Department of Paediatrics, Chang Gung Childrens Hospital, Kweishan 333, Taoyuan, Taiwan1
The Edward Jenner Institute for Vaccine Research, Compton, Newbury, Berkshire, UK2
Department of Microbiology and Immunology3, and Department of Paediatrics4, University of British Columbia, Vancouver, British Columbia, Canada
Author for correspondence: David P. Speert. Tel: +1 604 875 2438. Fax: +1 604 875 2226. e-mail: speert{at}interchange.ubc.ca
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ABSTRACT |
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Keywords: cystic fibrosis
Abbreviations: CF, cystic fibrosis
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INTRODUCTION |
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Recent taxonomic studies of isolates from human (CF and non-CF) and environmental origin indicate that the species B. cepacia is, in fact, highly heterogeneous, being composed of many subgroups, some of which might be reclassified as separate species (Govan et al., 1996 ; Vandamme et al., 1997
). There are at least five distinct genotypic species in B. cepacia, referred to as genomovars IV (Govan et al., 1996
; Vandamme et al., 1997
). All five genomovars have been isolated from CF patients; most of the epidemic strains are from genomovar III (Vandamme et al., 1997
).
Despite the progress in taxonomy and a better understanding of its evolving role in pulmonary infection in patients with CF, very little is known about the properties of B. cepacia that contribute to pulmonary infection. One of the most important microbial factors facilitating colonization and infection may be adhesion to host tissues, which seems to be mediated by bacterial pili (Kuehn et al., 1992 ; Goldstein et al., 1995
). Recent molecular studies have confirmed at least five different structural pilus types in different B. cepacia strains; one pilus type has been implicated in the enhanced transmissibility of one clone of B. cepacia (Goldstein et al., 1995
). This unique clone, expressing the specific cable-like pilus (Cbl), has been recovered from CF patients in Canada and Great Britain, many of whom have rapidly deteriorated. This clone exhibits specific in vitro binding with high affinity to carbohydrates of respiratory mucins (Sajjan & Forstner, 1992
); the mucin-binding adhesin is a 22 kDa protein present on the pili (Sajjan & Forstner, 1993
; Goldstein et al., 1995
). This Cbl pilus is the only genetically well characterized putative virulence factor associated with an epidemic B. cepacia strain type (Sajjan & Forstner, 1992
, 1993
; Sajjan et al., 1995
). Whereas another genetic element, the B. cepacia Epidemic Strain Marker (BCESM), is also associated with epidemic spread among patients with CF, its mechanism of action remains unexplained (Mahenthiralingam et al., 1997
).
B. cepacia appears to establish colonization by adhering first to respiratory tract epithelial cells (Kuehn et al., 1992 ). Once colonization with this pathogen is established, however, it is rarely if ever eradicated. Attempts to prevent bacterial colonization and infection in patients with CF by administering prophylactic antibiotics have been unsuccessful (Speert, 1989
). A potential explanation is that epidemic B. cepacia strains have adapted to the role of human intracellular pathogen with the ability to invade and survive within respiratory epithelial cells (Burns et al., 1996
) and professional phagocytes (Saini et al., 1999
). This virulence phenotype could conceivably protect them from actions of extracellular antibiotics. Since infection of CF patients with B. cepacia is associated with an adverse prognosis, a novel strategy for preventing respiratory tract colonization is urgently needed.
Dextrans are -1,6-linked homopolymers of glucose that have been used clinically as plasma volume expanders and antithrombotic agents. In previous studies, we have found that dextrans inhibit the adherence of P. aeruginosa to A549 pulmonary epithelial cells (Barghouthi et al., 1996) and prevent infection in neonatal mice when administered by aerosol (Bryan et al., 1999
). We reasoned that these agents might also interfere with the adherence of B. cepacia to epithelial cells. The present study was undertaken to evaluate the capacity of dextrans to inhibit the binding of B. cepacia to A549 pneumocytes. We also explored the characteristics of binding of selected Cbl+ and Cbl- B. cepacia strains in vitro to A549 cells and in vivo to murine respiratory tract epithelial cells to further elucidate the role for the cable-like pili in bacterial adherence.
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METHODS |
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Adherence assay.
Adherent cells were washed twice with warm PBS (pH 7·4) and incubated for 30 min in 0·5 ml binding buffer (138 mM NaCl, 8·1 mM Na2HPO4, 1·5 mM KH2PO4, 2·7 mM KCl, 1 mM MgCl2, 0·25 mM CaCl2 and 0·001% phenol red, pH 7·4) with or without dextran or another binding inhibitor. An aliquot of 25 µl of bacterial suspension (OD600=0·5) in L broth was then added and incubation was continued for 40 min at 37 °C. The coverslips were then washed four times with warm PBS and fixed with methanol for at least 15 min. The coverslips were mounted on slides, stained with fresh 3% Giemsa stain for 15 min and examined microscopically. If bacteria showed clump formation on the surface of the epithelial cells when they bound, each clump was counted as one bacterium. Approximately 50 cells per coverslip were examined to calculate the number of adherent bacteria per epithelial cell. Data were expressed as mean number of bound bacteria per cell±SEM. All experiments were done at least three times in duplicate.
