Adherence of Actinobacillus pleuropneumoniae to swine-lung collagen

Idalia Enríquez-Verdugo1, Alma L. Guerrero2, J. Jesús Serrano1, Delfino Godínez1, J. Luis Rosales3, Víctor Tenorio4 and Mireya de la Garza1

1 Departamento de Biología Celular, Centro de Investigación y de Estudios Avanzados del IPN, Ap. 14-740, México, DF 07000, Mexico
2 Departamento de Morfología, Centro de Ciencias Básicas, Universidad Autónoma de Aguascalientes, Blvd Universidad 940, Aguascalientes, Ags 20100, Mexico
3 Departamento de Patología Experimental, Centro de Investigación y de Estudios Avanzados del IPN, Ap. 14-740, México, DF 07000, Mexico
4 CENID-Microbiología, INIFAP, Carretera a Toluca Km 15.5, México, DF, Mexico

Correspondence
Mireya de la Garza
mireya{at}cell.cinvestav.mx


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Actinobacillus pleuropneumoniae serotype 1 adhered to immobilized swine-lung collagen. Bacteria bound to collagen type I, III, IV and V. At 5 min incubation, 30 % of bacteria adhered to collagen, reaching saturation in around 90 min. Treatment of bacteria with divalent-metal chelators diminished their attachment to collagen, and Ca2+ but not Mg2+ increased it, suggesting Ca2+ dependence for adherence. Proteolytic enzymes drastically reduced bacterial adherence to collagen, showing that binding involved bacterial surface proteins. Porcine fibrinogen, haemoglobin and gelatin partially reduced collagen adhesion. A 60 kDa outer-membrane protein of A. pleuropneumoniae recognized the swine collagens by overlay. This membrane protein was apparently involved in adhesion to collagen and fibrinogen, but not to fibronectin and laminin. Antibodies against the 60 kDa protein inhibited the adhesion to collagen by 70 %, whereas pig convalescent-phase antibodies inhibited it by only 40 %. Serotypes 1 and 7 were the most adherent to pig collagen (taken as 100 %); serotypes 6 and 11 were the lowest (~50 %), and neither showed the 60 kDa adhesin to biotinylated collagens. By negative staining, cells were observed initially to associate with collagen fibres in a polar manner, and the adhesin was detected on the bacterial surface. The results suggest that swine-lung collagen is an important target for A. pleuropneumoniae colonization and spreading, and that the attachment to this protein could play a relevant role in pathogenesis.


Abbreviations: ECM, extracellular matrix; OMP, outer-membrane protein; NBT/BCIP, nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate; TLCK, N{alpha}-p-tosyl-L-lysine chloromethyl ketone


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Actinobacillus pleuropneumoniae causes porcine pleuropneumonia, a worldwide lethal disease responsible for great losses to the pig industry. The disease generally progresses through hyperacute, acute and chronic stages (Bossé et al., 2002; Fenwick & Henry, 1994). A. pleuropneumoniae possesses several virulence factors: LPS, capsule, transferrin-binding proteins, and secreted Apx toxins and proteases (Baltes et al., 2000; Bossé et al., 2002; Frey, 1995; Negrete et al., 2000). Host factors are also involved in the disease; thus it has been considered multifactorial.

Extracellular matrix (ECM) is a structure found underneath epithelial and endothelial cells, and surrounding connective tissue cells (Westerlund & Korhonen, 1993). ECM can be reached by pathogenic micro-organisms that damage the superficial epithelia, leading to adhesion, colonization and invasion of the host (Olsen & Ninomiya, 1994; Patti et al., 1994). ECM is composed of polysaccharides and proteins, collagen being the most abundant ECM protein in mammals. In swine lung almost 60 % of the parenchyma connective tissue is collagen. Four types of collagen (I, III, IV and V) have been described in both human and swine lung (Mills & Haaworrth, 1987; Van-Kuppevelt et al., 1995). Collagen is one of the major targets for attachment of pathogenic and commensal bacteria, and this phenomenon generally occurs through specific adhesins (Mukai et al., 1997; Schulze-Koops et al., 1992; Schurts et al., 1998; Switalski et al., 1993; Trust et al., 1991; Westerlund et al., 1989). Collagen could be a receptor, or a ligand connecting bacteria to a host cell receptor. In the genus Actinobacillus, adhesion to human collagen through surface proteins has been reported in A. actinomycetemcomitans (Mintz & Fives, 1999).

