The Pseudomonas secretory product pyocyanin inhibits catalase activity in human lung epithelial cells

Yunxia Q. O'Malley,1,2 Krzysztof J. Reszka,1,3 George T. Rasmussen,1,2 Maher Y. Abdalla,3 Gerene M. Denning,1,2,4 and Bradley E. Britigan1,2,3,4

1Research Service and 4Department of Internal Medicine, Veterans Affairs Medical Center-Iowa City; 2Department of Internal Medicine and 3The Free Radical and Radiation Biology Program of the Department of Radiation Oncology, The Roy G. and Lucille A. Carver College of Medicine, The University of Iowa, Iowa City, Iowa 52242

Submitted 23 June 2003 ; accepted in final form 14 July 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Pyocyanin, produced by Pseudomonas aeruginosa, has many deleterious effects on human cells that relate to its ability to generate reactive oxygen species (ROS), such as superoxide and hydrogen peroxide. Human cells possess several mechanisms to protect themselves from ROS, including manganese superoxide dismutase (MnSOD), copper zinc superoxide dismutase (CuZnSOD), and catalase. Given the link between pyocyanin-mediated epithelial cell injury and oxidative stress, we assessed pyocyanin's effect on MnSOD, CuZnSOD, and catalase levels in the A549 human alveolar epithelial cell line and in normal human bronchial epithelial cells. In both cell types, CuZnSOD and MnSOD were unaltered, but over 24 h pyocyanin significantly decreased cellular catalase activity and protein content. Pyocyanin also decreased catalase mRNA. Overexpression of MnSOD in A549 cells prevented pyocyanin-mediated loss of catalase protein, but catalase activity still declined. Furthermore, pyocyanin decreased catalase activity, but not protein, in A549 cells overexpressing human catalase. These data suggest a direct effect of pyocyanin on catalase activity. Addition of pyocyanin to catalase in a cell-free system also decreased catalase activity. Mammalian catalase binds four NADPH molecules, helping maintain enzyme activity. Spin-trapping data suggest that pyocyanin directly oxidizes this NADPH, producing superoxide. We conclude that pyocyanin may decrease cellular catalase activity via both transcriptional regulation and direct inactivation of the enzyme. Decreased cellular catalase activity and failure to augment MnSOD could contribute to pyocyanin-dependent cytotoxicity.

hydrogen peroxide; superoxide dismutase; NADPH; cystic fibrosis; oxidant


PSEUDOMONAS AERUGINOSA is an important cause of acute nosocomial pneumonia, an infection with high morbidity and mortality due in part to the level of pulmonary tissue destruction that occurs (16). P. aeruginosa also chronically infects the lungs of individuals with cystic fibrosis (CF) (15). The persistence of P. aeruginosa in the airway of these patients is associated with the progressive loss of lung function that is responsible for most deaths that now occur in individuals with CF (15).

Many P. aeruginosa factors have been suggested to contribute to the pathogenesis of acute and chronic P. aeruginosa-associated lung injury (15). Among the factors identified in many model systems is the low-molecular-weight phenazine derivative pyocyanin (Fig. 1) (15, 38, 63). Pyocyanin, produced by most strains of P. aeruginosa (69), can be detected at concentrations of up to 100 µM in pulmonary secretions of CF patients and other individuals with chronic bronchiectasis who are infected with P. aeruginosa (71). It has a number of deleterious effects on eukaryotic cells, including epithelial cells, endothelial cells, and leukocytes (20, 28, 32, 33, 42, 45, 46, 52, 59, 65). These include reversible ciliary dysfunction in human respiratory and sheep tracheal epithelial cells (1, 28, 33, 70, 71), bronchoconstriction and neutrophilia in sheep (18, 37), and a decrease in in vivo tracheal mucus velocity in several animal models (13, 47, 56). In vitro, pyocyanin inhibits neutrophil, monocyte, and lymphocyte microbicidal functions (42-46, 48, 59, 60); enhances neutrophil inactivation of {alpha}1-protease inhibitor (53); inhibits endothelial cell and macrophage nitric oxide production (23, 27, 57, 68); blocks endothelial cell prostacyclin production (32); and promotes apoptosis in neutrophils (66).



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Fig. 1. Chemical structure of pyocyanin.

 

Most studies link the cytotoxic properties of pyocyanin with its ability to redox cycle, which under aerobic conditions leads to the production of reactive oxygen species (ROS), such as superoxide and hydrogen peroxide (H2O2) (19). Utilizing cellular reducing equivalents (e.g., NADH or NADPH), we find that addition of pyocyanin to cellular systems places them under increased oxidative stress (24).

