GBF-German Research Centre for Biotechnology, Division of Molecular Biotechnology, Mascheroder Weg 1, D-38124 Braunschweig, Germany
Correspondence
A.-P. Zeng
aze{at}gbf.de
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A phenomenon related to the formation of B-band LPS in P. aeruginosa is the occurrence of membrane vesicles (MVs) on the cell surface under certain conditions (Beveridge, 1999). MVs of P. aeruginosa contain mainly B-band LPS and play important roles in the release of many virulence factors (Beveridge, 1999
). The conditions that induce the occurrence and discharge of MVs in P. aeruginosa are not well understood, with the exception of the effect of the aminoglycoside antibiotic gentamicin (Kaduragamuwa & Beveridge, 1995
).
Several physical (e.g. temperature) and chemical (e.g. varying pH, and high phosphate, NaCl, MgCl2, glycerol and sucrose concentrations) conditions have been reported to cause alterations in the formation of LPS in P. aeruginosa PAO1 (Kropinski et al., 1987; McGroarty & Rivera, 1990
; Makin & Beveridge, 1996
). However, the possible effects of oxygen availability or oxidative stress on the formation of LPS and MVs in P. aeruginosa have not been investigated. Oxygen availability is one of the important influences in biofilms (Xu et al., 1998
), the preferred growth mode of P. aeruginosa, especially in the lung of CF patients (Costerton et al., 1999
). In a recent study, we showed that P. aeruginosa PAO1 possesses a remarkable ability to decrease the transport of oxygen in liquid culture, causing oxygen limitation or microaerobic conditions in the milieu (Sabra et al. 2002
). Oxygen limitation markedly affected growth and the formation of virulence factors and this may play an important role in the defence of this pathogen against oxidative stress. In this work, we investigated the effects of dissolved oxygen tensions (pO2) on the formation of LPS and MVs on the surface of P. aeruginosa PAO1 grown in a continuous chemostat culture under clearly defined conditions. Adhesion to hydrophilic surface (biofilm formation) of cells grown under different pO2 tensions and the overall toxicity of P. aeruginosa PAO1 culture supernatant for a mammalian cell line were also examined.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Continuous-flow biofilm culture.
The adhesion of PAO1 cells from steady states of the chemostat culture at different pO2 levels was determined using clean glass tubes as substratum in a continuous flow biofilm culture as shown in Fig. 1. A cell suspension (8 ml with an identical initial OD600 of 0·4) from the steady-state culture was poured into the glass tubes and incubated for 4 h at 37 °C. The tubes were then rinsed with a 1 : 10 dilution of glucose minimal medium (Sabra et al., 2002
) for 20 h at 37 °C at a dilution rate as high as 10 h-1 to prevent planktonic growth. The tubes were bubbled with pure oxygen. They were then washed with phosphate buffer solution (39 mM KH2PO4, 61 mM K2HPO4, pH 7·4) and frozen and thawed several times in the same buffer to resuspend the biofilm. The OD600 and protein content of the resuspended culture were measured as an indication of adhesion and biofilm formation.
|
Electron microscopy.
Cells from both microaerobic and oxidative stress steady states of the chemostat cultures were prepared for transmission electron microscopy (TEM) as described by Yakimov et al. (1998) and surface charge was detected using cationic colloidal thorium, as described by Winkler et al. (2001)
.
Overall cytotoxicity assay.
A toxicity assay was performed using cell-free supernatant from steady states of the chemostat culture at a constant growth rate, but different pO2 levels. The cytotoxicity was tested by applying different dilutions of the supernatant to the hybridoma cell line HyGPD YK-1-1 cultured in RPMI medium (Gibco-BRL) in freshly seeded 24-well plates. The plates were incubated at 37 °C under 5 % CO2 and 95 % relative humidity for 30 h. The proportion of hybridoma cells that survived was determined by staining with tetrazolium dye (MTT) as described by Denizot & Lang (1986). Untreated cell cultures were used as controls.
Other analytical methods.
