Pyocyanin alters redox homeostasis and carbon flux through central metabolic pathways in Pseudomonas aeruginosa PA14

The opportunistic pathogen Pseudomonas aeruginosa produces colorful, redox-active antibiotics called phenazines. Excretion of pyocyanin, the best-studied natural phenazine, is responsible for the bluish tint of sputum and pus associated with P. aeruginosa infections in humans. Although the toxicity of pyocyanin for other bacteria, as well as its role in eukaryotic infection, has been studied extensively, the physiological relevance of pyocyanin metabolism for the producing organism is not well understood. Pyocyanin reduction by P. aeruginosa PA14 is readily observed in standing liquid cultures that have consumed all of the oxygen in the medium. We investigated the physiological consequences of pyocyanin reduction by assaying intracellular concentrations of NADH and NAD+ in the wild-type strain and a mutant defective in phenazine production. We found that the mutant accumulated more NADH in stationary phase than the wild type. This increased accumulation correlated with a decrease in oxygen availability and was relieved by the addition of nitrate. Pyocyanin addition to a phenazine-null mutant also decreased intracellular NADH levels, suggesting that pyocyanin reduction facilitates redox balancing in the absence of other electron acceptors. Analysis of extracellular organic acids revealed that pyocyanin stimulated stationary-phase pyruvate excretion in P. aeruginosa PA14, indicating that pyocyanin may also influence the intracellular redox state by decreasing carbon flux through central metabolic pathways.     

Direct oxidation of 2',7'-dichlorodihydrofluorescein by pyocyanin and other redox-active compounds independent of reactive oxygen species production

Formation of dichlorofluorescein (DCF), the fluorescent oxidation product of 2',7'-dichlorodihydrofluorescein (DCFH2), in cells loaded with the latter compound is often used to detect ROS formation. We previously found that exposure of DCFH2-loaded A549 cells to the Pseudomonas aeruginosa secretory product pyocyanin results in DCF formation, consistent with ROS production. However, since pyocyanin directly accepts electrons from NAD(P)H, we hypothesized that pyocyanin might directly oxidize DCFH2 to DCF without an ROS intermediate. Incubation of DCFH2 with pyocyanin rapidly resulted in DCF formation, the rate of which was proportional to the [pyocyanin] and was not inhibited by SOD or catalase. Phenazine methosulfate, a pyocyanin analog, was more effective than pyocyanin in generating DCF. Mitoxantrone and ametantrone also produced DCF. However, menadione, paraquat, plumbagin, streptonigrin, doxorubicin, daunorubicin, and 5-iminodaunorubicin did not. Pyocyanin, phenazine methosulfate, mitoxantrone, and ametantrone also oxidized dihydrofluorescein and 5- (and 6-) -carboxy-2',7'-dichlorodihydrofluorescein, whereas dihydrorhodamine was oxidized only by pyocyanin or phenazine methosulfate. Under aerobic conditions, the interaction of DCFH2 with pyocyanin or phenazine methosulfate (but not mitoxantrone or ametantrone) produced superoxide, as detected by spin trapping. Direct oxidation of the fluorescent probes needs to be controlled for when employing these compounds to assess ROS formation by biological systems exposed to redox active compounds.     

Nitric oxide is inactivated by the bacterial pigment pyocyanin.

Pyocyanin is a phenazine pigment produced by the bacterium Pseudomonas aeruginosa and found in human lung secretions. Micromolar concentrations of pyocyanin inhibited the bioactivity of endothelium-derived relaxing factor (EDRF) generated from bovine pulmonary-artery endothelium in response to bradykinin. This inhibition was reversed by perfusing the EDRF-bioassay system with pyocyanin-free buffer for 15 min, but persisted in the presence of superoxide dismutase (20 units/ml). When nitric oxide, the major component of EDRF, was passed into an aqueous solution of pyocyanin in the absence of O2, a rapid colour change occurred from blue to pink; m.s. analysis of the products showed that the pyocyanin had been converted into a nitrosylated species.     