Human cells possess a variety of mechanisms to protect themselves from exposure to ROS. Included among these mechanisms is the presence of enzyme systems that catabolize superoxide and H2O2 (3, 25, 26, 51). In the case of superoxide, cells possess two distinct intracellular forms of the enzyme superoxide dismutase (SOD) (3, 25), which converts superoxide to H2O2 and water. One form, in which the active site contains Mn (MnSOD), is expressed in mitochondria, whereas a second form, where the Mn is replaced by Cu and Zn (CuZnSOD), is found in the cytosol. H2O2 removal involves the activity of a peroxisomal enzyme catalase, as well as other more complex enzymatic reaction cascades that center around the consumption and regeneration of cellular thiols such as glutathione and thioredoxin (26, 51).

Under conditions of oxidative stress, cells increase the levels of some of these antioxidant enzymes, most commonly MnSOD and catalase (3, 25, 26). Loss of, or failure to appropriately increase, these antioxidant enzymes is associated with increased susceptibility to oxidative injury (3, 25, 26).

Given the link between pyocyanin-mediated epithelial cell injury and oxidative stress, we examined the impact of pyocyanin exposure on airway epithelial cell antioxidant enzyme function using in vitro cultures of the human A549 alveolar type II epithelial cell line and normal human bronchial epithelial (NHBE) cells as models. In the present work, we report that pyocyanin fails to induce the expression of MnSOD and inactivates catalase. Implications of these observations on the susceptibility of human lung epithelium to pyocyanin-mediated alterations are discussed.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Cell culture. The human alveolar type II cell line A549 [CL-185; American Type Culture Collection (ATCC), Rockville, MD] was cultured in Dulbecco's modified Eagle's medium (Cellgro, Herndon, VA) containing 5% heat-inactivated fetal bovine serum (FBS), 2 mM glutamine, and 500 U/ml each of penicillin and streptomycin. Cell passages from 73 to 100 were used. Experiments were performed when the cell cultures were ~95% confluent. Growth rates of A549 were noted to slow in the presence of pyocyanin over 24 h of exposure. Consequently cellular protein was used as the denominator for all studies.

NHBE cells were obtained from Clonetics (San Diego, CA) and Cell Applications (San Diego, CA) and cultured as previously described (12) in basal epithelial growth medium (Clonetics). Cells were used between passages 2 and 4 when ~95% confluent.

Pyocyanin purification. Pyocyanin was extracted from the broth culture of P. aeruginosa as previously described (9). P. aeruginosa strain PAO1 (ATCC 15692) was grown in glycerol-alanine medium. The bacteria were removed by centrifugation. The culture supernatant was mixed with chloroform to remove most nonpyocyanin pigments. The blue pigments in chloroform were extracted by 10 mM HCl followed by neutral water. This process was repeated at least five times. The purity of the pyocyanin solution was confirmed by HPLC using a Beckman System Gold apparatus and a reverse-phase C18 column (Microsorb-MV-C18, 250 x 4.6 mm; Varian, Walnut Creek, CA). The solvent system consisted of 0.05% trifluoracetic acid (TFA) in water and 0.05% TFA in acetonitrile using 25- to 30-min runs.

Immunoblotting. Cells were rinsed twice with PBS, and lysed by Tris (50 mM, pH 7.4) containing 1% Nonidet P-40 (Amresco, Solon, OH). Cell lysate was collected into an Eppendorf tube, sonicated, and then centrifuged (14,000 g, 1 min). The protein content of each sample was determined by the bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL). Samples (20-30 µg of protein) were mixed 1:1 with Tris (1.25 M, pH 6.8), 20% glycerol, 4% SDS, 10% 2-mercaptoethanol, and 0.05% bromphenol blue. Proteins were separated using SDS-PAGE. The proteins were transferred to a nitrocellulose membrane overnight at 30 V. The membrane was blocked with 5% skim milk in Tris-buffered saline with 0.1% Tween (TBST) for 1 h and incubated with the primary antibody (1:1,000 dilution) for 1-2 h. Polyclonal antibody to human MnSOD, CuZnSOD, and catalase was kindly provided by Dr. Larry Oberley (Department of Radiation Oncology, University of Iowa, Iowa City, IA), and polyclonal antibody to heat shock protein (HSP) 70 was purchased from Affinity Bioreagents (Golden, CO). The blot was washed with TBST and incubated with a 1:10,000 dilution of the secondary antibody (horseradish peroxidase-conjugated anti-rabbit IgG; Amersham Pharmacia Biotech, Piscataway, NJ). The immunoreactive protein was detected by an enhanced chemiluminescence detection kit (Amersham Pharmacia Biotech).