Glucose concentrations were determined with immobilized glucose oxidase using a glucose analyser (Biochemistry Analyser; Yellow Springs Instruments). Alginate concentrations were determined in the culture supernatants as described previously (Sabra et al., 2002). Total extracellular protein content was determined in the cell-free supernatant by the Lowry method. The mannuronic acid concentration was determined using an isocratic HPLC system equipped with an HPX-87H column (Bio-Rad) and a differential refractive index detector. As a mobile phase, 0·005 M H2SO4 at a flow rate of 0·6 ml min-1 was used at a working temperature of 60 °C.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The LPS profiles of P. aeruginosa PAO1 grown at varying pO2 levels are shown in Fig. 2. Two major LPS bands were visible by PAGE at pO2 levels greater than 3 %, but only one band was detected under microaerobic or oxygen-limited conditions (Fig. 2a
). The band of higher mobility corresponded to A-band LPS, while the more slowly migrating band probably contains B-band LPS (McGroarty & Rivera, 1990
). When higher LPS concentrations were electrophoresed, the typical ladder pattern of the high-molecular-mass O-polymer-containing LPS was detected (Fig. 2b
). Generally, at oxygen-limited conditions, the LPS banding pattern changed significantly, especially with respect to the high-molecular-mass O-polymer-containing molecules. Furthermore, immunoblotting with mAb NIF10, which is specific for the common antigen, indicated that the size distribution of A-band LPS was minimally affected by the pO2 level (Fig. 2c
). However, the reaction with B-band-specific mAb MF15-4 was more intense under conditions of oxidative stress (Fig. 2d
). A very weak B-band LPS reaction was detected by immunostaining in cells grown under oxygen-limited conditions.
|
|
|
Toxicity of diluted cell-free supernatants of P. aeruginosa PAO1 cultures grown under different pO2 levels for a hybridoma cell line was assessed (Fig. 5). At dilutions below 1 : 8, the viability of hybridoma cells was greatly reduced after treatment with supernatants from cultures grown under conditions of oxidative stress. This concords with the observations of increased formation of MVs under oxidative conditions, as shown in Fig. 3
, and the suggestion that the release of virulence factors is associated with the formation of MVs.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We examined the toxicity of supernatants of P. aeruginosa cultures for a hybridoma cell line (Fig. 5). The toxicity significantly increased in samples taken from oxygen-stressed steady-state cultures. This correlates with the observed increase in MV formation. It may partially explain the observation that cells of P. aeruginosa in biofilms become less virulent (Xu et al., 1998
; Hassatt et al., 1999
), because in biofilms the environment is often predominantly microaerobic or oxygen-limited. However P. aeruginosa can secrete a number of extracellular virulence factors such as exoenzyme S, elastase, protease and phospholipase. Recently, we have shown differences in the release of virulence factors at varying pO2 levels in pO2-controlled batch cultures (Sabra et al., 2002
and unpublished data). The release of some of the virulence factors, such as elastase, was found to be strongly enhanced under oxygen-limited conditions. More detailed investigations are needed to understand the influence of varying oxidative conditions on the pathogenicity and the release of different virulence factors in P. aeruginosa.
The differences in production of B-band LPS and MVs in P. aeruginosa at different oxygen tensions can also significantly affect its adhesion to surfaces and thus biofilm formation, as shown in continuous-flow biofilm culture (Fig. 1). P. aeruginosa PAO1 grown at higher pO2 levels had an increased capacity to adhere to hydrophilic surfaces (Fig. 6
). This increased adhesion was not due to increased formation of alginate, but rather oxygen-dependent alterations in cell-surface components and properties (e.g. B-band LPS). Previous studies have shown that clinical isolates of P. aeruginosa that initially infect the lungs of CF patients have the non-mucoid phenotype (without alginate) typical of environmental isolates (Tatterson et al., 2001
). The initial adhesion of P. aeruginosa to the lungs of CF patients must therefore be mediated by other factors. It would be of interest to examine if B-band LPS plays a role in the initial adhesion of P. aeruginosa under in vivo conditions.
Considerable effort has been devoted to understanding the emergence of P. aeruginosa lacking the B-band LPS in chronically infected CF patients. However, the specific molecular mechanisms responsible for this change have not been fully elucidated (Knirel et al., 2001). Although B-band LPS may be necessary for initial attachment to hydrophilic surfaces in chronic infections, where a mature biofilm is normally formed in the lung, the expression of B-band LPS may be reduced due to the prevailing microaerobic environment in the biofilm (Xu et al., 1998
). Our work clearly shows that under such conditions the expression of B-band LPS is markedly diminished (Fig. 2
). Thus, this study provides a possible explanation for the lack of B-band LPS in clinical isolates of P. aeruginosa.