Construction of a glucose sensor based on a screen-printed electrode and a novel mediator pyocyanin from Pseudomonas aeruginosa

Pyocyanin is the blue phenazine pigment produced by Pseudomonas aeruginosa. Pyocyanin production using immobilized cells was investigated. The maximum production of pyocyanin was obtained using cells immobilized in kappa-carrageenan. Moreover, 0.01% PO4(3-), 0.2% Mg(2+), 0.001% Fe(2+), 1% glycerine, 0.8% leucine and 0.8% dl-alanine were also essential for pyocyanin production. Pyocyanin was purified by chloroform extraction and silica gel column chromatography. An amperometric biosensor system using a screen-printed electrode and pyocyanin as mediator were also developed for a more accurate determination of glucose concentration. Pyocyanin, which exists in the oxidated form, was reduced by the reaction between glucose oxidase and glucose. The reduced form was then converted back to the oxidized form by an oxidative reaction on the electrode. There was a linear relation ship between sensor output currents and glucose concentrations ranging from 1 to 20mM under the following conditions: -200 mV of the applied potential, pH 5.0, and 10 U of the immobilized enzyme. The coefficient of variation was below 3% (n = 5) for the glucose sensor.     

Oxidative stress caused by pyocyanin impairs CFTR Cl(-) transport in human bronchial epithelial cells

Pyocyanin (N-methyl-1-hydroxyphenazine), a redox-active virulence factor produced by the human pathogen Pseudomonas aeruginosa, is known to compromise mucociliary clearance. Exposure of human bronchial epithelial cells to pyocyanin increased the rate of cellular release of H(2)O(2) threefold above the endogenous H(2)O(2) production. Real-time measurements of the redox potential of the cytosolic compartment using the redox sensor roGFP1 showed that pyocyanin (100 microM) oxidized the cytosol from a resting value of -318+/-5 mV by 48.0+/-4.6 mV within 2 h; a comparable oxidation was induced by 100 microM H(2)O(2). Whereas resting Cl(-) secretion was slightly activated by pyocyanin (to 10% of maximal currents), forskolin-stimulated Cl(-) secretion was inhibited by 86%. The decline was linearly related to the cytosolic redox potential (1.8% inhibition/mV oxidation). Cystic fibrosis bronchial epithelial cells homozygous for DeltaF508 CFTR failed to secrete Cl(-) in response to pyocyanin or H(2)O(2), indicating that these oxidants specifically target the CFTR and not other Cl(-) conductances. Treatment with pyocyanin also decreased total cellular glutathione levels to 62% and cellular ATP levels to 46% after 24 h. We conclude that pyocyanin is a key factor that redox cycles in the cytosol, generates H(2)O(2), depletes glutathione and ATP, and impairs CFTR function in Pseudomonas-infected lungs.     

Pseudomonas aeruginosa pyocyanin directly oxidizes glutathione and decreases its levels in airway epithelial cells

Production of pyocyanin enhances Pseudomonas aeruginosa virulence. Many of pyocyanin's in vitro and in vivo cytotoxic effects on human cells appear to result from its ability to redox cycle. Pyocyanin directly accepts electrons from NADH or NADPH with subsequent electron transfer to oxygen, generating reactive oxygen species. Reduced glutathione (GSH) is an important cellular antioxidant, and it contributes to the regulation of redox-sensitive signaling systems. Using the human bronchial epithelial (HBE) and the A549 human type II alveolar epithelial cell lines, we tested the hypothesis that pyocyanin can deplete airway epithelial cells of GSH. Incubation of both cell types with pyocyanin led to a concentration-dependent loss of cellular GSH (up to 50%) and an increase in oxidized GSH (GSSG) in the HBE, but not A549 cells, at 24 h. An increase in total GSH, mostly as GSSG, was detected in the culture media, suggesting export of GSH or GSSG from the pyocyanin-exposed cells. Loss of GSH could be due to pyocyanin-induced H(2)O(2) formation. However, overexpression of catalase only partially prevented the pyocyanin-mediated decline in cellular GSH. Cell-free electron paramagnetic resonance studies revealed that pyocyanin directly oxidizes GSH, forming pyocyanin free radical and O(2)(-). Pyocyanin oxidized other thiol-containing compounds, cysteine and N-acetyl-cysteine, but not methionine. Thus GSH may enhance pyocyanin-induced cytotoxicity by functioning as an alternative source of reducing equivalents for pyocyanin redox cycling. Pyocyanin-mediated alterations in cellular GSH may alter epithelial cell functions by modulating redox sensitive signaling events.     