Antioxidant native activity gels. SOD and catalase activity were measured by native-gel staining (4, 6, 61). Cells were collected and sonicated with Tris buffer (50 mM, pH 7.4) containing protease inhibitor. Cell lysate was separated by centrifugation (14,000 g, 1 min). Cell protein (50 µg) in the supernatant, as quantitated in the BCA protein assay, was further separated by electrophoresis (10% polyacrylamide gel without SDS). For SOD activity determinations, the gel was incubated in potassium phosphate buffer (50 mM, pH 7.8) containing 2.6 µM nitroblue tetrazolium, 28 mM riboflavin, and 28 mM tetramethylethylenediamine for 20 min in the dark. The gel was soaked in H2O and illuminated under a fluorescent light. Both CuZnSOD and MnSOD enzyme activities were visualized as achromatic bands against a dark background. For analysis of catalase, following electrophoresis, the gels were extensively rinsed with deionized H2O. After a 10-min soak for in 0.003% H2O2, a staining solution consisting of 2% potassium ferricyanide and 2% ferric chloride was added (61). Areas of catalase activity appear as clear (negative staining) bands on a blue-green background (61).

Catalase activity assay. Catalase activity was measured as the consumption of H2O2 as monitored spectrophotometrically as previously described (5). Bovine catalase and catalase from Aspergillus niger were purchased from Sigma (St. Louis, MO).

Northern blot. The catalase and actin cDNA probes were provided by the laboratories of Drs. Larry Oberley and Gerene Denning, respectively. Total RNA was isolated from cells exposed to pyocyanin or vehicle with TRIzol reagent (Invitrogen, Carlsbad, CA) according to the protocol provided by the manufacturer. Northern blot analysis was based on the protocol of Ausubel et al. (2). RNA (20 µg) was loaded onto a 1% agarose-formaldehyde gel, and gel electrophoresis was performed in 1x MOPS buffer [20 mM 3-(N-morpholino)propanesulfonic acid, 8 mM sodium acetate, 1 mM EDTA, pH 7] at 80 V for 4 h. The RNA fractionated by gel electrophoresis was transferred to a nylon membrane (Schleicher & Schuell BioScience, Keene, NH) by capillary action with 10x SSC solution (NaCl 88 g/l, sodium citrate 44 g/l, pH 7) for 24 h. The RNA was cross-linked by exposure to UV irradiation and then incubated with prehybridization buffer (5x SSC, 1x Denhardt's, 1% SDS, 50% formamide, and 250 µg/ml salmon sperm DNA) for 4 h at 42°C. The cDNA probes were radiolabeled with {alpha}[32P]dCTP (Perkin Elmer Life Science Products, Boston, MA) using the Prime-It II Random Primer Labeling Kit (Stratagene Cloning Systems, La Jolla, CA). The blot was subsequently hybridized overnight at 42°C with radiolabeled probe in hybridization buffer (5x SSC, 1x Denhardt's, 1% SDS, 50% formamide, and 250 µg/ml salmon sperm DNA). Blots were washed as follows: 15 min x 2 at room temperature in SSPE (17.5 g/l NaCl, 2.76 g/l NaH2PO4, 0.74 g/l EDTA, pH 7.4) plus 0.1% SDS, and 0.1x SSPE x 1 for 30 min, 65°C. Blots were then exposed to film and processed for autoradiography.

SOD and catalase overexpression. Recombinant adenovirus (Ad) vectors [cytomegalovirus (CMV)] expressing MnSOD (AdCMVMnSOD), CuZnSOD (AdCMVCuZnSOD), catalase (AdCMVcatalase), and {beta}-galactosidase (AdCMV LacZ) were constructed by and purchased from the Vector Core Facility at The University of Iowa. Each adenoviral stock (4-6 x 1010 viral particles/ml) was stored in 3% sucrose at -80°C. Multiplicity of infection was used as the infectious unit. Cells were exposed to adenovirus in media containing 5% FBS at 37°C for 24 h. Successful transfection was confirmed by measuring enhanced expression of the desired enzyme at both the protein and activity level by immunoblot and by a native-gel activity assay (4, 6, 40).

