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Eur Respir J 2003; 21:932-938
Copyright ©ERS Journals Ltd 2003

Pseudomonas aeruginosa adherence to human basement membrane collagen in vitro

K.W. Tsang1, D.K. Shum2, S. Chan1, P. Ng1, J. Mak1, R. Leung1, I.H. Shum1, G.C. Ooi3, G.L. Tipoe4 and W.K. Lam1

University Depts of 1 Medicine, 2 Biochemistry, 3 Diagnostic Radiology and 4 Anatomy, Queen Mary Hospital, The University of Hong Kong, Hong Kong SAR, China

CORRESPONDENCE: K.W. Tsang, Division of Respiratory and Critical Care Medicine, University Dept of Medicine, Queen Mary Hospital, The University of Hong Kong, Pokfulam, Hong Kong SAR, China. Fax: 852 28725828. E-mail: kwttsang@hkucc.hku.hk

Keywords: bacterial adherence, basement membrane, collagen, Pseudomonas aeruginosa

Received: October 23, 2002
Accepted January 23, 2003

This study was supported by a research grant from the Hong Kong Research Grants Council, Hong Kong SAR, China.


   Abstract
TOP
 Abstract
Materials and methods
 Results
 Discussion
 References
 
The mechanisms for Pseudomonas aeruginosa colonisation in the airways of patients with bronchiectasis and cystic fibrosis are poorly understood. P. aeruginosa could evade mucociliary clearance by adhering to the basement membrane at areas denuded of intact respiratory epithelium.

The authors have developed an in vitro model to study P. aeruginosa adherence tohuman basement membrane type-IV collagen by using scanning electron microscopy. P. aeruginosa adherence density was determined as the number of P. aeruginosa per 20microscope fields (2,000x) to log inocular size after incubation at 37°C for 45 min.

The presence of phytohaemagglutinin (PHA)-E, which binds specifically to d-galactose-ß1–4-d-N-acetylglucosamine, significantly reduced P. aeruginosa adherence density compared with control. The presence of heparin and calcium also significantly reduced P. aeruginosa adherence density. P. aeruginosa adherence was not affected by the presence of proline, trans-hydroxyproline, glycine, galactose, N-acetylneuraminic acid, N-acetylglucosamine or Arachis hypogea.

Pseudomonas aeruginosa adherence probably acts via recognition of the d-galactose-ß1–4-d-N-acetylglucosamine sequence on type-IV collagen and this process could be inhibited by heparin and calcium. As persistent Pseudomonas aeruginosa colonisation is detrimental to patients with cystic fibrosis and bronchiectasis and there is currently no effective treatment for its eradication, these results could lead to novel therapy for persistent Pseudomonas aeruginosa infection.

Bronchiectasis, defined as pathological permanent dilatation of the bronchial tree, is a common respiratory disease among East Asians. There is no effective therapy for bronchiectasis and many severely affected patients are chronically infected with Pseudomonas aeruginosa, which accounts for significant morbidity and mortality 1. At present, the only treatment for P. aeruginosa lung infection is administration of antibiotics, which is ineffective in eradicating P. aeruginosa. Abetter understanding of the mechanism of P. aeruginosa persistence in the lungs of these patients holds the key to the development of potential new and novel therapies for this resistant infection.

It is widely believed that bacterial adherence to the target mucosal surface has an important role in the pathogenesis of disease, since adherence establishes anchorage for further interactions with the host 2. Bacteria may achieve this process by expressing surface adhesins, which bind to epithelial surface receptors in a specific fashion. P. aeruginosa adheres to a number of mammalian cell types including buccal epithelium 3, respiratory epithelium 4, respiratory mucin 5 and exposed collagen 6. An in vivo study recently showed that P.aeruginosa adhered to exposed bronchial connective tissue and intraluminal secretions rather than intact respiratory mucosa in patients with cystic fibrosis (CF) 7. Collagen-binding proteins have been identified for Streptococcus pneumoniae and Staphylococci, which mediate their adherence to mammalian extracellular matrix material 8. By using transmission electron microscopy, the present group has recently shown that P. aeruginosa has a high affinity for human basement membrane collagen fibrils in vitro 9. Adherence to basement membrane is, therefore, an important issue that has not been studied previously. Therefore, the authors have recently established a model to study bacterial adherence to basement membrane collagen and applied this to evaluate the effects of various chemicals on the adherence of P. aeruginosa to collagen in vitro 10.