Pyocyanin: production,applications, challenges and new insights

Pseudomonas aeruginosa is an opportunistic, Gram-negative bacterium and is one of the most commercially and biotechnologically valuable microorganisms. Strains of P. aeruginosa secrete a variety of redox-active phenazine compounds, the most well studied being pyocyanin. Pyocyanin is responsible for the blue-green colour characteristic of Pseudomonas spp. It is considered both as a virulence factor and a quorum sensing signalling molecule for P. aeruginosa. Pyocyanin is an electrochemically active metabolite, involved in a variety of significant biological activities including gene expression, maintaining fitness of bacterial cells and biofilm formation. It is also recognised as an electron shuttle for bacterial respiration and as an antibacterial and antifungal agent. This review summarises recent advances of pyocyanin production from P. aeruginosa with special attention to antagonistic property and bio-control activity. The review also covers the challenges and new insights into pyocyanin from P. aeruginosa.     

Pseudomonas aeruginosa-Candida albicans Interactions: Localization and Fungal Toxicity of a Phenazine Derivative

Phenazines are redox-active small molecules that play significant roles in the interactions between pseudomonads and diverse eukaryotes, including fungi. When Pseudomonas aeruginosa and Candida albicans were cocultured on solid medium, a red pigmentation developed that was dependent on P. aeruginosa phenazine biosynthetic genes. Through a genetic screen in combination with biochemical experiments, it was found that a P. aeruginosa-produced precursor to pyocyanin, proposed to be 5-methyl-phenazinium-1-carboxylate (5MPCA), was necessary for the formation of the red pigmentation. The 5MPCA-derived pigment was found to accumulate exclusively within fungal cells, where it retained the ability to be reversibly oxidized and reduced, and its detection correlated with decreased fungal viability. Pyocyanin was not required for pigment formation or fungal killing. Spectral analyses showed that the partially purified pigment from within the fungus differed from aeruginosins A and B, two red phenazine derivatives formed late in P. aeruginosa cultures. The red pigment isolated from C. albicans that had been cocultured with P. aeruginosa was heterogeneous and difficult to release from fungal cells, suggesting its modification within the fungus. These findings suggest that intracellular targeting of some phenazines may contribute to their toxicity and that this strategy could be useful in developing new antifungals.     

Glutathione modulates the toxicity of, but is not a biologically relevant reductant for, the Pseudomonas aeruginosa redox toxin pyocyanin

Pyocyanin is an important redox toxin produced by the common human pathogen Pseudomonas aeruginosa. It generates reactive oxygen species (ROS) that alter intracellular redox status and cell function. Reducing equivalents for pyocyanin are provided by intracellular NAD(P)H and, it has been reported, glutathione (GSH). Cellular GSH levels are at least 1-2 orders of magnitude greater than NAD(P)H; therefore GSH should represent the major reductant for pyocyanin and potentiate its toxicity. Paradoxically, GSH has been found to inhibit pyocyanin toxicity in cellular models. This study was undertaken to evaluate the potential of GSH as a biologically relevant reductant for pyocyanin. As observed using spectrophotometry, under aerobic conditions pyocyanin readily oxidized NADPH, whereas oxidation of GSH could not be detected. Under anaerobic conditions pyocyanin was reduced by NADPH, but reduction by GSH could not be detected. Reduction of molecular oxygen and the formation of ROS readily proceeded in the presence of pyocyanin and NADPH, whereas GSH was without effect. Finally, exposure of normal human dermal fibroblasts to subcytotoxic concentrations of pyocyanin did not lead to depletion of endogenous GSH, but exogenous GSH provided protection against the senescence-inducing effects of the toxin. In summary, GSH does not reduce pyocyanin under physiologically relevant conditions or contribute to pyocyanin toxicity. However, GSH does provide protection against the deleterious effects of this important bacterial toxin on mammalian cells.     