Spin trapping. Experiments were performed using a 5,5 dimethyl-1-pyrroline-N-oxide [DMPO, additionally purified with charcoal; Sigma (8)] spin-trapping system in which reaction mixtures containing 100 mM DMPO in the presence and absence of 50 µM pyocyanin and/or 80 µM catalase were prepared and then immediately placed into the cavity of the electron paramagnetic resonance (EPR) spectrometer (Brüker EMX, Karlsrühe, Germany). In some cases, reaction mixtures also contained CuZnSOD (300 units/ml, Sigma). Spectra shown are the result of seven signal-averaged scans and were obtained at room temperature. The magnitude of the EPR signal observed is directly proportional to the concentration of the spin adduct in the sample. Instrument settings were as follows: microwave power, 20 mW; modulation frequency, 100 kHz; modulation amplitude, 0.94 G; time constant, 82 ms; scan rate 80 G/42 s.

Statistical analyses. Results obtained under different experimental conditions were compared by Student's paired t-test when independent variables were being assessed or by analysis of variance when analyses of trends were being determined. For both types of analyses, results were considered significant at P <= 0.05.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Pyocyanin is felt to mediate much of its cytotoxicity in human cells through its ability to induce the generation of superoxide and H2O2 (24). In earlier studies, we observed that 24-48 h after exposure to pyocyanin many of its initial effects on the A549 cell line (aconitase inactivation and ATP depletion) resolved (49). One possible explanation for this was that the cellular antioxidant defenses were increased, as both MnSOD and catalase are frequently upregulated by sublethal oxidant stress (3, 26). Accordingly we examined the effect of pyocyanin on A549 cell levels of CuZnSOD, MnSOD, and catalase.

Pyocyanin decreases catalase but not SOD expression. A549 cells were incubated with pyocyanin concentrations reported to occur in vivo [up to 100 µM (71)], and cellular levels of CuZnSOD, MnSOD, and catalase were examined over time at the level of both protein and activity. As shown in Fig. 2, pyocyanin had no effect on A549 expression of CuZnSOD or MnSOD. This was somewhat unexpected as MnSOD levels usually increase in response to ROS formation (3, 26). More surprising was a dramatic decrease in catalase activity (Fig. 3) and protein levels (Fig. 4A) when the cells were exposed to >=5 µM pyocyanin. The decrease in catalase required >12 h of cellular exposure to pyocyanin (Fig. 4B), which is likely due to the fact that catalase protein has a half-life of 24-72 h in eukaryotic cells (22, 31, 41, 58).



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Fig. 2. Pyocyanin exposure does not alter A549 cell superoxide dismutase (SOD). A549 cells were incubated in medium alone or medium containing the indicated concentrations of pyocyanin for 24 h following which the cells were harvested and CuZnSOD and MnSOD activity (A) or protein (B) were determined by activity gel or immunoblot, respectively. Results shown are representative of 6 separate experiments that found no effect of pyocyanin exposure on either enzyme as assessed by densitometric analysis (P > 0.05).

 


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Fig. 3. Pyocyanin exposure decreases A549 cell catalase activity. A549 cells were incubated in medium alone or medium containing the indicated concentrations of pyocyanin for 24 h, following which the cells were harvested and catalase activity was measured via spectrophotometric assay. Results shown are the means ± SD of 12 separate experiments. At each concentration >=5 µM, pyocyanin exposure decreased catalase activity relative to untreated control (P < 0.001).

 


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Fig. 4. Pyocyanin exposure decreases A549 cell catalase protein. A: A549 cells were incubated in medium alone or medium containing the indicated concentrations of pyocyanin for 24 h, following which the cells were harvested, and catalase protein was measured by immunoblot. Shown is an immunoblot from a single experiment that is representative of 6 separate experiments and the means ± SD of the densitometric analyses of all 6 experiments. At concentrations of >=2.5 µM, pyocyanin exposure decreased catalase protein relative to untreated control (*P < 0.05, ***P < 0.001). B: A549 cells were incubated in medium alone or medium containing 50 µM pyocyanin for 2, 6, 12, and 24 h, following which the cells were harvested, and catalase protein was measured by immunoblot. Shown is an immunoblot from a single experiment that is representative of 6 separate experiments and the means ± SD of the densitometric analyses of all 6 experiments. Pyocyanin exposure decreased catalase protein relative to untreated control only at 24 h (P < 0.001).