The mechanisms involved in colonization of the swine respiratory tract by A. pleuropneumoniae are beginning to be known. LPS is considered an adhesin for vascular endothelia, lung mesenchyma and tracheal epithelium (Paradis et al., 1994). Recently, a 55 kDa outer-membrane protein (OMP) was reported as an adhesin to swine alveolar epithelial cells (Van Overbeke et al., 2002). Adhesins to pig respiratory-tract collagen may play a key role in early stages of A. pleuropneumoniae colonization, but to our knowledge, no research has been done on the attachment of this bacterium to pig-lung collagen. In this paper the in vitro adherence of A. pleuropneumoniae to swine-lung collagen is assessed. Additionally, a 60 kDa OMP is reported as a collagen adhesin.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains and growth conditions.
The 13 reference strains of A. pleuropneumoniae used were donated by E. M. Kamp (Department of Bacteriology, ID-DLO, The Netherlands) (serotypes in parentheses): S4074 (1a), 1536 (2), 1421 (3), M62 (4), K17 (5a), L20 (5b), Fem Ø (6), WF 53 (7), 405 (8), CVI 13261 (9), D 13039 (10), 56153 (11) and 6329 (12). A. pleuropneumoniae strains Fe/710 (serotype 6) and 56153 (11), donated by M. Gottschalk (Université de Montréal, Quebec, Canada), were also used. All A. pleuropneumoniae strains were grown in brain heart infusion broth (BHI, Difco) with 10 µg NAD ml–1 (Sigma) at 37 °C under shaking conditions (150 r.p.m.). Escherichia coli HB101 was grown in Luria–Bertani (LB) medium.

Collagen extraction.
Pig-lung collagens were obtained by a method based on those described for other tissues (Duance, 1990; Konomi et al., 1984; Serrano et al., 1994). Reagents were from Sigma, the procedure was carried out at 4 °C, and centrifugations were done for 1 h at 10 000 g. The lung was chilled, washed with distilled water, and minced with 4 mg sodium azide l–1. Tissue was digested with 0·25 M acetic acid containing 500 mg pepsin l–1 for 76 h on a stirrer, and centrifuged. The supernatant was neutralized to pH 7·0 with 1 M NaOH, and the final volume determined; it was adjusted to 0·7 M NaCl, stirred overnight, and centrifuged. Supernatant (collagens IV and V) was decanted and recovered, and the pellet (collagens I and III) was dissolved in 0·25 M acetic acid, neutralized, adjusted to 1·7 M NaCl and stirred overnight. The mixture was centrifuged; the pellet (collagen III) was recovered, and the supernatant adjusted to 2·5 M NaCl, stirred overnight, and centrifuged. The pellet (collagen I) was recovered and the supernatant (collagens IV and V) adjusted to 1·2 M NaCl, stirred overnight and centrifuged; the resulting pellet was diluted in 0·5 M acetic acid and dialysed against 0·01 M Tris/HCl pH 8·5, 0·02 M NaCl, and 2 M urea. After this, protein mixture was centrifuged. The supernatant contained collagen IV and the pellet collagen V.

The swine-lung collagens obtained (types I, III, IV and V) were compared with purified human-placenta collagen type I (Serrano et al., 1994), type I from Sigma and type IV (donated by Dr Muñoz, Cinvestav-IPN, Mexico), in 7·5 % (w/v) polyacrylamyde SDS-PAGE (Laemmli, 1970) stained with Coomassie blue or silver (Harper et al., 1995). Collagens were also checked by using antibodies; for this, the collagens were dropped onto a nitrocellulose membrane (Sigma), blocked for 2 h at 25 °C in 5 % (w/v) skimmed milk (Difco) with PBST [PBS+0·05 % (v/v) Tween 20], washed, and incubated for 2 h with mAbs against human-placenta collagen I, III or V, polyclonal Ab against human collagen IV (all diluted 1 : 500; ICN); also mAb against human fibronectin (donated by Dr Talamás, Cinvestav-IPN, Mexico), and mAb against human laminin (Sigma) (both diluted 1 : 5000). The membrane was then washed and incubated for 1 h with the secondary Ab (peroxidase-conjugated goat anti-mouse or anti-rabbit IgG) (Zymed). Reaction was revealed with 3,3'-diaminobenzidine.