Source of superoxide anion radical in aerobic mixtures consisting of NAD[P]H, 5-methylphenazinium methyl sulfate and nitroblue tetrazolium chloride

The source of superoxide anion radical (O2-.) in aerobic mixtures consisting of NAD[P]H, 5-methylphenazinium methyl sulfate (or its 1-methoxy derivative) and tetrazolium salt was investigated using superoxide dismutase (SOD), Mn(II), ferricytochrome-C, and epinephrine as probes. NAD[P]H + phenazine + O2 was found to reduce nitroblue tetrazolium, iodonitrotetrazolium, and thiazolyl blue in a manner sensitive to agents that dismutase O2-., viz., SOD and Mn(II). It also mediated the reduction of ferricytochrome-C, and augmented the autooxidation of epinephrine to the adrenochrome, without a tetrazolium salt present in the medium. The autooxidation of epinephrine, but not the reduction of ferricytochrome-C, was found to be sensitive to SOD. Nitroblue tetrazolium, either singly or in combination with SOD, did not stimulate the reduction of ferricytochrome-C. The oxidation of NADH, mediated by a catalytically low concentration of phenazine(+O2), was augmented two-fold by SOD. These observations are consistent with, and lend support to, a scheme of redox events (Scheme-3) wherein it is proposed that the source of O2-. in the NAD[P]H + phenazine + tetrazolium(+O2) system is the reduced phenazine, that the tetrazoinyl radical (a one-electron reduction product of tetrazolium) may not reduce O2 to O2-., that the redox reaction between semiquinone radicals of phenazine and O2 is reversible, and that the disproportionation of semiquinone radicals constitutes an important rate-limiting reaction in the expression of phenazine redox couple.     

Pyocyanin induced in vitro oxidative damage and its toxicity level in human,fish and insect cell lines for its selective biological applications

Pyocyanin is a redox active phenazine pigment produced by Pseudomonas aeruginosa, with broad antibiotic activity having pharmacological, aquaculture, agriculture and industrial applications. In the present work cytotoxicity induced by pyocyanin is demonstrated in a human embryonic lung epithelial cell line (L-132), a rainbow trout gonad cell line (RTG-2) and a Spodoptera frugiperda pupal ovarian cell line (Sf9). For toxicity evaluation, cellular morphology, mitochondrial function (XTT), membrane leakage of lactate dehydrogenase, neutral red uptake, affinity of electrostatic binding of protein with sulforhodamine B dyes, glucose metabolism, and reactive oxygen species, were assessed. Results showed that higher pyocyanin concentration is required for eliciting cytotoxicity in L-132, RTG-2 and Sf9. The microscopic studies demonstrated that the cell lines exposed to pyocyanin at higher concentrations alone showed morphological changes such as clumping and necrosis. Among the three cell lines L-132 showed the highest response to pyocyanin than the others. In short, pyocyanin application at concentrations ranging from 5 to 10 mg l^?1 were not having any pathological effect in eukaryotic systems and can be used as drug of choice in aquaculture against vibrios in lieu of conventional antibiotics and as biocontrol agent against fungal and bacterial pathogens in agriculture. This is besides its industrial and pharmacological applications.     

