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.     

Functional analysis of genes for biosynthesis of pyocyanin and phenazine-1-carboxamide from Pseudomonas aeruginosa PAO1

Two seven-gene phenazine biosynthetic loci were cloned from Pseudomonas aeruginosa PAO1. The operons, designated phzA1B1C1D1E1F1G1 and phzA2B2C2D2E2F2G2, are homologous to previously studied phenazine biosynthetic operons from Pseudomonas fluorescens and Pseudomonas aureofaciens. Functional studies of phenazine-nonproducing strains of fluorescent pseudomonads indicated that each of the biosynthetic operons from P. aeruginosa is sufficient for production of a single compound, phenazine-1-carboxylic acid (PCA). Subsequent conversion of PCA to pyocyanin is mediated in P. aeruginosa by two novel phenazine-modifying genes, phzM and phzS, which encode putative phenazine-specific methyltransferase and flavin-containing monooxygenase, respectively. Expression of phzS alone in Escherichia coli or in enzymes, pyocyanin-nonproducing P. fluorescens resulted in conversion of PCA to 1-hydroxyphenazine. P. aeruginosa with insertionally inactivated phzM or phzS developed pyocyanin-deficient phenotypes. A third phenazine-modifying gene, phzH, which has a homologue in Pseudomonas chlororaphis, also was identified and was shown to control synthesis of phenazine-1-carboxamide from PCA in P. aeruginosa PAO1. Our results suggest that there is a complex pyocyanin biosynthetic pathway in P. aeruginosa consisting of two core loci responsible for synthesis of PCA and three additional genes encoding unique enzymes involved in the conversion of PCA to pyocyanin, 1-hydroxyphenazine, and phenazine-1-carboxamide.     

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.     

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.     

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.     

Oxidation of pyocyanin, a cytotoxic product from Pseudomonas aeruginosa, by microperoxidase 11 and hydrogen peroxide

Pyocyanin (1-hydroxy-N-methylphenazine) is a cytotoxic pigment secreted by the bacterial species Pseudomonas aeruginosa, which frequently infects the lungs of immunosuppressed patients as well as those with cystic fibrosis. Pyocyanin toxicity results presumably from the ability of the compound to undergo reduction by NAD(P)H and subsequent generation of superoxide and H2O2 directly in the lungs. We report that in the presence of peroxidase mimics, microperoxidase 11, or hemin, pyocyanin undergoes oxidation by H2O2, as evidenced by loss of the pigment's characteristic absorption spectrum and by EPR detection of a free radical metabolite. The oxidation of pyocyanin is irreversible, suggesting an extensive modification of the pigment's phenazine chromophore. Oxidation of pyocyanin was observed also when exogenous H2O2 was replaced by a H2O2-generating system consisting of NADH and the pigment itself. That the oxidation involves the phenolate group of pyocyanin was verified by the observation that a related pigment, phenazine methosulfate, which is devoid of this group, does not undergo oxidation by microperoxidase 11/H2O2. In contrast to intact pyocyanin, oxidized pyocyanin was less efficient in NADH oxidation and stimulation of interleukin-8 release by human alveolar epithelial A549 cells in vitro, suggesting that oxidation of pyocyanin leads to its inactivation. This study demonstrates that pyocyanin may play a dual role in biological systems, first as an oxidant and ROS generator, and second as a substrate for peroxidases, contributing to H2O2 removal. This latter property may cause pyocyanin degradation and inactivation, which may be of considerable biomedical interest.     

