Departments of Microbiology and Immunology,1 and Medicine,2 The University of Western Ontario, London, Ontario, CanadaN6A 5C1
Author for correspondence: Miguel A. Valvano. Tel: +1 519 661 3996. Fax: +1 519 661 3499. e-mail: mvalvano{at}julian.uwo.ca
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ABSTRACT |
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Keywords: cystic fibrosis, chronic granulomatous disease, oxidative stress, hydrogen peroxide, superoxide anion
Abbreviations: CF, cystic fibrosis; CGD, chronic granulomatous disease; SOD, superoxide dismutase
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INTRODUCTION |
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Burkholderia cepacia is an aerobic, Gram-negative micro-organism that was originally described as the cause of soft rot in onions (Burkholder, 1950 ). Over the past 20 years the bacterium has emerged as an important opportunistic pathogen, primarily in chronic granulomatous disease (CGD) and cystic fibrosis (CF) patients (Burkholder, 1950
; Govan & Deretic, 1996
; Govan & Vandamme, 1998
). CGD is a rare genetic disorder (X-linked or autosomal recessive) resulting in a defect in the oxidative killing mechanism of phagocytic cells, thus making them unable to generate the toxic oxygen metabolites normally involved in inactivating engulfed bacteria (Cline, 1975
; Speert et al., 1994
). These cells still retain their non-oxidative bactericidal activities, mainly through the functions of defensins as well as other bactericidal cationic peptides and proteins (Odell & Segal, 1991
).
CF is an autosomal recessive genetic disorder that affects approximately 1 in 2500 people in the Caucasian population. The disorder compromises the transport of chloride ions through apical epithelial cell membranes and leads to pancreatic insufficiency and abnormally thick mucous secretion in the lungs and other organs (Govan et al., 1996 ; Govan & Deretic, 1996
; Govan & Vandamme, 1998
). The disease is characterized by bacterial colonization and chronic airway infection and persistent inflammation that progressively compromises the lung function (Bals et al., 1999
). Ultimately, chronic inflammation and tissue damage leads to a reduction in the rate of gas exchange at the cellular level and can be fatal. The most common pathogen responsible for the morbidity and mortality in CF patients is Pseudomonas aeruginosa (Koch & Hoiby, 1993
). However, in recent years B. cepacia has increasingly been isolated from the airways of CF patients, and is often associated with a rapid decay of the lung function accompanied with a sepsis-like syndrome. This condition, referred to as the cepacia syndrome, occurs in roughly 20% of infected patients (Govan & Deretic, 1996
; Govan & Vandamme, 1998
; Tablan et al., 1985
). The severity of B. cepacia infections is amplified by the broad resistance of these micro-organisms to most clinically useful antibiotics, and the fact that they can be effectively transmitted from patient to patient (Govan & Deretic, 1996
; LiPuma et al., 1990
). Recent taxonomic studies have shown that strains formerly identified as B. cepacia can be grouped into six genomovars, each containing phenotypically similar yet genotypically distinct organisms (Vandamme et al., 1997
). Collectively, these genomovars are referred to as the Burkholderia cepacia complex. Strains belonging to genomovar III have been more commonly linked to fatal infections and constitute most of the epidemic isolates (Vandamme et al., 1997
).
Recent investigations in our laboratory have shown that B. cepacia can survive intracellularly in macrophages and amoebae (Marolda et al., 1999 ; Saini et al., 1999
). Survival within professional phagocytes suggests that B. cepacia may avoid the bactericidal mechanisms employed by these cells. B. cepacia strains demonstrate resistance to non-oxidative killing pathways, as they are able to survive in the presence of neutrophils from CGD patients while the micro-organisms are killed by neutrophils of normal individuals (Speert et al., 1994
). During colonization and infection of the airways in CF patients, there is a pronounced inflammatory response that results in the release of toxic oxygen and toxic nitrogen compounds (Bals et al., 1999
). Survival of B. cepacia in a macrophage cell line, in the presence of cell activation and an oxidative burst (Saini et al., 1999
), suggests that bacterial resistance to oxidative damage may play a role in the infectivity and persistence of this opportunistic pathogen. B. cepacia isolates are known to produce catalase (Palleroni, 1992
) but the presence of SOD has not been investigated. However, it is likely that B. cepacia strains produce SOD since this family of enzymes is found in essentially all aerobic forms of life (Weisiger & Fridovich, 1973
).