 

Induction of HSP70 commonly occurs in cells placed under sublethal oxidative stress (29). Induction of this protein also contributes to increased resistance of the cells to oxidative injury (29, 72). However, as we observed with MnSOD, no induction of A549 cell HSP70 protein was seen by immunoblot analysis (not shown).

A549 cells are a lung cancer cell line and expression of some antioxidant enzymes is altered in malignant, relative to normal, cells (50). To be certain that the above results were not unique to this cell line, we repeated the experiments using monolayers of primary NHBE cells. As shown in Fig. 5, the results were similar: pyocyanin exposure had no effect on MnSOD or CuZnSOD (Fig. 5A) but resulted in a decrease in both catalase protein (Fig. 5A) and enzyme activity (Fig. 5C), and that was related to time of cell exposure to pyocyanin (Fig. 5, B and C). Of note, the duration of exposure to pyocyanin that resulted in a loss of catalase activity was much shorter than that needed to decrease catalase protein (Fig. 5, B vs. C).



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Fig. 5. Effect of pyocyanin on SOD and catalase expression in normal human bronchial epithelial (NHBE) cells. A: NHBE cells were incubated in medium alone or medium containing the indicated concentrations of pyocyanin for 24 h following which the cells were harvested, and MnSOD, CuZnSOD, or catalase protein was measured by immunoblot. Results shown in each blot are representative of 6 separate experiments. No effect of pyocyanin exposure on NHBE MnSOD or CuZnSOD protein levels was seen. Densitometry of catalase protein from the 6 catalase immunoblots, at each pyocyanin concentration, demonstrated that pyocyanin exposure decreased catalase protein relative to untreated control (*P < 0.05, **P < 0.01, ***P < 0.001). B: this shows that >12 h of exposure of NHBE to 50 µM pyocyanin was required to decrease NHBE catalase protein levels (n = 6). C: similarly, when NHBE cell catalase activity was measured, each concentration of pyocyanin >=1 µM decreased catalase activity relative to untreated control (P < 0.001) at 24 h (a) and the decrease appeared as early as 2 h after the beginning of exposure to 50 µM pyocyanin (b).

 

Catalase mRNA levels are decreased by pyocyanin. Given the dramatic decrease in catalase protein observed as a consequence of pyocyanin exposure, we measured the steady-state levels of catalase mRNA levels as a function of their exposure to increasing pyocyanin concentrations by Northern blot analysis. Exposure of the A549 cells to up to 100 µM pyocyanin for 24 h resulted in a decrease in catalase mRNA (Fig. 6). This was true regardless of whether 18S or 28S rRNA was used as a loading control (Fig. 6B, a) or if catalase mRNA was normalized to actin mRNA (Fig. 6B, c). The latter approach was somewhat problematic as pyocyanin exposure appeared to slightly, but significantly, decrease actin mRNA as well (Fig. 6B, b), leaving uncertain the appropriateness of using actin mRNA as a "loading control." Nevertheless, these data indicate that pyocyanin exposure results in a decrease in catalase mRNA.



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Fig. 6. Pyocyanin exposure decreases A549 cell catalase mRNA. A549 cells were incubated in medium alone or medium containing pyocyanin for 24 h following which the cells were harvested, and catalase and actin mRNA were measured by Northern blot analysis. A: a representative blot from a single experiment that is representative of 9 separate experiments. B: densitometry readings (means ± SD, n = 9) relative to untreated control cells for both catalase (a) and actin (b). At each concentration, pyocyanin exposure decreased catalase and actin mRNA relative to untreated control (*P < 0.05, ***P < 0.001), despite the fact that 18S and 28S rRNA levels from the pyocyanin-treated cells were not significantly different from untreated control (not shown), suggesting that pyocyanin may also cause a decrease in actin mRNA without affecting total cellular mRNA. Regardless, pyocyanin was also shown to decrease (P < 0.001) the amount of catalase mRNA when normalized to the amount of actin mRNA (c).

 

MnSOD limits pyocyanin-mediated inactivation of catalase. Much of pyocyanin's cytotoxicity occurs through its ability to induce cellular generation of superoxide (24). To test whether superoxide production contributed to pyocyanin-induced catalase inhibition in A549 cells, we transfected these cells with either human CuZnSOD or MnSOD using an adenoviral vector. This led to a marked increase in expression and activity of each enzyme (Fig. 7). Overexpression of CuZnSOD increases cytosolic SOD activity, whereas MnSOD targets mitochondria via a mitochondrial targeting sequence on the protein. Cells transfected with MnSOD were protected from pyocyanin-mediated loss of cellular catalase protein as monitored by immunoblot (Fig. 8A). CuZnSOD transfection had no effect, nor did transfection with a LacZ control (Fig. 8A). We have previously shown that a significant portion of pyocyanin redox cycling occurs in/or near cellular mitochondria (49). Given that pyocyanin appears to affect gene transcription, these results are best explained by an effect of MnSOD on pyocyanin's ability to modulate signal transduction mechanisms that could in turn impact on catalase gene expression. Unfortunately, there is limited understanding of the transcriptional regulation of mammalian catalase genes (21, 54, 55, 64, 67).



