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. 2009 Apr;53(4):1395-402.
doi: 10.1128/AAC.01087-08. Epub 2009 Feb 17.

Contribution of oxidative damage to antimicrobial lethality

Xiuhong Wang  1 , Xilin Zhao
Affiliations

Contribution of oxidative damage to antimicrobial lethality

Xiuhong Wang et al. Antimicrob Agents Chemother. 2009 Apr.

Abstract

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.

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Figures

FIG. 1.
FIG. 1.
Effects of superoxide dismutase deficiency on norfloxacin lethality. Exponentially growing E. coli cells were treated with 0.4 μg/ml (10 times the MIC) norfloxacin for various times (A) or with the indicated concentrations of norfloxacin (B) for 2 h. Symbols: filled circles, wild type; filled squares, sodA sodB mutant; open circles, sodA mutant; open squares, sodB mutant. Error bars indicate standard deviations for replicate samples. At least three replicate experiments were performed, and each had results similar to those shown.
FIG. 2.
FIG. 2.
Effects of catalase/peroxidase deficiency on norfloxacin lethality. Exponentially growing E. coli cells were treated with 10 times the MIC of norfloxacin (0.8 μg/ml for the katG katE double mutant and 0.4 μg/ml for other strains) for various times (A) or with the indicated concentrations of norfloxacin (B) for 2 h as in Fig. 1. Symbols: filled circles, wild type; open circles, katG mutant; open squares, katE mutant; triangles, katG katE mutant. Error bars indicate standard deviations for replicate samples. At least three replicate experiments were performed, and each had results similar to those shown.
FIG. 3.
FIG. 3.
Effects of a ferrous chelator and a hydroxyl radical scavenger on norfloxacin lethality. Exponentially growing E. coli cells were preincubated with 250 μM bipyridyl (A and B) or 100 mM thiourea (C and D) for 10 min before they were treated with 10 times the MIC of norfloxacin (0.4 μg/ml) for various times (A and C) or with the indicated concentrations of norfloxacin (B and D) for 2 h as in Fig. 1. Symbols: filled circles, wild type; open circles, wild type plus bipyridyl or thiourea; filled squares, katG mutant; open squares, katG mutant plus bipyridyl or thiourea. Error bars indicate standard deviations for replicate samples. At least three replicate experiments were performed, and each had results similar to those shown.
FIG. 4.
FIG. 4.
Effects of katG, sodA sodB, and ahpC deficiencies on ampicillin lethality. Exponentially growing E. coli cells were treated with five times the MIC of ampicillin (150 μg/ml for the katG katE mutant and 75 μg/ml for other strains) for various times (A) or with the indicated concentrations of ampicillin (B) for 90 min. Symbols: filled circles, wild type; squares, katG mutant; open circles, sodA sodB mutant; triangles, ahpC mutant. Error bars indicate standard deviations for replicate samples. At least three replicate experiments were performed, and each had results similar to those shown.
FIG. 5.
FIG. 5.
Effects of katG, sodA sodB, and ahpC deficiencies on kanamycin lethality. Exponentially growing E. coli cells were treated with five times the MIC of kanamycin (12 μg/ml for the ahpC mutant and 3 μg/ml for other strains) for various times (A) or with the indicated concentrations of kanamycin (B) for 45 min. Symbols: filled circles, wild type; squares, katG mutant; open circles, sodA sodB mutant; triangles, ahpC mutant. Error bars indicate standard deviations for replicate samples. At least three replicate experiments were performed, and each had results similar to those shown.
FIG. 6.
FIG. 6.
Scheme depicting pathway by which bactericidal antimicrobial stress modulates lethal oxidative damage. Lethal stress from antimicrobial treatment causes an undefined redox imbalance, such that intracellular superoxides accumulate (step a). Upon conversion to hydrogen peroxide by dismutases (step b), hydroxyl radicals are generated from elevated levels of hydrogen peroxide via the Fenton reaction (step c). Hydroxyl radical species cause cell death (step d). Hydrogen peroxide is normally decomposed/detoxified by catalase/peroxidase (step e). Suppression of the generation of peroxide by a deficiency of both sodA and sodB protects cells from antimicrobial lethality, while inhibition of detoxification of peroxide via deficiencies in katG (in the case of norfloxacin) or ahpC (in the case of ampicillin and kanamycin) enhances cell death. Both an iron chelator (bipyridyl), which inhibits the Fenton reaction, and thiourea, a potent hydroxyl radical scavenger, protect cells from death.
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