Since the release of many virulence factors is known to be strongly associated with LPS structure (Michel et al., 2000) and MVs (Kadurugamuwa & Beveridge, 1995
; Beveridge, 1999
), the alterations in the formation of LPS and MVs caused by changes in oxygen tension deserve more attention in studies of the pathogenicity of this bacterium. The significance of this is emphasized by the recent discovery of a mechanism in P. aeruginosa to autogenously reduce oxygen availability in cultures under conditions of oxygen stress (Sabra et al., 2002
) and the importance of oxidative stress responses in many hostpathogen interactions (Nathan & Shilon, 2000
). Furthermore, MVs have interesting potential medical applications in drug delivery and in development of novel vaccines. A detailed characterization of the induction of MVs by oxygen stress to determine the mechanisms involved in formation and discharge, their composition, function and potential applications is desirable.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Berger, M. (2002). Inflammatory mediators in cystic fibrosis lung disease. Allerg Asthma Proc 23, 1925.
Beveridge, T. J. (1999). Structure of Gram-negative cell walls and their derived membrane vesicles. J Bacteriol 181, 47254733.
Boyd, A. & Chakrabarty, A. M. (1995). Pseudomonas aeruginosa biofilms: role of the alginate exopolysaccharide. J Ind Microbiol 15, 162168.[Medline]
Burrows, L. L. & Lam, J. S. (1999). Effect of wzx (rfbX) mutations on A-band and B-band lipopolysaccharide biosynthesis in Pseudomonas aeruginosa O5. J Bacteriol 181, 973980.
Chayabutra, C., Wu, J. & Ju, L. K. (2001). Rhamnolipid production by Pseudomonas aeruginosa under denitrification: effect of limiting nutrients and carbon substrate. Biotechnol Bioeng 72, 2533.[CrossRef][Medline]
Costerton, J. W., Cheng, K. J., Geesey, G. G., Ladd, P. I., Nickel, J., Dasgupta, M. & Marrie, T. J. (1987). Bacterial biofilms in nature and disease. Annu Rev Microbiol 41, 435464.[CrossRef][Medline]
Costerton, J. W., Stewart, P. S. & Greenberg, E. P. (1999). Bacterial biofilms: a common cause of persistent infections. Science 284, 13181322.
Denizot, F. & Lang, R. (1986). Rapid colorimetric assay for cell growth and survival: modification to the tetrazolium dye procedure giving improved sensitivity and reliability. J Immunol Methods 89, 271277.[CrossRef][Medline]
Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A. & Smith, F. (1956). Colorimetric method for determination of sugars and related substances. Anal Chem 28, 350356.
Fomsgaard, A., Freudenberg, M. A. & Galanos, C. (1990). Modification of the silver staining technique to detect lipopolysaccharide in polyacrylamide gels. J Clin Microbiol 28, 26272631.[Medline]
Guerra-Santos, A., Kaeppeli, L. H. & Fiechter, O. (1986). Dependence of Pseudomonas aeruginosa continuous culture biosurfactant production on nutritional and environmental factors. Appl Microbiol Biotechnol 24, 443448.
Hancock, R. E. W., Mutharia, L. M., Chan, L., Darveau, R. P., Speert, D. P. & Pier, G. B. (1983). Pseudomonas aeruginosa isolates from patients with cystic fibrosis: a class of serum sensitive, nontypable strains deficient in lipopolysaccharide O side chain. Infect Immun 42, 170177.[Medline]
Hassatt, D. J., Ma, J. F., Elkins, J. G. & 10 other authors (1999). Quorum sensing in Pseudomonas aeruginosa controls expression of catalase and superoxide dismutase genes and mediates biofilm susceptibility to hydrogen peroxide. Mol Microbiol 34, 10821093.[CrossRef][Medline]
Hitchcock, P. J. & Brown, T. M. (1983). Morphological heterogeneity among Salmonella lipopolysaccharide chemotypes in silver stained polyacrylamide gel. J Bacteriol 154, 269277.[Medline]
Kadurugamuwa, J. & Beveridge, T. J. (1995). Virulence factors are released from Pseudomonas aeruginosa in association with membrane vesicles during normal growth and exposure to gentamycin: a novel mechanism of enzyme secretion. J Bacteriol 177, 39984008.[Abstract]
Knirel, Y. A., Bystrova, O. V., Shashkov, A. S. & 7 other authors (2001). Structural analysis of the lipopolysaccharide core of a rough, cystic fibrosis isolate of Pseudomonas aeruginosa. Eur J Biochem 268, 47084719.