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Fig. 7. Overexpression of CuZnSOD or MnSOD in A549 cells is not altered by pyocyanin. Shown is CuZnSOD and MnSOD activity following transfection of A549 cells using an adenoviral vector expressing LacZ, CuZnSOD, or MnSOD [100 multiplicities of infection (MOI)] relative to control cells as a function of whether they were or were not also subsequently exposed to 50 µM pyocyanin. Results are representative of 6 separate experiments.

 


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Fig. 8. Overexpression of MnSOD prevents the loss of A549 cell catalase protein, but not activity, resulting from exposure to pyocyanin. Shown is A549 cell catalase protein (A) or enzymatic activity (B) following transfection of A549 cells using an adenoviral vector expressing LacZ, CuZnSOD, or MnSOD (100 MOI) relative to control cells as a function of whether they were or were not also subsequently exposed to 50 µM pyocyanin for 24 h. Results are representative of 8 separate experiments. A: a representative immunoblot and combined densitometry data from all experiments for catalase protein as a function of the presence or absence of exposure to 50 µM pyocyanin for 24 h following each of the noted transfections. Significant decrease from the respective control after pyocyanin treatment, **P < 0.01 and ***P < 0.001, respectively. B: catalase activity as a function of the presence or absence of exposure to 50 µM pyocyanin for 24 h following each of the noted transfections. Regardless of the nature of the transfection employed, all cells exhibited a significant (P < 0.05) decrease in catalase activity after 24 h of exposure to pyocyanin. Thus despite preventing the pyocyanin-mediated decrease in catalase protein (A), MnSOD expression did not prevent the ability of pyocyanin to inhibit catalase activity (B) in these cells.

 

Exposure of catalase to pyocyanin directly inhibits enzyme activity. In contrast to the ability of MnSOD overexpression to prevent the pyocyanin-mediated decrease in catalase protein content of A549 cells, it failed to prevent the pyocyanin-mediated decrease in catalase enzyme activity (Fig. 8B). This, along with the observations that the extent of pyocyanin-mediated inhibition of A549 cell catalase activity (Fig. 3) was somewhat greater than the decrease in catalase protein and mRNA levels (Figs. 5 and 6), suggested the possibility of an additional effect by pyocyanin on catalase activity independent of protein expression.

To provide additional insight into a second mechanism of pyocyanin-mediated inactivation of cellular catalase, we overexpressed catalase in A549 cells using a previously developed adenoviral vector expression system in which the human catalase gene is under the regulation of a CMV promoter. This would allow catalase enzyme expression that is independent of normal cellular catalase gene regulation and would negate any inhibitory effects of pyocyanin exposure on catalase activity at the level of protein expression. However, it would not prevent effects of pyocyanin directly on protein stability and/or enzyme activity.

Pyocyanin concentrations up to 100 µM had no effect on catalase protein levels in catalase-overexpressing A549 cells (Fig. 9). However, pyocyanin exposure led to a significant decrease in cellular catalase activity (Fig. 9), supporting an effect by pyocyanin on catalase activity that is independent of catalase protein expression.



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Fig. 9. Pyocyanin inhibits catalase activity in cells overexpressing human catalase. A549 cells were transfected with the indicated MOI of an adenoviral vector expressing catalase, and catalase protein expression (immunoblot) and activity were measured in the cells after 24-h exposure to medium alone or medium containing 50 µM pyocyanin. A is representative of 3 separate experiments and shows that catalase protein expression increased dramatically over nontransfected cells or those transfected with LacZ alone. Catalase activity of the cells increased 150-180-fold at the highest MOI, as measured by densitometry. Catalase protein expression was unaffected by pyocyanin (A), whereas catalase activity at 50 MOI decreased 2-3-fold with pyocyanin treatment (B, P < 0.05).