Effect of pyocyanin on a crude-oil-degrading microbial community

Pseudomonas aeruginosa is an n-alkane degrader that is frequently isolated from petroleum-contaminated sites and produces factors that enhance its competitiveness and survival in many environments. In this study, one such factor, pyocyanin, has been detected in an oil-degrading culture containing P. aeruginosa and is a redox-active compound capable of inhibiting microbial growth. To examine the effects of pyocyanin further, an oil-degrading culture was grown with and without 9.5 microM pyocyanin and microbial community structure and oil degradation were monitored for 50 days. Denaturing gradient gel electrophoresis (DGGE) analysis of cultures revealed a decrease in the microbial community diversity in the pyocyanin-amended cultures compared to that of the unamended cultures. Two members of the microbial community in pure culture exhibited intermediate and high sensitivities to pyocyanin corresponding to intermediate and low levels of activity for the antioxidant enzymes catalase and superoxide dismutase, respectively. Another member of the community that remained constant in the DGGE gels over the 50-day culture incubation period exhibited no sensitivity to pyocyanin, corresponding to a high level of catalase and superoxide dismutase when examined in pure culture. Pyocyanin also affected the overall degradation of the crude oil. At 50 days, the culture without pyocyanin had decreased polycyclic aromatic hydrocarbons compared to the pyocyanin-amended culture, with a specific reduction in the degradation of dibenzothiophenes, naphthalenes, and C(29) and C(30) hopanes. This study demonstrated that pyocyanin influenced the diversity of the microbial community and suggests the importance of understanding how interspecies interactions influence the degradation capability of a microbial community.     

Identification of Pseudomonas aeruginosa Phenazines that Kill Caenorhabditis elegans

Pathogenic microbes employ a variety of methods to overcome host defenses, including the production and dispersal of molecules that are toxic to their hosts. Pseudomonas aeruginosa, a Gram-negative bacterium, is a pathogen of a diverse variety of hosts including mammals and the nematode Caenorhabditis elegans. In this study, we identify three small molecules in the phenazine class that are produced by P. aeruginosa strain PA14 that are toxic to C. elegans. We demonstrate that 1-hydroxyphenazine, phenazine-1-carboxylic acid, and pyocyanin are capable of killing nematodes in a matter of hours. 1-hydroxyphenazine is toxic over a wide pH range, whereas the toxicities of phenazine-1-carboxylic acid and pyocyanin are pH-dependent at non-overlapping pH ranges. We found that acidification of the growth medium by PA14 activates the toxicity of phenazine-1-carboxylic acid, which is the primary toxic agent towards C. elegans in our assay. Pyocyanin is not toxic under acidic conditions and 1-hydroxyphenazine is produced at concentrations too low to kill C. elegans. These results suggest a role for phenazine-1-carboxylic acid in mammalian pathogenesis because PA14 mutants deficient in phenazine production have been shown to be defective in pathogenesis in mice. More generally, these data demonstrate how diversity within a class of metabolites could affect bacterial toxicity in different environmental niches.     

Induction of neutrophil apoptosis by the Pseudomonas aeruginosa exotoxin pyocyanin: a potential mechanism of persistent infection

Pseudomonas aeruginosa colonizes and infects human tissues, although the mechanisms by which the organism evades the normal, predominantly neutrophilic, host defenses are unclear. Phenazine products of P. aeruginosa can induce death in Caenorhabditis elegans. We hypothesized that phenazines induce death of human neutrophils, and thus impair neutrophil-mediated bacterial killing. We investigated the effects of two phenazines, pyocyanin and 1-hydroxyphenazine, upon apoptosis of neutrophils in vitro. Pyocyanin induced a concentration- and time-dependent acceleration of neutrophil apoptosis, with 50 microM pyocyanin causing a 10-fold induction of apoptosis at 5 h (p < 0.001), a concentration that has been documented in sputum from patients colonized with P. aeruginosa. 1-hydroxyphenazine was without effect. In contrast to its rapid induction of neutrophil apoptosis, pyocyanin did not induce significant apoptosis of monocyte-derived macrophages or airway epithelial cells at time points up to 24 h. Comparison of wild-type and phenazine-deleted strains of P. aeruginosa showed a highly significant reduction in neutrophil killing by the phenazine-deleted strain. In clinical isolates of P. aeruginosa pyocyanin production was associated with a proapoptotic effect upon neutrophils in culture. Pyocyanin-induced neutrophil apoptosis was not delayed either by treatment with LPS, a powerfully antiapoptotic bacterial product, or in neutrophils from cystic fibrosis patients. Pyocyanin-induced apoptosis was associated with rapid and sustained generation of reactive oxygen intermediates and subsequent reduction of intracellular cAMP. Treatment of neutrophils with either antioxidants or synthetic cAMP analogues significantly abrogated pyocyanin-induced apoptosis. We conclude that pyocyanin-induced neutrophil apoptosis may be a clinically important mechanism of persistence of P. aeruginosa in human tissue.     