On the mechanism of production of superoxide radical by reaction mixtures containing NADH, phenazine methosulfate, and nitroblue tetrazolium

In aerobic reaction mixtures containing NADH, phenazine methosulfate, and nitroblue tetrazolium, O2- production is mediated by the tetrazolium, not the phenazine. Thus, superoxide dismutase inhibited reduction of the tetrazolium, but when ferricytochrome c was substituted for the tetrazolium its reduction was not affected by this enzyme. Furthermore, NADH plus the phenazine did not accelerate the oxidation of epinephrine to adrenochrome unless the tetrazolium was present, and under those circumstances superoxide dismutase did inhibit adrenochrome formation. When the tetrazolium and ferricytochrome c were present simultaneously, addition of superoxide dismutase was seen to accelerate the reduction of the cytochrome. This is explainable by the reduction of O2- by the reduced phenazine, which thus competes with cytochrome c for the available O2-. When the O2- was eliminated by superoxide dismutase, more of the reduced phenazine was available for the direct reduction of cytochrome c.     

Crystal structure of the pyocyanin biosynthetic protein PhzS

The human pathogen Pseudomonas aeruginosa produces pyocyanin, a blue-pigmented phenazine derivative, which is known to play a role in virulence. Pyocyanin is produced from chorismic acid via the phenazine pathway, nine proteins encoded by a gene cluster. Phenazine-1-carboxylic acid, the initial phenazine formed, is converted to pyocyanin in two steps that are catalyzed by the enzymes PhzM and PhzS. PhzM is an adenosylmethionine dependent methyltransferase, and PhzS is a flavin dependent hydroxylase. It has been shown that PhzM is only active in the physical presence of PhzS, suggesting that a protein-protein interaction is involved in pyocyanin formation. Such a complex would prevent the release of 5-methyl-phenazine-1-carboxylate, the putative intermediate, and an apparently unstable compound. Here, we describe the three-dimensional structure of PhzS, solved by single anomalous dispersion, at a resolution of 2.4 A. The structure reveals that PhzS is a member of the family of aromatic hydroxylases characterized by p-hydroxybenzoate hydroxylase. The flavin cofactor of PhzS is in the solvent exposed out orientation typically seen in unliganded aromatic hydroxylases. The PhzS flavin, however, appears to be held in a strained conformation by a combination of stacking interactions and hydrogen bonds. The structure suggests that access to the active site is gained via a tunnel on the opposite side of the protein from where the flavin is exposed. The C-terminal 23 residues are disordered as no electron density is present for these atoms. The probable location of the C-terminus, near the substrate access tunnel, suggests that it may be involved in substrate binding as has been shown for another structural homologue, RebC. This region also may be an element of a PhzM-PhzS interface. Aromatic hydroxylases have been shown to catalyze electrophilic substitution reactions on activated substrates. The putative PhzS substrate, however, is electron deficient and unlikely to act as a nucleophile, suggesting that PhzS may use a different mechanism than its structural relatives.     

Inactivation of the potent Pseudomonas aeruginosa cytotoxin pyocyanin by airway peroxidases and nitrite