 

We next explored a possible mechanism whereby pyocyanin could lead to direct inactivation of catalase activity. Four molecules of NADPH are bound to eukaryotic catalase, and the presence of this NADPH protects the protein from inactivation by H2O2 (35, 36). Because pyocyanin reacts directly with NADPH, we examined whether direct interaction of pyocyanin with catalase would lead to a loss of catalase activity. Purified bovine catalase (8.3 nM) was incubated with pyocyanin (100 µM) for 2 h, following which catalase activity was measured. Incubation of catalase with 100 µM pyocyanin reduced its activity 14.8 ± 1.4% (mean ± SD, n = 12, P <= 0.0005). Catalase from the fungus A. niger does not bind NADPH (35). Incubation of this A. niger-derived catalase with pyocyanin did not decrease its activity, as it was 112.5 ± 2.9% of untreated A. niger catalase (mean ± SD, n = 8, P >= 0.05). These data suggest that the interaction of pyocyanin with catalase-associated NADPH can decrease catalase activity.

We next sought additional evidence that pyocyanin-mediated inactivation of catalase could involve the reaction of pyocyanin with catalase-bound NADPH leading to formation of superoxide. Consistent with this possibility, when pyocyanin was added to a solution of catalase, formation of superoxide was detected by spin-trapping methodology (Fig. 10). The spectrum A in Fig. 10 is that of DMPO/·OH formed presumably by decomposition of the superoxide-derived spin adduct DMPO/·OOH (17). That formation of this signal was mediated by superoxide is confirmed by measurement of a sample similar to that in Fig. 10A, but in the presence of SOD, in which the EPR signal from DMPO/·OH was abolished.



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Fig. 10. Pyocyanin reacts with bovine catalase to form superoxide. Tracing A is a signal-averaged (7 scans) electron paramagnetic resonance (EPR) scan, representative of 3 separate experiments that was obtained following the addition of pyocyanin (50 µM) to HBSS containing bovine catalase (80 µM), DMPO (100 mM), and diethylenetriaminepentaacetic acid (0.1 mM) at 25°C EPR. The spectrum is that of the DMPO/·OH spin adduct (aH = aN{beta} = 14.9 G). Tracing B shows the EPR scan obtained under the same conditions as in tracing A except that the reaction mixture also contained SOD (300 units/ml). Omission of either catalase or pyocyanin from the reaction mixture yielded a spectrum similar to that of tracing B (not shown).

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Antioxidant enzyme levels play a key role in protecting cells from oxidative injury. Expression of several of these enzymes, most notably MnSOD and catalase, is often upregulated by cells placed under increased oxidative stress (25). This response plays an important adaptive role in maintaining cell viability and function under such conditions (3, 25, 26, 51). Similarly, cellular exposure to ROS also increases expression of HSP70, a process that protects the cell against oxidative injury (29).

In the present work, we report that exposure of A549 airway epithelial cells to the P. aeruginosa-derived secretory product pyocyanin, a compound known to increase the production of ROS in these and other cells (24), failed to cause the expected increase in a variety of antioxidant responses. We found no evidence that 24 h of pyocyanin exposure, at concentrations we have previously shown lead to production of superoxide in this cell line (12) and that are present in the respiratory secretions of P. aeruginosa-infected patients (7, 11, 71), leads to alterations in the cellular levels of MnSOD, CuZnSOD, or HSP70.

More remarkably, we observed that pyocyanin exposure led to a marked concentration-dependent diminution in the levels of both catalase activity and steady-state protein in these cells. These results were not related to the fact that A549 cells are derived from a human cancer cell, as NHBE cell catalase was found to be even more susceptible to inhibition by pyocyanin. The effect on catalase protein required >12 h of pyocyanin exposure, which is not surprising given the very long half-life (days) that has been observed for mammalian catalase protein (22, 31, 41, 58). Airway cells of CF patients are continuously exposed to pyocyanin during chronic P. aeruginosa infection of their lungs. Thus even a delayed effect after initial exposure in vitro to pyocyanin is almost certainly of relevance in this form of P. aeruginosa lung infection.

Catalase concentrations are most often regulated at a transcriptional level. As assessed by Northern blot analysis, we found that pyocyanin exposure led to a decrease in steady-state levels of catalase mRNA. Thus it appears likely that the effect of pyocyanin on cellular catalase levels occurs, at least in part, at the level of gene transcription.