Collagen-binding assay.
Bacteria grown to exponential phase were harvested by centrifugation (10 min, 10 000 g), washed, and suspended in 10 mM HEPES pH 7·4 at an OD590 of 1. Pig-lung collagen was used at a concentration of 1 mg ml–1 in 0·25 M acetic acid, and neutralized (1 M Tris, pH 7·4). Collagen (100 µl) was immobilized as films on flat-bottomed microtitre plate wells (Nunc-Immuno) and sterilized overnight with UV light (Serrano et al., 1994). Films were blocked with BSA (1 %, 1 h) and washed with 10 mM HEPES pH 7·4; then 100 µl bacterial suspension was added to each well, and incubated at 37 °C. Non-adherent bacteria were removed by three washes, and those adhered to films were stained with methylene blue (0·4 %, 15 min), rinsed and dried. Finally, 200 µl 95 % ethanol was added to each well. Plates were read at 595 nm in a Micro-Plate Reader model 450 (Bio-Rad). Collagen, BSA, collagen plus BSA, or bacteria alone were used as negative controls; they were placed onto wells, incubated, washed, and stained like the samples. Adhesion to type III collagen was determined in A. pleuropneumoniae serotype 1 incubated for different times (5–120 min and 24 h) and at various concentrations of collagen (0·05–200 µg ml–1), and for 90 min with 100 µg collagen ml–1 for the other serotypes and E. coli. All experiments on adherence to collagens were done at least in triplicate.

Effect of putative inhibitors on the A. pleuropneumoniae serotype 1 adherence to pig-lung collagen III.
The compounds and concentrations used for these determinations are in Table 1. The effect of the following treatments was evaluated: proteins, proteolytic enzymes, carbohydrates (100 mM of glucose, galactose, mannose, N-acetylglucosamine and N-acetylgalactosamine), metaperiodate, CaCl2, MgCl2, chelating agents and antibodies [pig preimmune serum, and pig convalescent-phase serum (PCPS) from a farm animal affected by pleuropneumonia]. Serum without IgG was also tested in collagen-adherence inhibition by PCPS. Adherence inhibition was determined by preincubating bacteria with each compound at 37 °C for 1 h. Also, bacteria were strongly vortex-mixed for 1 min, grown at 18 °C for 18 h, or treated at 56 °C for 1 h, before assaying adhesion.


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Table 1. Effect of various treatments on A. pleuropneumoniae adherence to swine-lung collagen type III

 
Extraction of OMPs and detection of collagen adhesin.
Biotinylated pig-lung collagens were prepared as described for other proteins and exhaustively dialysed to remove free biotin (Savage et al., 1992). OMPs were obtained from A. pleuropneumoniae serotype 1 using 1 % (w/v) sarcosyl (Rapp et al., 1986). Protein concentration was determined by the method of Bradford (1976). OMPs were separated by 10 % SDS-PAGE, blotted onto a nitrocellulose membrane (Sigma) (Towbin et al., 1979), blocked for 2 h in skimmed milk-PBST, washed, and incubated for 2 h with 100 µg biotinylated collagen I, III, IV or V, or biotinylated fibrinogen, or fibronectin and laminin. The membrane was then washed and treated for 1 h with horseradish peroxidase–streptavidin for biotinylated proteins (diluted 1 : 1000). Collagen and fibrinogen adhesins were detected with Super Signal Chemiluminescent Substrate Western Blotting (Pierce), and exposed to an X-OmatV UV film (Kodak). Fibronectin- and laminin-binding proteins from the outer membrane were detected with anti-fibronectin and anti-laminin mAbs, incubated with secondary Abs (peroxidated anti-mouse IgG) and revealed with 3,3'-diaminobenzidine. In experiments with all A. pleuropneumoniae serotypes, collagen adhesin was detected by using alkaline phosphatase–streptavidin (diluted 1 : 3000), and revealed with NBT/BCIP.