We hypothesize that SOD and catalase protect B. cepacia from oxidative damage and may contribute to bacterial survival and persistence in infected CF lung and airways. To our knowledge, these enzymes have not been systematically characterized in B. cepacia genomovars. As a step towards examining the role of these enzymes in infection, we have investigated SOD and catalase activities of cell-free cell extracts of isolates representing each genomovar. Native PAGE analysis of cell-free extracts was performed to probe for the presence of multiple isozymes at various growth stages. We also compared the survival rate of B. cepacia strains following challenge with both extracellular and intracellular and H2O2 at concentrations equal or higher than those normally found in vivo, as a consequence of the oxidative burst in phagocytic cells. Our data show that isolates of genomovar III display a uniformly high level of survival in vitro following oxidative stress mediated by extracellular reactive species.
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METHODS |
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Enzymic activity assays.
The specific catalase activity was determined spectrophotometrically by following the disappearance of H2O2 over time at 240 nm, upon the addition of cell-free extract (Katsuwon & Anderson, 1992 ). One unit of catalase will decompose 1 µmol H2O2 (mg protein)-1 min-1 at 25 °C (Katsuwon & Anderson, 1989
). The specific peroxidase activity was determined spectrophotometrically by following the oxidation of o-dianisidine at 460 nm in a reaction buffer containing 50 mM potassium phosphate and 1 mM H2O2 upon the addition of cell-free extract. One unit of peroxidase activity equals 1 µmol H2O2 reduced min-1, with
M=11·3x104 M-1 cm-1 (Schnell & Steinman, 1995
). The specific SOD activity was determined spectrophotometrically by measuring the inhibition of the initial rate of auto-oxidation of 6-hydroxydopamine at 490 nm following the addition of cell-free extract (Heikkila & Cabbat, 1976
). A standard curve using known units of SOD activity was generated in order to determine the units of activity in each sample. One unit of SOD activity corresponded to 50% inhibition of the initial rate. Bovine liver catalase and both iron- and manganese-containing SOD enzymes (Fe-SOD and Mn-SOD) from Escherichia coli were used as positive controls in the respective assays.
Native PAGE for identification of catalase, peroxidase and SOD.
We used native PAGE to identify bands of catalase (Katsuwon & Anderson, 1992 ). Lanes were loaded with 1020 µg total protein and the electrophoresis was carried out using 10% Novex gels. Gels were washed with distilled water for 30 min to remove any traces of running buffer. To identify bands of catalase activity the gel was soaked in 200 ml of a 3 mM solution of H2O2 for 10 min and then rinsed in distilled H2O. Following the addition of a 1% potassium ferricyanide/1% ferric chloride (w/v) solution for 10 min in darkness, the gel stained dark blue except at sites showing catalase activity, which appeared clear. Bovine liver catalase was used as a positive control. Bands of peroxidase activity were visualized using the method described by Wayne & Diaz (1986)
. Briefly, following electrophoresis as described above, gels were washed for 30 min in phosphate-buffered saline (PBS). To identify bands of peroxidase activity the gels were soaked in a 200 ml PBS solution containing 1·0 mM H2O2 and 2·0 mM 3,3'-diaminobenzidine tetrahydrochloride for 30 min. Sites of peroxidase activity appeared brown on a clear background. Type II horseradish peroxidase was used as a positive control. In order to determine whether bands showing peroxidase activity were due to bifunctional catalase-peroxidase enzymes, gels were washed in distilled water and counter-stained for catalase activity as described above. Heat inactivation was performed by incubating samples at 65 °C for 1 min immediately prior to loading (Wayne & Diaz, 1986
). Chemical inactivation of catalase activity was performed by incubating extracts with 3-amino-1,2,4-triazole for 20 min. Bands of SOD activity were identified by the native PAGE method described by Beauchamp & Fridovich (1971)
. Lanes were routinely loaded with 2040 µg total protein. After electrophoresis, under the conditions as described above, gels were washed for 30 min in distilled water, and incubated with shaking in the dark for 30 min in a solution of 250 µM nitro blue tetrazolium (NBT) dissolved in 200 ml distilled water. Gels were then incubated in a developing solution containing 50 mM potassium phosphate, pH 7·8, 1 mM EDTA, pH 8·0, 20 mM N,N,N',N'-tetramethylethylenediamine (Bio-Rad) and 30 µM riboflavin in the dark with shaking for 20 min. Bands of SOD activity were visualized by exposing the gels to light for 10 min or until sufficient contrast with the background developed. Sites with SOD activity appeared clear on a purple background. Both Fe-SOD and Mn-SOD purified from E. coli were used as positive controls in this assay.
In vitro protection assays.
