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

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 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 supraphysiological levels of H₂O₂. 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.The 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 (7, 8). 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 antibiotic-induced ROS generation is a more complex process than originally suggested, likely involving additional means of production.The redox stress component of antibiotic lethality is hypothesized to derive from alterations to multiple core aspects of cellular physiology and stress response activation. Specifically, this component includes alterations to central metabolism, cellular respiration, and iron metabolism initiated by drug-mediated disruptions of target-specific processes and resulting cellular damage (Fig. 1A). Important support for this hypothesis can be found in pathogenic clinical isolates whose drug tolerance involves mutations in oxidative stress response and defense genes and not exclusively in drug target mutagenesis (40–48).Open in a separate windowFig. 1.Bactericidal antibiotics promote the generation of toxic reactive species. (A) Bactericidal antibiotics of different classes are capable of inducing cell death by interfering with their primary targets and corrupting target-specific processes, resulting in lethal cellular damage. Target-specific interactions trigger stress responses that induce redox-related physiological alterations resulting in the formation of toxic reactive species, including ROS, which further contribute to cellular damage and death. (B) Treatment of wild-type E. coli with ampicillin (Amp, 5 μg/mL), gentamicin (Gent, 5 μg/mL), or norfloxacin (Nor, 250 ng/mL) induces ROS, detectable by several chemically diverse fluorescent dyes with ranging specificity. One-way ANOVA was performed to determine statistical significance against the no-dye autofluorescence control. The dyes used were 5/6-carboxy-2'',7''-dichlorodihydrofluorescein diacetate (Carboxy-H₂DCFDA); 5/6-chloromethyl-2'',7''-dichlorodihydrofluorescein diacetate (CM-H₂DCFDA); 4-amino-5-methylamino-2´,7´-diflurorescein diacetate (DAF-FM); 2'',7''-dichlorodihydrofluorescein diacetate (H₂DCFDA); 3′-(p-hydroxyphenyl) fluorescein (HPF); OxyBURST Green (Oxyburst); and Peroxy-Fluor 2 (PF2). (C) Treatment of a quinolone-resistant strain (gyrA17) with norfloxacin does not produce detectable ROS. Data shown reflect mean ± SEM of three or more technical replicates. Where appropriate, statistical significance is shown and computed against the no-treatment control or no-dye control (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001).Recent critiques of this evolving model have misinterpreted an essential aspect of the hypothesis. Specifically, these recent studies (49–51) are predicated on the notion that ROS are the sole arbiters of antibiotic lethality, thereby implying that the model suggests that antibiotics do not kill by disrupting their well-established, target-specific processes. However, the evolving model is completely consistent with the literature indicating that bactericidal antibiotics are capable of inducing lethal cellular damage via interference with target-specific processes, ultimately resulting in cell death. Rather than refute this traditional view of antibiotic action, the hypothesis extends it by suggesting that an additional component of toxicity results from ROS, which are generated as a downstream physiological consequence of antibiotics interacting with their traditional targets. In this respect, reactive species are thought to contribute causatively to drug lethality.However, an important gap exists in our general understanding of how bacteria respond physiologically to antibiotic–target interactions on a system-wide level, how these responses contribute to antibiotic killing, and how the extracellular environment protects or exacerbates the intracellular contributions to cell death. Here, we use a multidisciplinary set of biochemical, enzymatic, biophysical, and genetic assays to address these issues and expand our understanding of antibiotic-induced physiological responses and factors contributing to antibiotic lethality. Data from the present work indicate that antibiotic lethality is accompanied by ROS generation and that such reactive species causatively contribute to antibiotic lethality.