Kropinski, A. M. B., Lewis, V. & Berry, D. (1987). Effect of growth temperature on the lipids, outer membrane proteins, and lipopolysaccharides of Pseudomonas aeruginosa PAO1. J Bacteriol 169, 19601966.[Medline]
Makin, S. A. & Beveridge, T. G. (1996). Pseudomonas aeruginosa PAO1 ceases to express serotype-specific lipopolysaccharide at 45 °C. J Bacteriol 178, 33503352.[Abstract]
McGroarty, E. J. & Rivera, M. (1990). Growth dependent alterations in production of serotype specific and common antigen lipopolysaccharides in Pseudomonas aeruginosa PAO1. Infect Immun 58, 10301037.[Medline]
Michel, G., Ball, G., Goldberg, J. B. & Lazdunski, A. (2000). Alteration of the lipopolysaccharide structure affects the functioning of the Xcp secretory system in Pseudomonas aeruginosa. J Bacteriol 182, 696703.
Nathan, C. & Shiloh, M. U. (2000). Reactive oxygen and nitrogen intermediates in the relationship between mammalian hosts and microbial pathogen. Proc Natl Acad Sci U S A 97, 88418848.
Pier, G. B. (1998). Pseudomonas aeruginosa: a key problem in cystic fibrosis. Missing or defective CFTR receptors may allow this pathogen to evade specific host cell defence mechanisms. ASM News 64, 339347.
Sabra, W., Zeng, A.-P., Lünsdorf, H. & Deckwer, W.-D. (2000). Effect of oxygen on the formation and structure of Azotobacter vinelandii alginate and its role in protecting nitrogenase. Appl Environ Microbiol 66, 40374044.
Sabra, W., Kim, E. J. & Zeng, A.-P. (2002). Physiological responses of Pseudomonas aeruginosa PAO1 to oxidative stress in controlled microaerobic and aerobic cultures. Microbiology 148, 31953202.
Smith, A. R. W., Munro, S. M., Wait, R. & Hignett, R. C. (1994). Effect on lipopolysaccharide structure of aeration during growth of a plum isolate of Pseudomonas syringae pv. morsprunorum. Microbiology 140, 15851593.[Abstract]
Tatterson, L. E., Poschet, J. F., Firoved, A., Skidmore, J. & Deretic, V. (2001). CFTR and Pseudomonas infections in cystic fibrosis. Front Biosci 6, 890897.
Winkler, J., Lünsdorf, H., Wirbelauer, C., Reinhardt, D. P. & Laqua, H. (2001). Immunohistochemical and charge-specific localization of anionic constituents in pseudoexfoliation deposits on the central anterior lens capsule from individuals with pseudoexfoliation syndrome. Graefe's Arch Clin Exp Ophthalmol 239, 952960.[Medline]
Xu, D., Stewart, K. S., Xia, P. F., Huang, C. T. & McFeters, G. (1998). Spatial physiological heterogenicity in Pseudomonas aeruginosa biofilm is determined by oxygen availability. Appl Environ Microbiol 64, 40354039.
Yakimov, M. M., Golyshin, P. N., Lang, S., Moore, E. R. B., Abraham, W. R., Lünsdorf, H. & Timmis, K. N. (1998). Alcanivorax borkumensis gen. nov., sp. nov., a new, hydrocarbon degrading and surfactant-producing marine bacterium. Int J Syst Bacteriol 48, 339348.
Received 28 April 2003;
revised 14 July 2003;
accepted 24 July 2003.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |