An electron spin resonance study of oxyradical generation in superoxide dismutase- and catalase-deficient mutants of Escherichia coli K-12

IntroductionPyocyanin, a secretory product of Pseudomonas aeruginosa, has the capacity to undergo redox cycling under aerobic conditions with resulting generation of superoxide and hydrogen peroxide. By using spin trapping techniques in conjunction with electron paramagnetic resonance spectrometry (EPR), superoxide was detected during the aerobic reduction of pyocyanin by NADH or porcine endothelial cells. No evidence of hydroxyl radical formation was detected. Chromium oxalate eliminated the EPR spectrum of the superoxide-derived spin adduct resulting from endothelial cell exposure to pyocyanin, suggesting superoxide formation close to the endothelial cell plasma membrane. We have previously reported that iron bound to the P. aeruginosa siderophore pyochelin (ferripyochelin) catalyzes the formation of hydroxyl free radical from superoxide and hydrogen peroxide via the Haber-Weiss reaction. In the present study, spin trap evidence of hydroxyl radical formation was detected when NADH and pyocyanin were allowed to react in the presence of ferripyochelin. Similarly, endothelial cell exposure to pyocyanin and ferripyochelin also resulted in hydroxyl radical production which appeared to occur in close proximity to the cell surface. As assessed by 51Cr release, endothelial cells which were treated with pyocyanin or ferripyochelin alone demonstrated minimal injury. However, endothelial cell exposure to the combination of pyochelin and pyocyanin resulted in 55% specific 51Cr release. Injury was not observed with the substitution of iron-free pyochelin and was diminished by the presence of catalase or dimethyl thiourea. These data suggest the possibility that the P. aeruginosa secretory products pyocyanin and pyochelin may act synergistically via the generation of hydroxyl radical to damage local tissues at sites of pseudomonas infection. (J. Clin. Invest. 1992. 90:2187-2196

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“…Consistent with previous work with pyocyanin (5,25,34,50,51) and other redox active compounds (27,52,53), O2…”

Section: Discussionsupporting

confidence: 92%

“…In contrast, a small amount of hydroxyl radical has been reportedly spin trapped with addition of pyocyanin to E. coli, particularly a catalase-deficient strain (50). These data suggest that the reduction of pyocyanin by NADH or cellular enzyme systems does not generate hydroxyl radical within the limits ofsensitivity of spin trapping.…”

Section: Discussionmentioning

confidence: 89%

Interaction of the Pseudomonas aeruginosa secretory products pyocyanin and pyochelin generates hydroxyl radical and causes synergistic damage to endothelial cells. Implications for Pseudomonas-associated tissue injury.

Britigan¹,

Roeder²,

Rasmussen³

et al. 1992

J. Clin. Invest.

135

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“…The results described so far indicate that the enzyme catalase-peroxidase can contribute to the protection of logphase R. capsulatus against H202, but this function is manifest only when hydrogen peroxide concentrations in vivo are higher than those present in cells grown in air (37). There are indications in the literature that aged cells are more vulnerable to oxidative damage, both because their metabolic activities produce more deleterious oxygen products and because they have a lower potential for protection (15,16,34).…”

Section: Methodsmentioning

confidence: 90%

Physiological functions of hydroperoxidases in Rhodobacter capsulatus

1992

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Rhiodobacter capsulatus Jl has two hydroperoxidases: a catalase-peroxidase and a peroxidase. A mutant strain, AH18, that had no catalase-peroxidase was isolated. The growth rate under aerobic and photosynthetic conditions, respiration, superoxide dismutase and peroxidase activities, and pigment content of the mutant were similar to those of the wild type. AH18 was more susceptible to killing and to inhibition of nitrogenase by H202 but not by molecular oxygen. The incidences of spontaneous mutations were similar in both strains. Viable counts in aerobic but not anaerobic cultures of AH18 started to decline as soon as the cultures reached the stationary phase, and the rate of cell death was much higher in AH18 than in the wild type. It is inferred that the peroxidase provides protection against H202 in log-phase cells and that the catalase-peroxidase provides protection under the oxidative conditions that prevail in aging cultures. This protective function might be related to the dual activity of the latter as a catalase and a peroxidase or to its capacity to oxidize NADH, NADPH, and cytochrome c.Metabolic activation of molecular oxygen very often results in the production of hydrogen peroxide, which has been shown to be deleterious to most cellular components (12,29). The potential damage is especially significant in cells faced with stresses characteristic of special conditions. For example, photosynthetic organisms are challenged by the photodynamic reactions that enhance partial reduction of 02 (12); in nitrogen-free medium, diazotrophs are vulnerable because of the extreme lability to oxygen of the nitrogen fixation system (35); and elevated steady-state concentrations of toxic oxygen products play a significant role in aging of cells and tissues (15,16,35). Hydrogen peroxide is metabolized by three different types of hydroperoxidases: catalase, peroxidase, and catalase-peroxidase. The typical catalases, which catalyze the dismutation of H202 to 02 and H20, have been isolated from animals, plants, and microorganisms (7, 10). It is generally accepted that the major physiological role of the typical catalase is protection of the cells against the damaging effect of hydrogen peroxide (7). Peroxidase, which catalyzes oxidation of H202 by a large variety of substrates, also may function in detoxifying hydrogen peroxide as well as in various cellular activities of biosynthesis and degradation (12). The catalase-peroxidases, which were only recently classified as a distinct group of enzymes (19,33) tochrome c (unpublished data). These enzymes share biochemical and physicochemical properties with both catalases and peroxidases. Like the typical catalases they are tetramers, with a combined molecular mass of about 240,000 Da, and they have hydrophobic properties exhibited by their binding to phenyl-Sepharose. Unlike typical catalases and similar to peroxidases, the catalase-peroxidases are inactivated by ethanol-chloroform and are not inhibited by 3-amino-1,2,4-triazole, they have a sharp dependence of their activity...

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“…EPR: spin trapping. To measure oxygen-centered radicals formed after exposure to pyocyanin, a spin-trapping tech-nique similar to that described by Schellhorn et al was employed (38). Bacteria were grown in TSY to mid-logarithmic phase, collected by centrifugation (10,000 x g for 10 min), and suspended in cold Chelex 100 (Bio-Rad)-treated 50 mM KPi (pH 7.0)-0.1 mM diethylenetriaminepentaacetic acid (Sigma).…”

Section: Methodsmentioning

confidence: 99%

“…Glucose (0.5%), 0.1 M 5,5-dimethyl-1-pyrroline-1-oxide (DMPO; provided by Ron Mason, National Institute of Environmental Health Sciences, Cary, N.C.), and pyocyanin (20 FxM) were then added. DMPO is a nitroxide which reacts with 02 or HO to form spin-trapped adducts with characteristic spectra which can be visualized by using EPR (20,38). Thorough reviews of spin trapping in biological systems with DMPO are documented elsewhere (34,37).…”

Section: Methodsmentioning

confidence: 99%

Response of Pseudomonas aeruginosa to pyocyanin: mechanisms of resistance, antioxidant defenses, and demonstration of a manganese-cofactored superoxide dismutase

et al. 1992

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Pseudomonas aeruginosa produces a blue pigment, pyocyanin. Pyocyanin is a redox-active phenazine compound that kills mammalian and bacterial cells through the generation of reactive oxygen intermediates. We examined the mechanisms by which P. aeruginosa resists pyocyanin. [14C]pyocyanin was taken up by both Escherichia coli and P. aeruginosa, though more slowly by the latter. Cyanide-insensitive respiration, used as an indicator of intracellular superoxide and/or hydrogen peroxide production, was 50-fold less in pyocyanintreated P. aeruginosa than in E. coli. P. aeruginosa showed less cyanide-insensitive respiration than E. coli upon exposure to other redox-active compounds (paraquat, streptonigrin, and plumbagin). Electron paramagnetic resonance spectrometry and spin trapping showed that P. aeruginosa generated less pyocyanin radical and superoxide than E. coli. Cell extracts from E. coli contained an NADPH:pyocyanin oxidoreductase which increased the rate of reduction of pyocyanin by NADPH. Conversely, cell extracts from P. aeruginosa contained no NADPH:pyocyanin oxidoreductase activity and actually decreased the rate of pyocyanin-mediated NADPH oxidation. Antioxidant defenses could also reduce the sensitivity of P. aeruginosa to pyocyanin. Under culture conditions of limited phosphate, both pyocyanin production and catalase activity were enhanced. Superoxide dismutase activity was also increased under low-phosphate conditions. When cells were grown in a high-phosphate succinate medium, P. aeruginosa formed a previously described iron-superoxide dismutase as well as a manganese-cofactored superoxide dismutase. These results demonstrate that P. aeruginosa resists pyocyanin because of limited redox cycling of this compound and that under conditions favoring pyocyanin production, catalase and superoxide dismutase activities increase.

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An electron spin resonance study of oxyradical generation in superoxide dismutase- and catalase-deficient mutants of Escherichia coli K-12

Cited by 11 publications

References 26 publications

Interaction of the Pseudomonas aeruginosa secretory products pyocyanin and pyochelin generates hydroxyl radical and causes synergistic damage to endothelial cells. Implications for Pseudomonas-associated tissue injury.

Interaction of the Pseudomonas aeruginosa secretory products pyocyanin and pyochelin generates hydroxyl radical and causes synergistic damage to endothelial cells. Implications for Pseudomonas-associated tissue injury.

Physiological functions of hydroperoxidases in Rhodobacter capsulatus

Response of Pseudomonas aeruginosa to pyocyanin: mechanisms of resistance, antioxidant defenses, and demonstration of a manganese-cofactored superoxide dismutase

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