   Materials and methods
TOP
 Abstract
 Materials and methods
Results
 Discussion
 References
 
Inoculation of Pseudomonas aeruginosa
A clinical isolate of a nonmucoid and piliated strain of P. aeruginosa (PACS001) was stored in brain/heart infusion that contained 20% glycerol in liquid nitrogen. P. aeruginosa was retrieved on brain/heart infusion agar (Oxoid, Basingstoke, UK) plates and incubated overnight at 37°C. Passage was limited to three times prior to experiments. Following overnight incubation, a colony of P. aeruginosa was agitated in 4 mL of brain/heart infusion in a 6 mL clear plastic tube mounted on a roller stage for 24 h at 37°C. The resultant bacterial suspension was then centrifuged for 10 min at 2,000xg. The supernate was discarded and replaced with 4 mL of phosphate-buffered saline (PBS; Oxoid). This was repeated three times to wash the bacteria, which were finally resuspended in PBS. The final P. aeruginosa suspension was used for incubation with the Eppendorf lids (see description below).

Collagen coating
Sterile human type-IV collagen (Sigma, St. Louis, MO, USA) solution (2 mg·mL–1 in 1% acetic acid) was prepared immediately before each experiment. According to the manufacturer, the collagen had three major bands after sodium dodecylsulphate-polyacrylamide gel electrophoresis under reducing conditions consistent with basement membrane collagen 11. Lids of plastic Eppendorf (microcentrifuge) tubes (Sorenson, Salt Lake City, UT, USA) were carefully trimmed and removed from the body of the tubes and sterilised by autoclaving. Collagen solution (50 µL) was added to the inside of an inverted Eppendorf lid and allowed to air-dry in an incubator maintained at 37°C for 24 h. Collagen-coated lids were washed by immersing in sterile PBS three times and air-dried for 30 min in an unhumidified incubator at 37°C. This protocol provided consistent and uniform coating of type-IV collagen onto the Eppendorf lids (fig. 1Go).



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  		Fig. 1.— A scanning electron micrograph showing the test surface coated with a smooth layer of human type-IV collagen, the edges of which became slightly wrinkled after processing. Scale bar=500 µm.

 
Incubation of Pseudomonas aeruginosa with collagen-coated lids
P. aeruginosa suspension (50 µL in PBS), which contained either none or various concentrations of test agents, was carefully added onto the collagen-coated lids by gentle pipetting. Viable count of the inoculating P. aeruginosa suspension was also performed to determine the bacterial concentration and purity. The lids were then incubated in the P. aeruginosa suspension for 45 min at 37°C in an unhumidified atmosphere. The authors had previously determined that 45 min was optimal for maximal adherence without any significant alteration in P. aeruginosa viable count. After incubation, the P. aeruginosa suspension was carefully decanted from the collagen-coated lids. The lids were rinsed in sterile PBS solution (5 mL) three times to remove nonadherent bacteria. Following that, the lids were fixed in 4% glutaraldehyde and stored at 4°C until processing for electron microscopy.

Scanning electron microscopy processing
Collagen-coated lids incubated with P. aeruginosa were fixed in 4% glutaraldehyde for 24 h before rinsing in sodium cacodylate buffer, and postfixed in 1% osmium tetroxide for 1 h. Standard dehydration in graded ethanol then followed (three times in 50%, three times in 70%, three times in 90%, and three times in 100% for 5 min each) to 100% acetone. Specimens were then critically dried in carbon dioxide and mounted on aluminium stubs before being sputter-coated with gold. These specimens were randomly coded and stored in individual desiccated tubes prior to scanning electron microscopy examination by an observer who was unaware of the treatment.