Immunohistological staining.
To characterize the adherence patterns of various B. cepacia strains to A549 pneumocytes, the following was done. After completion of the adhesion assay, as described above, coverslips were washed with PBS and incubated with polyclonal rabbit antiserum to B. cepacia strain JTC (dilution of 1:5000) at 25 °C for 0·5 h. Slides were then washed and incubated with biotinylated goat anti-rabbit IgG, followed by the avidinbiotin complex (Signet Laboratories). Finally, the substrate diaminobenzidine was applied and the specimens counterstained with haematoxylin. Sections were covered with aqueous mountant and dried on a warming plate at 60 °C until the mountant was polymerized. Slides were then examined by light microscopy.
Mice.
Female C57BL/6 mice were purchased from Charles River Breeding Laboratories, St-Constant, Quebec, Canada. Mice were maintained in a specific pathogen-free environment until challenge with B. cepacia, after which they were housed in a biohazard room. Mice were used between 6 and 8 weeks of age. The animal procedures were approved by the University of British Columbia Committee on Animal Care, Vancouver, BC, Canada.
In vivo adherence.
Bacterial inocula were prepared by seeding five colonies of each B. cepacia strain to 5 ml Luria broth (L broth) and allowing them to grow for 18 h at 37 °C. Bacteria were collected by centrifugation and resuspended in 1 ml 1% gelatin-Hanks balanced salt solution (gel-HBSS; Gibco BRL). All mice were challenged intratracheally with approximately 5x108 bacteria in 50 µl gel-HBSS and sacrificed by cervical dislocation 1 h after the infection. Immunohistologically stained lung sections were examined microscopically for bacterial adherence.
Dextrans and oligosaccharides.
Dextrans of nominal molecular mass 4000 and 6000 Da were provided by Polydex Pharmaceuticals. Dextran of nominal molecular mass 10000 Da and three oligosaccharides, including isomaltose (Glc1
6Glc), isomaltotriose (Glc
1
6Glc
1
6Glc) and isomaltotetraose [(Glc
1
6Glc)3Glc], were obtained from Sigma.
Gel filtration of dextrans.
Dextran mixtures of each nominal molecular mass were separated first according to size by gel filtration chromatography using Bio-Gel P4 (Bio-Rad). Conditions were essentially as described by Ashford et al. (1987) . Several milligrams of material was loaded on to a 1·5x100 cm column. The solvent used was water. The eluant was monitored by changes in refractive index, the eluted fractions were combined into several pools and small amounts of these pools were analysed further by HPLC and MS.
HPLC and MS analysis of dextrans.
Small samples from each pool were fluorescently labelled by reductive amination with 2-aminobenzamide, according to the method of Bigge et al. (1995) , using a Signal Labelling Kit (Oxford GlycoSciences). HPLC analysis was then performed by procedures described by Guile et al. (1996)
using a Waters 2690XE separations module and a Jasco FP-920 fluorescence detector. The mixtures of labelled dextran oligomers were separated by normal phase HPLC on a 4·6x250 mm Oxford GlycoSciences Glycosep-N column. A binary gradient system using 50 mM ammonium formate, pH 4·4 (solvent A), and acetonitrile (solvent B) was used. Initial conditions of 35% solvent A and a flow rate of 0·4 ml min-1 were followed by a linear gradient of 3558% solvent A over the next 92 min. The flow rate was then increased to 1 ml min-1 over the next 2 min and the column washed in 100% solvent A for 5 min before being re-equilibrated in 35% solvent A for the next injection. Column temperature was maintained at 30 °C and total run time between samples was 120 min. The eluant was monitored by fluorescence (excitation at 330 nm, emission at 420 nm).
Matrix-assisted laser desorption/ionization MS was performed using a PerSeptive Biosystems Voyager elite reflection spectrometer as described by Kuster et al. (1997) . Samples were loaded on to the mass spectrometer target in 1 µl water, mixed with 1 µl 2,5-dihydroxybenzoic acid (10 mg ml-1 in acetonitrile) and allowed to dry. Oligosaccharides were observed as [M+Na]+ ions accompanied by a smaller signal of the respective [M+K]+ ions in the positive ion spectra.
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RESULTS |
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HPLC and MS
Dextran, nominally 4000 Da, was actually a mixture of glucose and its oligomers, ranging in size from 1 to 19 glucose units with the majority of the sugars in the very low molecular mass range: 36 glucose units (5271013 Da) (Fig. 6). The expectation of a peak corresponding to 23 glucose units for nominally 4000 Da dextran was not found by normal phase HPLC, nor by MS of underivatized dextran. Moreover, dextran, nominally 6000 Da, appeared very similar to dextran of 4000 Da, with a mean size of approximately 5 glucose units (828 Da). Dextran, nominally 10000 Da, was more uniform, but the mean molecular mass was low and far less than 10000 Da; the mean size detected was 12 glucose units (1962 Da).