The level of protection against H2O2 exposure in the strains used in this study was measured as described by Katsuwon & Anderson (1989) . Briefly, cultures of B. cepacia were grown with shaking at 37 °C in LB broth until late stationary phase. Samples containing 1x108 cells ml-1 of each strain were treated with various H2O2 concentrations and incubated with shaking at 25 °C for 30 min. Control samples received H2O in place of H2O2 treatment. Appropriate serial dilutions were plated in triplicate on LB agar plates. Colonies were counted after 48 h incubation and percentage survival calculated by comparison with colony counts obtained from untreated samples. The level of protection of strains to extracellular
exposure was measured as described elsewhere (Schnell & Steinman, 1995
) with some modifications. Late stationary phase culture samples containing 1x108 cells ml-1 were incubated with shaking at 37 °C in a mixture containing 250 µM xanthine (X) and 0·14 units of xanthine oxidase (XO); 100 U catalase ml-1 was added to each sample prior to addition of XO to protect cells from the toxicity of any H2O2 produced as a consequence of SOD activity. Aliquots were removed at 0, 30, 60 and 120 min and serially diluted in 200 mM phosphate buffer, pH 7·4. Time 0 aliquots were removed before addition of the
generating components. Appropriate dilutions were plated in triplicate on LB agar plates, incubated for 2448 h at 37 °C and the colonies counted. Percentage survival was determined using the following equation: % Survival=(No. of colonies, treated sample/No. of colonies at time 0)x100. Survival after intracellular
exposure was measured using the liquid assay. The liquid assay was performed as described by Membrillo-Hernandez et al. (1999)
with some modifications. Cultures of B. cepacia strains were grown to late stationary phase. Samples containing 1x108 cells ml-1 were incubated with shaking at 37 °C in a mixture containing 0, 2·5, 5 or 10 mM paraquat. Aliquots were removed after 45 min, serially diluted and plated in triplicate on LB agar plates. Following incubation at 37 °C for 2448 h, colonies were counted and percentage survival was calculated using the equation described above.
Statistical analysis.
Data were analysed using the Fisher least significant difference (LSD) method, using 95% confidence intervals.
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RESULTS |
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SOD production by strains of the B. cepacia complex
Cell-free extracts were also used to determine the specific SOD activity by measuring the inhibition of the initial rate of auto-oxidation of 6-hydroxydopamine at 490 nm. This assay was chosen because it does not suffer any interference from other components in the crude cell-free lysates (Bannister & Calabrese, 1987 ). The E. coli K-12 sodA sodB mutant QC779 was used as a negative control for the assay. Units of SOD activity were calculated by generating a standard curve with Fe-SOD from E. coli as a control, where 1 unit represented 50% inhibition of auto-oxidation of the substrate. Table 4
shows the SOD activities for each strain at the early exponential and late stationary growth stages. As with catalase activities, the level of SOD activity measured increased dramatically in the extracts from cultures at stationary phase. Likewise, genomovar III strains also appeared to possess, on average, higher levels of SOD activity than the strains belonging to the other genomovars (Table 4
). However, following statistical analysis, the levels of SOD activity observed for strains from the various genomovars were not significantly different.
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DISCUSSION |
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Bifunctional catalase/peroxidase enzymes were detected in all strains examined from genomovars III, IV and VI, but only in one genomovar I isolate, which also expressed a monofunctional catalase. These enzymes have been found to be associated with virulence in a wide range of lung pathogens such as Mycobacterium tuberculosis (Manca et al., 1999 ) and Legionella pneumophila (Bandyopadhyay & Steinman 1998
). Although in this study a high level of survival to H2O2 exposure in vitro could not be correlated with the amount of catalase activity, 7 out of the 11 strains showing the highest survival rates produced bifunctional catalase/peroxidase and belonged to genomovar I, III and VI. The prevalence of catalase/peroxidase in the genomovar III strains, which are most commonly associated with the cepacia syndrome in CF patients, and their apparent increased resistance to H2O2 exposure in vitro, provides justification for further study into the role of this enzyme in the pathogenesis of B. cepacia infections. Monofunctional catalases were found in the B. cepacia complex isolates from genomovars I, II and V. Isolates of closely related species such as Pseudomonas aeruginosa, P. putida, P. syringae and Xanthomonas possess multiple catalase isozymes (Brown et al., 1995
; Katsuwon & Anderson, 1992
; Mongkolsuk et al., 1996
). Under our experimental conditions, however, we have found only one enzyme class in most isolates, suggesting either that B. cepacia isolates have fewer catalase forms or, alternatively, that additional catalases may be expressed under conditions of stress not examined in this study.