Scanning electron microscope assessment of Pseudomonas aeruginosa adherence to collagen-coated Eppendorf lids
Each lid was placed on the stage of a scanning electron microscope (SEM) and viewed at low magnification (200x) to confirm uniform collagen coating, as shown in figure 1Go; otherwise the specimen would be rejected. For each specimen, 20 random SEM fields were examined at 2,000x magnification at the centre of the lid. The number of bacilli was counted manually for each of the SEM fields. The total number of P. aeruginosa bacilli was then calculated as P. aeruginosa density on collagen surface, which was a reflection of P. aeruginosa affinity towards collagen under the specific experimental condition. Adherence density was calculated as the total number of P. aeruginosa bacilli detected in 20 SEM fields divided by the logarithm of inocular size of P. aeruginosa, determined by viable counting as colony-forming units.

Effects of lectins, cations, sugars and other reagents on Pseudomonas aeruginosa adherence
A number of reagents, purchased from Sigma, unless otherwise stated, which were previously shown to affect P. aeruginosa adherence or were biochemically appropriate inthe form of collagen constituents, were mixed with the P. aeruginosa suspension to evaluate their effects on P. aeruginosa adherence. Ca2+ was presented as calcium chloride (CaCl2·H2O) solution (Merck, Berlin, Germany). The concentrations of each of these reagents used in the P. aeruginosa suspension are shown in tables 1–4GoGoGoGo.


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  		Table 1 The effects of lectins on the adherence densities of Pseudomonas aeruginosa adherence to human collagen type-IV

 

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  		Table 2 The effects of charge on the adherence densities of Pseudomonas aeruginosa adherence to collagen type-IV

 

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  		Table 3 The effects of collagen component amino acids on the adherence of Pseudomonas aeruginosa adherence to collagen type-IV

 

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  		Table 4 The effects of sugars on the adherence densities of Pseudomonas aeruginosa adherence to collagen type-IV

 
Statistical analysis
Data are expressed as mean±sem, unless otherwise stated. Wilcoxon-signed rank tests were employed to compare paired data from the same experiments. A p-value of <0.05 was taken as a statistically significant difference between two groups of data.


   Results
TOP
 Abstract
 Materials and methods
 Results
Discussion
 References
 
General observation
There was a consistent pattern on the SEM examination ofP. aeruginosa adherence to collagen surface. The vast majority of SEM fields examined showed singular identical bacilli adherent to the collagen, usually with the long axis of the bacilli in direct contact with the latter (fig. 2Go).



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  		Fig. 2.— Scanning electron micrographs showing the collagen-coated surface with adherent Pseudomonas aeruginosa bacilli after 45 min of incubation in phosphate-buffered saline containing a) no phytohaemagglutinin (PHA)-E and b) 0.1 mg·mL–1 of PHA-E, which significantly reduced P. aeruginosa adherence. Scale bars=10 µm.

 
In <1% of the SEM fields examined, the P. aeruginosa bacilli appeared in a cluster, like a bunch of grapes. There was little evidence of detachment of originally adherent bacilli, asthere were no bacterial "footprints" or other tell-tale distortion of the collagen surface. Bacterial polar pili were also found to be attached to the collagen surface (fig. 3Go).



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  		Fig. 3.— A high-power scanning electron micrograph showing the adherence of Pseudomonas aeruginosa bacilli to the collagen surface, usually on the bacterial long axis. There were polar pili on the bacillus, which appeared to be attached to the collagen surface. Scale bar=1 µm.

 
Effects of lectins on Pseudomonas aeruginosa adherence density
Table 1Go shows that phytohaemagglutinin (PHA)-E had inhibitory effects on P. aeruginosa adherence density. PHA-E at concentrations of 0.1 and 1 mg·mL–1 significantly reduced P. aeruginosa adherence density when compared with absence of PHA-E (p<0.05). The presence of Arachis hypogea appeared to decrease P. aeruginosa adherence density, although there was no statistical significance (p>0.05).