Pyocyanin (1-hydroxy-N-methylphenazine, PCN) is a cytotoxic pigment and virulence factor secreted by the human bacterial pathogen, Pseudomonas aeruginosa. Here, we report that exposure of PCN to airway peroxidases, hydrogen peroxide (H(2)O(2)), and NaNO(2) generates unique mononitrated PCN metabolites (N-PCN) as revealed by HPLC/mass spectrometry analyses. N-PCN, in contrast to PCN, was devoid of antibiotic activity and failed to kill Escherichia coli and Staphylococcus aureus. Furthermore, in contrast to PCN, intratracheal instillation of N-PCN into murine lungs failed to induce a significant inflammatory response. Surprisingly, at a pH of ～7, N-PCN was more reactive than PCN with respect to NADH oxidation but resulted in a similar magnitude of superoxide production as detected by electron paramagnetic resonance and spin trapping experiments. When incubated with Escherichia coli or lung A549 cells, PCN and N-PCN both led to superoxide formation, but lesser amounts were detected with N-PCN. Our results demonstrate that PCN that has been nitrated by peroxidase/H(2)O(2)/NO(2)(-) systems possesses less cytotoxic/proinflammatory activity than native PCN. Yield of N-PCN was decreased by the presence of the competing physiological peroxidase substrates (thiocyonate) SCN(-) (myeloperoxidase, MPO, and lactoperoxidase, LPO) and Cl(-) (MPO), which with Cl(-) yielded chlorinated PCNs. These reaction products also showed decreased proinflammatory ability when instilled into the lungs of mice. These observations add important insights into the complexity of the pathogenesis of lung injury associated with Pseudomonas aeruginosa infections and provide additional rationale for exploring the efficacy of NO(2)(-) in the therapy of chronic Pseudomonas aeruginosa airway infection in cystic fibrosis.     

Premature cellular senescence induced by pyocyanin, a redox-active Pseudomonas aeruginosa toxin

Pseudomonas aeruginosa is an important nosocomial pathogen that can cause acute and chronic infection, particularly of the respiratory system. Pyocyanin is a major P. aeruginosa virulence factor that displays redox activity and induces oxidative stress in cellular systems. The effect of pyocyanin on replicating human pulmonary epithelial (A549) cells was investigated. Cells were exposed to pyocyanin for 24 h and their subsequent growth and development were followed for 7 days. Pyocyanin (5-10 microM) arrested cell growth and resulted in the development of a morphological phenotype consistent with cellular senescence, that is, an enlarged and flattened appearance. The senescent nature of these cells was supported by positive staining for increased lysosomal content and senescence-associated beta-galactosidase activity. All cells treated with pyocyanin (10 microM) converted to the senescent phenotype, which remained stable for up to 7 days. Exposure to pyocyanin at 25 microM or greater resulted in cell death due to apoptosis. A549 cells exposed to pyocyanin generated hydrogen peroxide in a dose-dependent manner and the senescence-inducing effect of pyocyanin was inhibited by the antioxidant, glutathione, suggesting the involvement of reactive oxygen species. The induction of premature cellular senescence by redox-active bacterial toxins may be a hitherto unrecognized aspect of infection pathology and a limiting factor in the tissue repair response to infection.     

Pyocycanin,a Contributory Factor in Haem Acquisition and Virulence Enhancement of Porphyromonas gingivalis in the Lung

Several recent studies show that the lungs infected with Pseudomonas aeruginosa are often co-colonised by oral bacteria including black-pigmenting anaerobic (BPA) Porphyromonas species. The BPAs have an absolute haem requirement and their presence in the infected lung indicates that sufficient haem, a virulence up-regulator in BPAs, must be present to support growth. Haemoglobin from micro-bleeds occurring during infection is the most likely source of haem in the lung. Porphyromonas gingivalis displays a novel haem acquisition paradigm whereby haemoglobin must be firstly oxidised to methaemoglobin, facilitating haem release, either by gingipain proteolysis or capture via the haem-binding haemophore HmuY. P. aeruginosa produces the blue phenazine redox compound, pyocyanin. Since phenazines can oxidise haemoglobin, it follows that pyocyanin may also facilitate haem acquisition by promoting methaemoglobin production. Here we show that pyocyanin at concentrations found in the CF lung during P. aeruginosa infections rapidly oxidises oxyhaemoglobin in a dose-dependent manner. We demonstrate that methaemoglobin formed by pyocyanin is also susceptible to proteolysis by P. gingivalis Kgp gingipain and neutrophil elastase, thus releasing haem. Importantly, co-incubation of oxyhaemoglobin with pyocyanin facilitates haem pickup from the resulting methemoglobin by the P. gingivalis HmuY haemophore. Mice intra-tracheally challenged with viable P. gingivalis cells plus pyocyanin displayed increased mortality compared to those administered P. gingivalis alone. Pyocyanin significantly elevated both methaemoglobin and total haem levels in homogenates of mouse lungs and increased the level of arginine-specific gingipain activity from mice inoculated with viable P. gingivalis cells plus pyocyanin compared with mice inoculated with P. gingivalis only. These findings indicate that pyocyanin, by promoting haem availability through methaemoglobin formation and stimulating of gingipain production, may contribute to virulence of P. gingivalis and disease severity when co-infecting with P. aeruginosa in the lung.     