An integrated workflow for phenazine-modifying enzyme characterization

Increasing availability of new genomes and putative biosynthetic gene clusters (BGCs) has extended the opportunity to access novel chemical diversity for agriculture, medicine, environmental and industrial purposes. However, functional characterization of BGCs through heterologous expression is limited because expression may require complex regulatory mechanisms, specific folding or activation. We developed an integrated workflow for BGC characterization that integrates pathway identification, modular design, DNA synthesis, assembly and characterization. This workflow was applied to characterize multiple phenazine-modifying enzymes. Phenazine pathways are useful for this workflow because all phenazines are derived from a core scaffold for modification by diverse modifying enzymes (PhzM, PhzS, PhzH, and PhzO) that produce characterized compounds. We expressed refactored synthetic modules of previously uncharacterized phenazine BGCs heterologously in Escherichia coli and were able to identify metabolic intermediates they produced, including a previously unidentified metabolite. These results demonstrate how this approach can accelerate functional characterization of BGCs.     

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.     

Pyocyanin biosynthesis protects Pseudomonas aeruginosa from nonthermal plasma inactivation

Pseudomonas aeruginosa is an important opportunistic human pathogen, which raises a worldwide concern for its increasing resistance. Nonthermal plasma, which is also called cold atmospheric plasma (CAP), is an alternative therapeutic approach for clinical infectious diseases. However, the bacterial factors that affect CAP treatment remain unclear. The sterilization effect of a portable CAP device on different P. aeruginosa strains was investigated in this study. Results revealed that CAP can directly or indirectly kill P. aeruginosa in a time-dependent manner. Scanning electron microscopy and transmission electron microscope showed negligible surface changes between CAP-treated and untreated P. aeruginosa cells. However, cell leakage occurred during the CAP process with increased bacterial lactate dehydrogenase release. More importantly, pigmentation of the P. aeruginosa culture was remarkably reduced after CAP treatment. Further mechanical exploration was performed by utilizing mutants with loss of functional genes involved in pyocyanin biosynthesis, including P. aeruginosa PAO1 strain-derived phzA1::Tn, phzA2::Tn, ΔphzA1/ΔphzA2, phzM::Tn and phzS::Tn, as well as corresponding gene deletion mutants based on clinical PA1 isolate. The results indicated that pyocyanin and its intermediate 5-methyl phenazine-1-carboxylic acid (5-Me-PCA) play important roles in P. aeruginosa resistance to CAP treatment. The unique enzymes, such as PhzM in the pyocyanin biosynthetic pathway, could be novel targets for the therapeutic strategy design to control the growing P. aeruginosa infections.     

Synthesis and biological evaluation of thiazolidinedione derivatives with high ligand efficiency to P. aeruginosa PhzS

The thiazolidinone ring is found in compounds that have widespan biology activity and there is mechanism-based evidence that compounds bearing this moiety inhibit P. aeruginosa PhzS (PaPzhS), a key enzyme in the biosynthesis of the virulence factor named pyocyanin. Ten novel thiazolidinone derivatives were synthesised and screened against PaPhzS, using two orthogonal assays. The biological results provided by these and 28 other compounds, whose synthesis had been described, suggest that the dihydroquinazoline ring, found in the previous hit (A- Kd = 18 µM and LE = 0.20), is not required for PaPzhS inhibition, but unsubstituted nitrogen at the thiazolidinone ring is. The molecular simplification approach, pursued in this work, afforded an optimised lead compound (13- 5-(2,4-dimethoxyphenyl)thiazolidine-2,4-dione) with 10-fold improvement in affinity (Kd= 1.68 µM) and more than 100% increase in LE (0.45), which follows the same inhibition mode as the original hit compound (competitive to NADH).