Effects of charge on Pseudomonas aeruginosa adherence density
Table 2Go shows that the presence of Ca2+ at concentrations of 1, 5, and 10 mM, but not 0.1 mM, significantly reduced P. aeruginosa adherence density when compared with the absence of Ca2+ (p<0.05). There appeared to be no dose-dependent inhibition of P. aeruginosa adherence to collagen in the range of Ca2+ tested. Heparin also reduced P. aeruginosa adherence density significantly at concentrations of 10 and 100 International Units (IU)·mL–1, but not 1,000 IU·mL–1, when compared with no heparin (p<0.05).

Effects of collagen components on Pseudomonas aeruginosa adherence density
Table 3Go shows that proline, trans-hydroxyproline and glycine at concentrations of 0.1, 1, and 10 mg·mL–1 did not have any significant effects on P. aeruginosa adherence to collagen when compared with absence of test reagent (p>0.05).

Effects of sugars on Pseudomonas aeruginosa adherence density
Table 4Go shows that galactose, N-acetylneuraminic acid, andN-acetylglucosamine at concentrations of 0.01, 0.1, and1 mg·mL–1 did not have any significant effects on the adherence of P. aeruginosa to collagen when compared with absence of test agent (p>0.05).


   Discussion
TOP
 Abstract
 Materials and methods
 Results
 Discussion
References
 
The authors have described a new model to directly study bacterial adherence to basement membrane using scanning electron microscopy 10. By using direct manual counting of surface adherent P. aeruginosa bacilli with scanning electron microscopy, they have determined the exact number of adherent bacteria on the collagen surface. This could be a more direct and specific, albeit more laborious, method to determine bacteria adherence than previous indirect assays ofbacterial adherence, such as radiolabelling techniques. Byrecent use of this model, the authors have shown that P. aeruginosa adherence to basement membrane collagen is reduced in the presence of low-dose erythromycin, probably partly due to alteration of bacterial morophology 10. These results showed that the lectin PHA-E, but not A. hypogea, significantly inhibited P. aeruginosa adherence to collagen. PHA-E appeared to inhibit P. aeruginosa adherence at 0.01, 0.1 and 1 mg·mL–1, although only the latter two concentrations inhibited adherence significantly. Ca2+ inhibited P. aeruginosa adherence at a concentration of >0.1 mM, although there wasno obvious dose-dependent effect. Heparin only inhibited P. aeruginosa adherence at 10 and 100 IU·mL–1 but not at 1,000 IU·mL–1. Major amino acid constituents of collagen, namely proline, trans-hydroxylproline and glycine, did not affect P. aeruginosa adherence significantly. Similarly, sugars, including galactose, N-acetylneuraminic acid and N-acetylglucosamine, did not alter P. aeruginosa adherence significantly.

Deoxyribonucleic acid fingerprinting techniques suggest that most CF patients harbour genetically related P. aeruginosa strains in their respiratory tract over long periods of time 12. However, little is known of the mechanism(s) of P. aeruginosa persistence in the bronchiectatic airway. The preferential adherence of P. aeruginosa to damaged tissue is also largely unexplained, although damaged airway epithelial cells express asialo-GM1 oligosaccharide, which could be a P. aeruginosa receptor 13. It is possible that P. aeruginosa bacilli evade mucociliary clearance by adhering to basement membrane at mucosal sites denuded of intact ciliated epithelium. P. aeruginosa exotoxins, such as pyocyanin, 1-hydroxylphenazine and rhamnolipid, can also expose the basement membrane to P. aeruginosa bacilli through slowing of ciliary beating, separation of epithelial tight junctions, and sloughing of damaged respiratory mucosa 7, 9. As many intraluminal bacteria are adherent to respiratory mucus, many workers believe that this could be a reservoir for persistent airway pathogens, such as P. aeruginosa and Haemophilus influenzae 7, 14. However, respiratory mucus is eventually expectorated and cannot subsequently retain these pathogens in the airways. The hypothesis described above could, therefore, better explain the persistent airway colonisation by respiratory pathogens, such as P. aeruginosa and nontypable H. influenzae. However, the mechanism(s) of P. aeruginosa adherence to basement membrane have not been studied systematically.