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DISCUSSION |
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The authentic molecular masses of dextrans utilized in this study were at variance with those reported by the manufacturer. The concentration of dextran preparations used in our previous studies were based on the manufacturers quoted molecular mass (Barghouthi et al., 1996; Bryan et al., 1999 ). The mean molecular masses of dextrans used, as determined by gel filtration and MS, were approximately 10-fold lower than that indicated. Thus the reported apparent molarities must be adjusted according to the authentic molecular mass determined. It is not practical to determine the molecular mass of all dextrans used in studies such as these (Barghouthi et al., 1996
; Bryan et al., 1999
), but our observations should provide a note of caution in interpreting results.
All data from the present as well as previous studies (Barghouthi et al., 1996 ) support the conclusion that dextran blocks the adherence of B. cepacia in a non-specific fashion; that is, it does not interfere with a single type of receptorligand interaction. Observations in favour of this conclusion are: the inhibitory effect was readily reversible; oligosaccharides composed of 24 glucose units with the same
-1,6 linkage were only minimally inhibitory; dextran did not bind specifically to either P. aeruginosa or epithelial cells (Barghouthi et al., 1996
); and dextran blocked attachment of other respiratory tract pathogens (Staphylococcus aureus, group A streptococcus and Haemophilus influenzae) as well (Barghouthi et al., 1996
).
The mechanism by which dextran inhibits adhesion of bacteria to epithelial cells remains incompletely understood. However, it has been recognized for years that dextran has pervasive effects on cellcell interactions. For instance, it enhances erythrocyte clumping and is used as an agent for facilitating sedimentation in vitro. Furthermore, it inhibits platelet adhesiveness and has anticoagulant activity (Cronberg et al., 1966 ). Dextran could have exerted its inhibitory effect by coating both the epithelial cells and the bacteria. One possible target for this antiadhesive effect was the Cbl pilus on the bacteria.
After their discovery in 1995, Cbl pili have been identified as one of the adhesins of a specific epidemic B. cepacia clone (Goldstein et al., 1995 ; Sajjan et al., 1995
). Sajjan et al. (2000)
recently found that cytokeratin 13 (CK13) may be the target for the binding of cable-piliated B. cepacia; however, the expression of CK13 in normal human bronchial epithelial cells is low. In the current study, we demonstrated that epidemic B. cepacia isolates, irrespective of cable piliation, are capable of attaching both in vitro and in vivo to respiratory tract epithelial cells. These data suggest that the Cbl pilus is not the only factor required for adherence of epidemic B. cepacia strains to respiratory tract epithelial cells. Furthermore, we found that the Cbl+ strains of B. cepacia formed clumps when they bound to A549 pneumocytes, whereas the Cbl- strains bound as single bacteria. It was also the case when B. cepacia were bound to murine respiratory tract epithelial cells in vivo. A previous study indicated that most Cbl+ B. cepacia isolates from CF patients exhibited a rough morphotype that were subject to autoagglutination (Butler et al., 1994
). This rough LPS morphotype could contribute to clump formation when bacteria bind to epithelial cells. The observations of this study strongly support the speculation that the Cbl+ strains are able to co-aggregate via tangling with similar fibres from neighbouring bacteria, thus enhancing the attachment and the survival of the bacterial microcolonies on the respiratory tract epithelial cells. This autoaggregation may play a role in enhancing the virulence of B. cepacia in the lung of patients with CF. As shown in this study, dextran can effectively inhibit the bacterial binding as well as change the specific binding pattern associated with the Cbl pili.
Dextran has several features that make it an attractive candidate therapeutic agent for preventing respiratory tract infections in patients with CF. It is inexpensive, non-toxic and, most importantly, of low viscosity, even at a high concentration. It therefore can be aerosolized readily (Bryan et al., 1999 ; Finlay et al., 2000
). The ability of aerosolized dextran to protect mice from pneumonia due to P. aeruginosa has been documented (Bryan et al., 1999
). Our data further confirmed the in vitro effect of dextran in another important CF pathogen, B. cepacia. Other studies have demonstrated that dextran improves mucociliary clearance of CF sputum as monitored using a frog palate mucociliary transportability assay (Feng et al., 1996
). The combined effect on sputum rheology and inhibitory effect on bacterial adherence together suggest that dextran delivered by aerosol may be useful in patients with CF to prevent colonization and infection with P. aeruginosa and B. cepacia.
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ACKNOWLEDGEMENTS |
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Received 12 April 2000;
revised 19 June 2001;
accepted 27 June 2001.