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

Abstract

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.

Keywords: DNA repair; mutagenesis; reactive oxygen species.

Fig. 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).

Fig. 2.

Antibiotics trigger physiologically relevant generation of H₂O₂. (A) Treatment of wild-type E. coli with ampicillin (Amp, 5 μg/mL), gentamicin (Gent, 5 μg/mL) or norfloxacin (Nor, 250 ng/mL) induces H₂O₂ production, detected by the intracellular enzymatic sensor APX, using Amplex Red fluorescence as an output. Antibiotic induction was compared with an exogenous H₂O₂ dose-range control. (B) Fold change of antibiotic-induced, H₂O₂-mediated Amplex Red fluorescence at 1 h and 2 h posttreatment, compared with the no-treatment control with basal H₂O₂ production. (C) Antibiotic-induced ROS trigger endogenous oxidative stress responses. Treatment with ampicillin (5 μg/mL) or norfloxacin (250 ng/mL) induces GFP expression from promoters regulated by H₂O₂-sensitive OxyR (pOxyS-gfp) and superoxide-sensitive SoxR (pSoxS-gfp) in wild-type E. coli. Data from additional GFP promoter reporters are included in SI Appendix. (D) Antibiotics induce physiologically relevant levels of oxidative stress. Antibiotic induction of GFP expression from pOxyS was compared with an exogenous H₂O₂ dose-range control. (E) Antibiotic-induced redox stress is comparable to physiologically relevant oxidative stress perturbations at the transcriptional level. Microarrays were performed on cells treated with H₂O₂ (10 μM), ampicillin (5 μg/mL), gentamicin (5 μg/mL), or norfloxacin (250 ng/mL) to quantify altered expression. Data shown reflect mean ± SEM o three or more technical replicates. Where appropriate, statistical significance is shown (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001). In each instance, an untreated control was used for normalization and determination of statistical significance.

Fig. 3.

Antibiotic stress induces redox-related alterations to cell physiology. Treatment of wild-type E. coli with bactericidal ampicillin (Amp, 5 μg/mL), gentamicin (Gent, 5 μg/mL), or norfloxacin (Nor, 250 ng/mL), but not bacteriostatic chloramphenicol (Chlor, 10 μg/mL), elevates respiratory activity as indicated by elevated OCR, measured by the Seahorse Extracellular Flux Analyzer. Data shown reflect mean ± SEM of three or more technical replicates.

Fig. 4.

Antibiotic-induced ROS damage DNA nucleotides. Overexpression of the DNA mismatch repair protein MutS inhibits killing by ampicillin (Amp, 5 μg/mL), kanamycin (Kan, 5 μg/mL), or norfloxacin (Nor, 250 ng/mL). Overexpression of recognition (F36A) or ATPase (K620A) mutants reduces MutS’s ability to suppress killing, indicating oxidative nucleotide damage. Data shown reflect mean ± SEM of three or more technical replicates for all data points. Where SEM is small, error bars are present but are inside symbols.

Fig. 5.

ROS contribute to the lethality elicited by bactericidal antibiotics. (A) Overexpression of the bifunctional peroxidase/catalase KatG inhibits killing by ampicillin (Amp, 10 μg/mL), gentamicin (Gent, 5 μg/mL), or norfloxacin (Nor, 125 ng/mL). Overexpression of heme cofactor-bearing mutant (H106Y), with markedly decreased catalase activity, reduces KatG’s ability to suppress killing. (B) Pretreatment with H₂O₂ (5 mM) for 15 min induces transient protection against killing by ampicillin (5 μg/mL), gentamicin (5 μg/mL), or norfloxacin (250 ng/mL). (C) Preincubation with glutathione (GSH, 50 mM), a natural antioxidant, inhibits killing by ampicillin (10 μg/mL), gentamicin (5 μg/mL), or norfloxacin (250 ng/mL). (D) Preincubation with ascorbic acid (AsA), another natural antioxidant, inhibits antibiotic killing by ampicillin (10 μg/mL, 1 mM AsA), gentamicin (5 μg/mL, 50 mM AsA), or Nor (250 ng/mL, 50 mM AsA). Transient protection of killing by ampicillin with 50 mM AsA pretreatment is depicted in SI Appendix, Fig. S8D. Data shown reflect mean ± SEM of three or more technical replicates for all data points. Where SEM is small, error bars are present but are inside symbols.

Fig. 6.

Antibiotic killing efficacy is sensitive to the availability of molecular oxygen and to alternative electron acceptors. (A) Strict, fully anaerobic treatment and plating reduces killing by 1–4 log over fully aerobic treatment and plating. Survival upon treatment (at indicated concentrations) with ampicillin (Amp), gentamicin (Gent), or norfloxacin (Nor) was assessed in nitrate-free Neidhardt complete defined medium. (B) Strict anaerobic conditions significantly inhibit antibiotic killing for high-concentration treatments with ampicillin (15 μg/mL), gentamicin (1 μg/mL), norfloxacin (400 ng/mL), and clinically relevant β-lactams [meropenem (Mero, 900 ng/mL)], ceftriaxone (Ceft, 3.5 μg/mL), or fluoroquinolones [moxifloxacin (Moxi, 275 ng/mL)]. (C) Exposure of anaerobically treated cells to environmental oxygen enhances antibiotic lethality. Cells were treated with ampicillin (25 μg/mL), gentamicin (1.25 μg/mL), or norfloxacin (650 ng/mL) under strict anaerobic conditions and then were diluted, plated, and incubated under either strict anaerobic or aerobic conditions. (D) Alternative electron acceptors enhance killing under strict anaerobic conditions by ampicillin (15 μg/mL, 10 mM KNO₃), gentamicin (5 μg/mL, 5 mM KNO₃), or norfloxacin (250 ng/mL, 5 mM KNO₃). Cells were supplemented with up to 10 mM nitrate, similar to concentrations found in LB (98). Data shown reflect mean ± SEM of three or more technical replicates for all data points. Where SEM is small, error bars are present but are inside symbols. Where appropriate, statistical significance is shown (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001).