The adhesion of P. aeruginosa to respiratory mucosa is complex and multiple P. aeruginosa adhesins and epithelial receptors appear to be involved. P. aeruginosa pili are highly strain-specific proteinaceous appendages, which are adhesins mediating adherence to human tracheal mucosa 15. Pili present on the surface of P. aeruginosa recognise the d-N-acetylgalactosamine-ß1–4-d-galactose (GalNAcß1–4Gal) disaccharide of asialo-GM1 and -GM2 receptors 16. Mucoid strains of P. aeruginosa produce an exopolysaccharide that forms a loose capsule of organised linear strands of polysaccharide radiating outwards from the cell surface. This has been shown to mediate attachment to human respiratory epithelium 9, 17. The authors have also observed a direct apposition of P. aeruginosa polar pili to the collagen surface in many SEM fields, although in many instances the P. aeruginosa bacilli were also directly attached to the collagen surface themselves.

P. aeruginosa and other common respiratory pathogens, such as nontypable H. influenzae and S. pneumoniae, bind to glycoconjugates on glycolipids and mucins. Specifically, the GalNAcß1–4Gal disaccharide found in glycosphingolipid of epithelial cell surfaces of human lung explants is a candidate receptor 18. Cell surface sialic acid has been identified as a vital component of epithelial receptors for P. aeruginosa adhesin(s) 19. Several other respiratory pathogens, such as Mycobacterium pneumoniae utilise sialic acid-containing glycoconjugates as receptors 20. Surface-bound neuraminidase could play a part in the initial recognition system, in addition to its removal of sialic acid residues to allow increased binding affinity between adhesin and asialo-terminal residues of cell surface receptors 21. Available data also show that sialic acids and N-acetylglucosamine arecomponents of mucin receptor(s), and both type 1 d-galactose-ß1-3-d-N-acetylglucosamine (Galß1–3GlcNAc) and type 2 d-galactose-ß1–4-d-N-acetylglucosamine (Galß1–4GlcNAc) disaccharide units are involved in the binding to P. aeruginosa 22. Recently, P. aeruginosa has also been shown to possess high-affinity binding sites for sialyl-Lewis X conjugate, an N-acetylneuraminic acid {alpha}2–3-d-galactose-ß1–4(d-fucose{alpha}1-3)-d-N-acetylglucosamine oligosaccharide sequence that is commonly found in the mucins of CF patients 23. This suggests that in addition to the recognition of neutral carbohydrate determinants, there are P. aeruginosa adhesins specific to acidic glycoconjugates produced as a response to local inflammation of the airway mucosa.

Basement membranes are predominantly comprised of type-IV collagen, laminin, fibronectin, and heparan sulphate proteoglycans. They underlie epithelial and endothelial cells and surround peripheral nerve and muscle cells 24. Type-IV collagen is the most abundant nonfibril-forming collagen within the lung and provides the scaffolding for other basement membrane components to attach to. P. aeruginosa adheres to type-I collagen matrix 25, fibronectin 26, and laminin via a nonpilus-mediated mechanism 27. P. aeruginosa adherence to type-I and -II collagen is inhibited by d-galactose, d-mannose and N-acetylneuraminic acid 28, and this suggests that saccharides could play a role in P. aeruginosa adherence to type-I and -II collagen. However, the adherence of P. aeruginosa to type-IV collagen, the most abundant framework of basement membrane, has not been studied previously.

