, inferred from hydroxyphenyl fluorescein dye measurements, and bacterial survival, nor was there any significant protective effect engendered by thiourea. The results do not support a common mode of action for bactericidal a...
The lipid membrane is a fundamental part of life. However, the effects of different stresses on membranal integrity and physiology are less understood. Using novel 4-aminophthalimide-based membrane-specific dyes (4AP-Cn: n is carbon chain-length), aided with confocal microscopy, fluorescence spectroscopy, molecular dynamics simulations, and flow cytometry, we have studied stress-mediated changes in E. coli membranes. By exploiting the depth-dependent positioning and subsequent environmental sensitivity of the dyes, we have proposed a measure of antibiotic-induced membrane damage: the fluorescence Peak Maxima Difference (PMD) between 4AP-C9 and 4AP-C13. The ROS-influenced PMD quantifies cytoplasmic membrane thickness and measures sensitivity against most bactericidal antibiotics, depending upon the extent of lipid peroxidation. Importantly, we have verified this observation using antibiotic-sensitive and resistant clinical isolates of E. coli and ESKAPE pathogens like K. pneumoniae a...
Moving forward with reactive oxygen species involvement in antimicrobial lethality
The Journal of antimicrobial chemotherapy, 2015
Support for the contribution of reactive oxygen species (ROS) to antimicrobial lethality has been refined and strengthened. Killing by diverse antimicrobials is enhanced by defects in genes that protect against ROS, inhibited by compounds that block hydroxyl radical accumulation, and is associated with surges in intracellular ROS. Moreover, support has emerged for a genetic pathway that controls the level of ROS. Since some antimicrobials kill in the absence of ROS, ROS must add to, rather than replace, known killing mechanisms. New work has addressed many of the questions concerning the specificity of dyes used to detect intracellular ROS and the specificity of perturbations that influence ROS surges. However, complexities associated with killing under anaerobic conditions remain to be resolved. Distinctions among primary lesion formation, resistance, direct lesion-mediated killing and a self-destructive stress response are discussed to facilitate efforts to potentiate ROS-mediated...
The involvement of oxidative stress in the mechanism of antibiotics-meditated cell death is unclear and subject to debate. The kinetic profile and a quantitative relationship between the release of reactive oxygen species (ROS), bacteria and antibiotic type remain elusive. Here we report direct measurements and analytical quantification of the release of superoxide radicals (O 2 Á À ), a major contributor to ROS, in antibiotics-treated bacterial cultures using a cytochrome c electrochemical biosensor. The specificity of electrochemical measurements was established by the addition of superoxide dismutase (SOD) which decreased the O 2 Á À signal. Measurements using a general ROS-specific fluorescence dye and colony forming units (CFU) assays were performed side-by-side to determine the total ROS and establish the relationship between ROS and the degree of lethality. Exposure of Escherichia coli and Listeria monocytogenes cultures to antibiotics increased the release of O 2 Á À radicals in a dose-dependent manner, suggesting that the transmembrane generation of ROS may occur as part of the antibiotic action. The study provides a quantitative methodology and fundamental knowledge to further explore the role of oxidative stress in antibiotics-meditated bacterial death and to assess physiological changes associated with the complex metabolic events related to oxidative stress and bacterial resistance.
Lipid Peroxidation in Membranes: The Peroxyl Radical Does Not “Float”
The Journal of Physical Chemistry Letters, 2014
Lipid peroxidation is a fundamental phenomenon in biology and medicine involved in a wide range of diseases. Some key microscopic aspects of this reaction in cell membranes are still poorly studied. In particular, it is commonly accepted that the propagation of the radical reaction in lipid bilayers is hampered by the rapid diffusion of peroxyl intermediates toward the water interface, that is, out of the reaction region. We investigated the validity of this "floating peroxyl radical" hypothesis by means of molecular modeling. Combining quantum calculations of model systems and atomistic simulations of lipid bilayers containing lipid oxidation products, we show that the peroxyl radical does not "float" at the surface of the membrane. Instead, it remains located quite deep inside the bilayer. In light of our findings, several critical aspects of biological membranes' peroxidation, such as their protection mechanisms, need to be revisited. Our data moreover help in the design of more efficient antioxidants, localized within reach of the reaction propagating radical. SECTION: Biomaterials, Surfactants, and Membranes 1B). An important aspect of this reaction is that the Pl • intermediate is a pretty stable entity with a half-life of ∼1 s. 7
F1000 - Post-publication peer review of the biomedical literature, 2013
Prolonged antibiotic treatment can lead to detrimental side effects in patients, including ototoxicity, nephrotoxicity, and tendinopathy, yet the mechanisms underlying the effects of antibiotics in mammalian systems remain unclear. It has been suggested that bactericidal antibiotics induce the formation of toxic reactive oxygen species (ROS) in bacteria. We show that clinically relevant doses of bactericidal antibiotics-quinolones, aminoglycosides, and β-lactams-cause mitochondrial dysfunction and ROS overproduction in mammalian cells. We demonstrate that these bactericidal antibiotic-induced effects lead to oxidative damage to DNA, proteins, and membrane lipids. Mice treated with bactericidal antibiotics exhibited elevated oxidative stress markers in the blood, oxidative tissue damage, and up-regulated expression of key genes involved in antioxidant defense mechanisms, which points to the potential physiological relevance of these antibiotic effects. The deleterious effects of bactericidal antibiotics were alleviated in cell culture and in mice by the administration of the antioxidant N-acetyl-L-cysteine or prevented by preferential use of bacteriostatic antibiotics. This work highlights the role of antibiotics in the production of oxidative tissue damage in mammalian cells and presents strategies to mitigate or prevent the resulting damage, with the goal of improving the safety of antibiotic treatment in people.