Electrophoretic analysis also revealed the presence of two electrophoretotypes of SOD in B. cepacia, one of which corresponded to a larger protein aggregate. While SOD is commonly found in three major forms, Fe, Mn and Cu/Zn co-factored enzymes, it is not uncommon for multiple bands of SOD to be detected (Bannister & Calabrese, 1987 ). Initial attempts to classify the SOD activities using specific inhibitors yielded inconclusive results, possibly due to interference from other components found in cell-free cell lysates. As H2O2 is the standard inhibitor of Fe-SOD enzymes, any catalase activity present in the samples would probably limit the effectiveness of the inhibition. Therefore, following inactivation of the catalase activities we were able to identify that the SOD electrophoretotypes in B. cepacia corresponded to Fe-SODs. Using PCR amplification with degenerate primers, we have recently been able to clone a portion of the B. cepacia SOD gene that shows features of a typical Fe-SOD and that hybridized with all the strains tested (unpublished results). Gene knockout experiments in combination with studies under different growth conditions, currently under way in our laboratory, will permit us to determine whether the Fe-SOD is unique to B. cepacia or other SOD forms are also present in these isolates.
The level of expression of catalase and SOD was associated with the growth stage of the cultures. In all strains the activity of both enzymes increased in the cells entering stationary phase as shown by the band intensities in native gels as well as by the specific activity determinations. This may be important in vivo as it would provide a higher level of protection to those cells able to establish a chronic infection where the bacteria would find themselves under a physiological situation similar to the late stationary phase of growth. Alternatively, other conditions of stress, including tissue inflammation, may stimulate a bacterial response similar to that of stationary phase and induce a high level of expression of these enzymes. The susceptibility of B. cepacia to oxidative killing is suggested by the observation that neutrophils from CGD patients fail to inactivate B. cepacia (Speert et al., 1994 ). However, this conclusion contrasts with the apparent tolerance of B. cepacia to the highly oxidative environment in the CF lung, where the inflammatory response is dominated by neutrophils. Furthermore, B. cepacia strains isolated from CF patients, especially those of genomovar III, are strong catalase/peroxidase and SOD producers. One possibility to explain this paradox could be that the strains causing infections in CGD individuals are different from those found in CF patients. This may be true, at least in part, since all genomovar III isolates reported by Vandamme et al. (1997)
were obtained exclusively from CF patients. In addition, the unique nature of the CF lung and airways may also contribute to the possible escape of B. cepacia from oxidative killing. Studies into the pathology of bacterial infections in the respiratory airways of CF patients have identified chronic inflammation as a major cause of tissue damage and loss of gas-exchange function (Tager et al., 1998
). Recruitment of neutrophils to the site of infection is associated with a release of reactive oxygen species and other toxic compounds. Ongoing release of reactive oxygen species may in turn overwhelm the cellular antioxidant defences and lead to the accumulation of toxic levels of these compounds, which may damage the lung cells (Bals et al., 1999
; Suttorp & Simon, 1982
). Lipopolysaccharide (LPS) has been shown to elicit a powerful inflammatory response in neutrophils (Forehand et al., 1993
; Worthen et al., 1988
). Interestingly, it has been shown that the LPS of B. cepacia elicits a much greater inflammatory response as measured by
release than the LPS of P. aeruginosa, another common CF pathogen (Hughes et al., 1997
; Zughaier et al., 1999
). These observations are consistent with our own findings indicating that activation of macrophages following phagocytosis of B. cepacia is primarily due to LPS release (Saini et al., 1999
). Therefore, it is possible that bacterial antioxidant defences allow B. cepacia to survive and persist despite the oxidative stress in the lung promoted by LPS-mediated stimulation of inflammatory cells. Experimental evidence showing that B. cepacia strains can survive within macrophages in the presence of macrophage cell activation supports these conclusions (Saini et al., 1999
). The resistance of B. cepacia strains to toxicity by reactive oxygen species in lung tissue, in association with the ability of these isolates to survive intracellularly (Burns et al., 1996
; Marolda et al., 1999
; Martin & Mohr, 2000
; Saini et al., 1999
) may explain, at least in part, their persistence as well as their ability to continually elicit an inflammatory response. Furthermore, a recent study showed that nitric oxide acts synergistically with reactive oxygen species to kill B. cepacia in vitro (Smith et al., 1999
) and suggested that persistence of B. cepacia in CF patients may be associated with a defect in the inducible nitric oxide synthase activity in these patients (Kelley & Drumm, 1998
). Taken together, these factors would explain the high level of tissue damage and lethality commonly associated with B. cepacia infection in CF patients, especially with genomovar III isolates. Confirmation of this pathogenic model awaits further experiments using animal models of lung infection as well as the generation of genetically defined catalase- and SOD-deficient mutants.
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ACKNOWLEDGEMENTS |
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Received 3 April 2000;
revised 10 July 2000;
accepted 28 September 2000.