As noted above, pyocyanin induces the production of both superoxide and H2O2 in target cells. Transfection of A549 cells with MnSOD but not CuZnSOD was found to inhibit the ability of pyocyanin to decrease cellular catalase expression. MnSOD is targeted to the mitochondria, whereas CuZnSOD is expressed predominantly in the cytosol. CuZnSOD has been reported to be detectable in peroxisomes, the site at which catalase is found, in some cell types (34). To our knowledge, there is no evidence that MnSOD is present in peroxisomes, and thus it seems unlikely that MnSOD is acting at this site. We have previously shown that redox cycling of pyocyanin occurs in or near mitochondria (49). It is possible that elevated SOD levels at that site prevent escape of superoxide from the mitochondria and subsequent interaction of it, or H2O2 derived from it, with catalase-containing peroxisomes. However, since pyocyanin appears to affect catalase gene transcription, it seems more likely that overexpression of MnSOD prevents pyocyanin-mediated alterations in transcription factors that regulate cellular catalase expression. Regulation of the human catalase gene remains poorly defined (55). Initial review of the genomic sequence for the human catalase promoter (human genomic contig NT_009237 [GenBank] ) and a transcription factor binding site search with Transcription Element Search System using the TransFac database version 4.0 (http://www.cbil.upenn.edu/tess) indicate that the human catalase promoter is a CpG island that contains binding sites for SP1 (many), NF-{kappa}B, activator protein (AP)-2 (many), CCAAT/enhancer-binding protein, and a slightly degenerate AP-1 site. We have obtained preliminary data indicating that pyocyanin alters both AP-1 and NF-{kappa}B activity in A549 cells, and changes in MnSOD levels have been shown to alter transcription factor activity induced by other forms of cellular oxidative stress (39, 73).

Pyocyanin also appears to have the ability to inhibit catalase activity independently of its effect on catalase protein expression. Although the overexpression of MnSOD prevented loss of catalase protein in A549 cells exposed to pyocyanin, it failed to prevent the loss of enzymatic activity. Furthermore, we found that pyocyanin decreased catalase enzyme activity, but not protein levels, in A549 cells overexpressing catalase under the control of the CMV promoter following adenoviral-mediated transfection.

NADPH binds to mammalian catalase, which in turn protects the molecule from H2O2-mediated inactivation (35, 36). Using a cell-free assay system, we found that incubation of purified bovine catalase with pyocyanin leads to a modest, but significant, decrease in the activity of the enzyme. This is not seen using catalase from A. niger, which does not bind NADPH (35). Spin-trapping data were consistent with formation of superoxide upon exposure of catalase to pyocyanin. Given that pyocyanin is able to accept electrons directly from NADPH, we postulate that the ability of pyocyanin to inhibit catalase activity is due in part to its consumption of catalase-associated NADPH with resultant production of superoxide and/or H2O2.

The cytotoxic effects of pyocyanin have been repeatedly linked to its ability to induce the formation of ROS in target cells. Cellular induction of antioxidant defenses plays an important role in protecting cells from exposure to conditions of increased ROS in their environment (25). For reasons that are unclear, A549 cells and NHBE cells fail to increase their levels of MnSOD and/or HSP70 (A549 cells) following pyocyanin exposure. This is not the case with other factors that alter the redox state of A549 cells (10, 14, 30). Even more dramatic is the loss of catalase activity in response to pyocyanin exposure. This lack of cellular response to a compound that enhances exposure to ROS may play a role in enhancing susceptibility of airway epithelial cells to various pyocyanin-mediated alterations in their function. The role of these events in the pathogenesis of P. aeruginosa-associated lung injury in which local production of ROS by pyocyanin, as well as by other sources, including activated phagocytes (62), clearly warrants further study.


    DISCLOSURES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
This work was supported in part by Research Service of the Department of Veterans Affairs and the Public Health Service Grants RO1AI-3954 and P30 DK-4759 (to B. E. Britigan) and grants from the Heartland Affiliate of the American Heart Association (to K. J. Reska and G. M. Denning).


    ACKNOWLEDGMENTS
 
We thank Laynez Ackerman for helpful advice regarding the Northern blot analyses, Dr. Larry Oberley for reagents and helpful advice, and Dr. Frederick Domann for discussions and insight regarding the transcriptional regulation of human catalase.


    FOOTNOTES
 

Address for reprint requests and other correspondence: B. E. Britigan, Univ. of Iowa Hospitals and Clinics, Dept. of Internal Medicine, Div. of Infectious Diseases, 200 Hawkins Dr., SW54, GH, Iowa City, IA 52242 (E-mail: bradley-britigan{at}uiowa.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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 ABSTRACT
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 DISCLOSURES
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