A new method of preparation of pyocyanin and demonstration of an unusual bacterial sensitivity.

Pyocyanin was prepared in 60% yield from phenazine methoxysulfate by a photooxidation procedure and purification by silica gel chromatography. Monitoring was performed by thin-layer chromatography. Approximately 50% of clinical Pseudomonas aeruginosa isolates were found to produce pyocyanin at 37°C. Among Proteus strains, P. morganii strains were sensitive to concentrations of pyocyanin 16 to 64 times lower than concentrations that inhibited the growth of P. mirabilis and P. vulgaris strains.     

Subcellular localization of Pseudomonas pyocyanin cytotoxicity in human lung epithelial cells

The Pseudomonas aeruginosa secretory product pyocyanin damages lung epithelium, likely due to redox cycling of pyocyanin and resultant superoxide and H(2)O(2) generation. Subcellular site(s) of pyocyanin redox cycling and toxicity have not been well studied. Therefore, pyocyanin's effects on subcellular parameters in the A549 human type II alveolar epithelial cell line were examined. Confocal and electron microscopy studies suggested mitochondrial redox cycling of pyocyanin and extracellular H(2)O(2) release, respectively. Pyocyanin decreased mitochondrial and cytoplasmic aconitase activity, ATP levels, cellular reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, and mitochondrial membrane potential. These effects were transient at low pyocyanin concentrations and were linked to apparent cell-mediated metabolism of pyocyanin. Overexpression of MnSOD, but not CuZnSOD or catalase, protected cellular aconitase, but not ATP, from pyocyanin-mediated depletion. This suggests that loss of aconitase activity is not responsible for ATP depletion. How pyocyanin leads to ATP depletion, the mechanism of cellular metabolism of pyocyanin, and the impact of mitochondrial pyocyanin redox cycling on other cellular events are important areas for future study.     

Reactions of Pseudomonas aeruginosa pyocyanin with reduced glutathione

Pseudomonas aeruginosa is the most common cause of chronic and recurrent lung infections in patients with cystic fibrosis (CF) whose sputa contain copious quantities of P. aeruginosa toxin, pyocyanin. Pyocyanin triggers tissue damage mainly by its redox cycling and induction of reactive oxygen species (ROS). The reactions between reduced glutathione (GSH) and pyocyanin were observed using absorption spectra from spectrophotometry and the reaction products analysed by nuclear magnetic resonance imaging. Pyocyanin reacted with GSH non-enzymatically at 37 degrees C resulting in the production of red-brown products, spectophotometrically visible as a 480 nm maximum absorption peak after 24 h of incubation. The reaction was concentration-dependent on reduced glutathione but not on pyocyanin. Minimizing the accessibility of oxygen to the reaction decreased its rate. The anti-oxidant enzyme catalase circumvented the reaction. Proton-NMR analysis demonstrated the persistence of the original aromatic ring and the methyl-group of pyocyanin in the red-brown products. Anti-oxidant agents having thiol groups produced similar spectophotometrically visible peaks. The presence of a previously unidentified non-enzymatic GSH-dependent metabolic pathway for pyocyanin has thus been identified. The reaction between pyocyanin and GSH is concentration-, time-, and O(2)-dependent. The formation of H(2)O(2) as an intermediate and the thiol group in GSH seem to be important in this reaction.