Purification of the 60 kDa A. pleuropneumoniae OMP that recognizes pig-lung collagen.
The protein that recognized swine-lung collagen by overlay was purified by affinity chromatography, coupling 10 mg collagen III to Sepharose 4B (1 g, Sigma) activated with CNBr. OMPs (10 mg protein solubilized with 2 mM Tris/HCl, pH 7·4) were obtained as described above, and passed through the column (4 °C). The charged column was gently washed with 10 mM HEPES containing 0·1 M KCl, and then eluted with 10 mM HEPES containing 1 M KCl. A second elution was done with 10 mM HEPES containing 40 % formamide. The whole procedure was performed in the presence of 10 mM TLCK. The eluted protein was precipitated overnight with ethanol (protein : ethanol 1 : 5, v/v, 4 °C). The 60 kDa OMP was also obtained by electroelution (Miniprotean III electroelution system, Bio-Rad) from 10 % SDS-PAGE. The protein was checked by binding to biotinylated pig-collagen III.

Polyclonal Abs raised against the 60 kDa OMP, and inhibition of adherence to collagen III by these Abs.
To determine the specificity of A. pleuropneumoniae serotype 1 adherence to pig collagen III through the 60 kDa OMP, specific polyclonal antibodies against this adhesin (anti-OMP60) were prepared. This was done after cutting and electroeluting the band from the gel (see above). Abs were induced in two female New Zealand rabbits. After collecting the preimmune serum, the protein was inoculated four times at intervals of a week (100 µg each). The first inoculation was by the subcutaneous route with complete Freund's adjuvant. Boosters were applied with Al(OH)3 by the intramuscular route. Sera were collected 7 days after the last immunization. To determine whether anti-OMP60 Abs inhibited the adherence of A. pleuropneumoniae to collagen, dilutions of the anti-OMP60 were incubated with bacteria for 1 h at 37 °C, washed, and placed onto collagen III immobilized in microtitre plates as in the adhesion assay.

Electron microscopy.
To demonstrate the adherence of A. pleuropneumoniae serotype 1 to swine collagen III fibres, this protein was adsorbed on a Formvar-covered nickel grid (10 min, 25 °C), and washed with 10 mM HEPES pH 7·4. Bacterial suspension was added, incubated (1 h, 37 °C), and the grid washed again. Samples were fixed with glutaraldehyde (EMS; 1 %, 10 min), washed with bidistilled water, and contrasted with 0·5 % uranyl acetate (EMS). Collagen III or bacteria, which were adsorbed to grids, washed, fixed and stained, were used as controls. To show the 60 kDa OMP on the cell surface, an immunodetection method was used (Li et al., 1996). Briefly, one drop of bacterial suspension was placed on a Formvar-covered nickel grid, blocked for 30 min with 0·5 % glycine and 1 % BSA, incubated with the anti-OMP60 serum (1 h, 25 °C), and washed with bidistilled water. Colloidal gold–protein A (CGPA) (10 nm diameter, diluted 1 : 25) was then applied. Grids were washed with distilled water, and contrasted with uranyl acetate. As negative controls, we used bacteria blocked with glycine and BSA, treated either with CGPA or preimmune serum plus CGPA, in both cases without anti-OMP60. Observations were made with a JEM 2000 EX electron microscope (Japan Electron Optics Labs).

Statistical analysis.
Adhesion assay data are expressed as means±SD. Student's t-test was used to establish statistical significance.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Collagen analysis
Swine-lung collagens were enriched by selective precipitation with NaCl (2·5 M for type I, 1·7 M for III, and 1·2 M for IV and V). These collagens showed a similar pattern to human collagen, and did not show contaminant bands after being electrophoresed and stained with silver (Fig. 1a). To determine the specifity of the pig collagen types obtained, immunoblots with commercial Abs reported to specifically recognize collagen I, III, IV or V were performed (Fig. 1b). The mAb against type I recognized the fibrillar pig collagens (I, III and V) and also human collagen I. The mAb against human collagen III only recognized pig collagen III, and the mAb against human collagen V specifically recognized pig collagen V. Thus, pig collagens I and IV were not contaminated with the other collagens tested but collagens III and V could have contained a small proportion of type I, as has been reported for these proteins. The polyclonal Ab against human basal-membrane collagen IV recognized all collagens tested (data not shown); thus this Ab was not considered further. No reaction was observed with the secondary Abs. As A. pleuropneumoniae is able to adhere to swine fibronectin (Hamer et al., 2004), we checked that pig collagens were not contaminated with fibronectin; Fig. 1(b) shows that neither fibronectin nor laminin was in the samples.