Executive summary

• PhzS is a key enzyme in the pyocyanin biosynthesis pathway in P. aeruginosa.
• Orthogonal assays (TSA and FITC) show that fragment-like thiazolidinedione derivatives bind to PaPhzS with one-digit micromolar affinity.
• Fragment-like thiazolidinedione derivatives bind to the cofactor (NADH) binding site in PaPhzS.
• The molecular simplification optimised the ligand efficiency and affinity of the lead compound.

     

Pyocyanin and its precursor phenazine-1-carboxylic acid increase IL-8 and intercellular adhesion molecule-1 expression in human airway epithelial cells by oxidant-dependent mechanisms

Pseudomonas aeruginosa secretes numerous factors that alter host cell function and may contribute to disease pathogenesis. Among recognized virulence factors is the redox-active phenazine pyocyanin. We have recently demonstrated that the precursor for pyocyanin, phenazine-1-carboxylic acid (PCA), increases oxidant formation and alters gene expression in human airway epithelial cells. We report in this work that PCA and pyocyanin increase expression of ICAM-1 both in vivo and in vitro. Moreover, phenazines enhanced cytokine-dependent increases in IL-8 and ICAM-1. Antioxidant intervention studies indicated both similarities and differences between PCA and pyocyanin. The thiol antioxidant N-acetyl cysteine, extracellular catalase, and inducible NO synthase inhibitors inhibited ICAM-1 and IL-8 increases in response to both phenazines. However, pyocyanin was significantly more sensitive to N-acetylcysteine inhibition. Interestingly, hydroxyl radical scavengers inhibited the response to pyocyanin, but not to PCA. These studies suggest that P. aeruginosa phenazines coordinately up-regulate chemokines (IL-8) and adhesion molecules (ICAM-1) by mechanisms that are, at least in part, oxidant dependent. However, results indicate that the mechanisms by which PCA and pyocyanin exert their effects are not identical, and not all antioxidant interventions are equally effective in inhibiting phenazine-mediated proinflammatory effects.     

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.     

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.     

Dual activity of pyocyanin from Pseudomonas aeruginosa--antibiotic against phytopathogen and signal molecule for biofilm development by rhizobia

The purified pyocyanin from Pseudomonas aeruginosa TO3 was investigated for its antagonistic activity against Macrophomina phaseolina and as a signaling molecule for development of biofilm by rhizobial strain Ca2. The antagonistic activity of purified pyocyanin, as determined by a dry mass method, showed inhibition of M.?phaseolina. Biofilm formation by strain Ca2 was performed by crystal violet assay. There was an increase in biofilm development by Ca2 with an increase in pyocyanin concentration up to 0.12?nmol·L(-1), followed by a reduction. Using a well-diffusion method, we determined the effect of pyocyanin on disease suppression and biofilm formation by strain Ca2 on radicles of groundnut ( Arachis hypogaea L. ) placed in three concentric whorls on water agar plates. Pyocyanin suppressed disease better at high concentration; however, at lower concentrations increased colony-forming units of Ca2 on radicles of seedlings was observed. A field study in soil infested with M. phaseolina showed that a coinoculant of P.?aeruginosa TO3 and rhizobial strain Ca2 enhanced nodule mass and nitrogenase activity by 264.38% and 269.06%, respectively, over that of the control. This study reports that application of pyocyanin-producing pseudomonads together with rhizobia contributes to the enhancement of nodulation ability and better sustains the growth and productivity of groundnut even in the presence of M.?phaseolina.     

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.