The lectins PHA-E and A. hypogea were used to antagonise the adherence of P. aeruginosa to type-IV collagen in the present model. PHA-E specifically binds Galß1–4GlcNAc linked to the Man{alpha}1–6 arm of complex-type N-glycans 11, and is likely to compete with the P. aeruginosa adhesin that recognises receptors bearing this disaccharide unit 22. As the 7S domain of type-IV collagen bears loci for asparagine-linked glycans of the bi- and triantennary type with terminal ß1–4-d-galactose-d-N-acetylgalactosamine (Galß1–4GalNAc) 29 and the current results showed that PHA-E reduced P. aeruginosa adherence to collagen, P. aeruginosa adherence to type-IV collagen could involve the Galß1–4GalNAc sequence. A. hypogea agglutinin binds specifically to ß1–3-d-galactose-d-N-acetylgalactosamine (Galß1–3GalNAc), which is the terminal sequence of gangliotetrosylceramide. The binding of P. aeruginosa to the latter suggests that this glycolipid might be an epithelial receptor for P. aeruginosa 18. However, the lack of effects of A. hypogea on P. aeruginosa adherence in this study suggests that the terminal Galß1–3GalNAc sequence is not involved in P. aeruginosa adherence to type-IV collagen. The lack of effect on adherence by the sugars, including galactose, N-acetylneuraminic acid, and N-acetylglucosamine (table 4Go), suggests that the three-dimensional structure of the determinant disaccharide or clusters of these are more important for adhesin recognition than individual sugars, and is consistent with the findings from the lectin studies.

Heparin is a glycosaminoglycan similar to heparan sulphate in disaccharide repeats of d-glucuronic acid-d-N-acetylglucosamine but different in extensive domains where disaccharide repeats are substituted with N- and O-sulphates. Heparin probably acts via competition with heparan sulphate moieties of proteoglycans present in the tissue, inhibiting adherence ofurinary pathogens to bladder mucosa 30. Thepresent results show that heparin significantly inhibited P. aeruginosa adherence between 10–100 IU·mL–1. It is possible that heparan sulphate was present as part of the proteoglycans in the large molecular aggregate component of the type-IV collagen preparation and therefore played a part in P. aeruginosa adherence. This interesting phenomenon should be further evaluated, as this low concentration of heparin should be achievable in the airways by nebulisation of a low dosage of heparin without systemic anticoagulative effect. A higher level of heparin, namely 1,000 IU·mL–1, was also associated with a lower P. aeruginosa adherence density compared with control, although this difference was not statistically significant (table 2Go). This lack of dose-dependent response is puzzling and cannot be explained by the current patchy understanding of the antiadherence effects of heparin. Further studies areclearly warranted as these could provide insights on P. aeruginosa adherence mechanisms and clues for designing experimental novel therapy for P. aeruginosa infection using heparin.

Marcus et al. 31 showed that supraphysiological concentrations of Ca2+ (15mM) enhance P. aeruginosa adherence to hamster tracheal epithelium, suggesting the involvement of metal ions in adhesin-oligosaccharide binding. In contrast, the present authors found that physiological concentrations of Ca2+ inhibited P. aeruginosa adherence (table 2Go). In view of the possibility of heparan sulphate proteoglycans associated with type-IV collagen, Ca2+ binding to heparan sulphate moieties could have altered the available heparan sulphate for P. aeruginosa adherence. The major constituent amino acid components of collagen, namely proline, trans-hydroxyproline and glycine, had no effects on P. aeruginosa adherence to type-IV collagen (table 3Go). This suggests that these amino acids are not directly involved in the adherence process between P. aeruginosa and type-IV collagen. This is consistent with the above findings that the adherence process is more likely to involve Galß1–4GlcNAc but not amino acids. It is also highly possible that these component amino acids are locked in the collagen skeleton and not directly exposed for the adherence process.

The results from this study show that Pseudomonas aeruginosa adherence to type-IV collagen probably acts via specific mechanism(s) involving adhesin recognition of the d-galactose-ß1–4-d-N-acetylglucosamine sequence. In addition, heparin and Ca2+ also appear to be inhibitory for Pseudomonas aeruginosa adherence to proteoglycan components associated with basement membrane type-IV collagen. As persistent Pseudomonas aeruginosa colonisation is detrimental to patients with cystic fibrosis and bronchiectasis and there is currently no effective treatment for its eradication, these results could lead toa novel approach to treatment of persistent Pseudomonas aeruginosa infection. Further research should be pursued using this model on Pseudomonas aeruginosa adherence to other basement membrane components.


   References
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 Abstract
 Materials and methods
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 Discussion
 References
 

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