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Fig. 1. Identification of swine-lung collagens. (a) SDS-PAGE (7·5 %) of silver-stained collagens. MM, molecular mass markers; pig I, III, IV, V, pig-lung collagen type I, III, IV and V, respectively; human I and IV, purified human-placenta collagen type I and IV. (b) Immuno dot-blot. The first Ab was commercial mAb against human placenta collagen (Cn) I, III or V, or mAbs against human fibronectin (Fn) and laminin (Ln). The secondary Ab was peroxidase-labelled goat anti-mouse or anti-rabbit IgG.

 
A. pleuropneumoniae adheres in vitro to swine-lung collagen
To determine the adherence of A. pleuropneumoniae to swine-lung collagen, experiments were designed using a solid-phase binding assay with immobilized collagen. The bacteria adhered in similar numbers to collagen types I and IV (P>0·05), indicating that A. pleuropneumoniae is able to bind to fibrillar and basal-membrane collagen. Adhesion to collagen was measured at 2 h of interaction, the time reported for adherence to this protein in other bacteria. As A. pleuropneumoniae adherence to the four types of collagen did not differ significantly, collagen III at that time was arbitrarily chosen as 100 % for this assay (Fig. 2a), giving binding means of 95, 91 and 96 % for collagens I, IV, and V, respectively; there was no bacterial adhesion to a plastic surface, or to BSA. Considering that collagen III is the most abundant in pig lung, this type was selected as a representative target for A. pleuropneumoniae adherence to collagen. An adhesion kinetics study was done to initially characterize the binding of bacteria to collagen III (Fig. 2b). Bacterial adherence occurred immediately, with a value of 30 % at 5 min (A595 0·150). Saturation was reached in around 90 min (A595 0·485), and bacteria stayed attached at least for 24 h. A. pleuropneumoniae adhered avidly to swine collagen III, with adhesion observed in 0·05 µg collagen ml–1 (Fig. 2c). Fig. 2(d) shows that serotypes 1 and 7 adhered similarly (taken as 100 %). The other serotypes adhered at 70–80 % (P<0·05), except for 6 and 11, which showed low adherence (50 and 60 %, respectively).



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Fig. 2. Adherence of A. pleuropneumoniae to swine-lung collagen. Collagen films were prepared on microtitre wells. Collagen (100 µg per well) was blocked with 1 % BSA, washed, and bacteria (OD590 1) were added and incubated for 2 h at 37 °C. (a) Bacterial adherence to: Bact., plastic; BSA+bact., BSA; I, III, IV and V, the types of purified collagen. Adherence to collagen III was taken as 100 % adhesion. (b, c) Adhesion kinetics of A. pleuropneumoniae at different incubation times (b) and to several concentrations of collagen III (c). (d) Adhesion of different A. pleuropneumoniae reference serotypes to collagen III. E.c., E. coli (negative control). The data represent the means of at least three independent experiments in triplicate.

 
A. pleuropneumoniae serotype 1 can attach to collagen fibres
Electron microscopy negative-staining experiments were done to directly observe the interaction between A. pleuropneumoniae and pig-lung collagen III fibres. Fig. 3(a, b) shows bacteria are shown in exponential phase. Collagen was observed in classical striated fibres (Fig. 3c). Interestingly, in the first minutes most bacteria bound to the fibres in a polar manner (Fig. 3d); however, bacteria were later observed longitudinally bound to fibres, apparently contacting them via the whole cell surface (Fig. 3e). Also, there were abundant cells bound to or covered by collagen networks (Fig. 3f–i).



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Fig. 3. Electron microscopy of A. pleuropneumoniae adhered to swine-lung collagen III. (a, b) Bacteria in exponential phase; bars, 1 µm. (c) Pig-lung collagen III fibre; bar, 20 nm. (d) Polarized adhesion of bacteria to a collagen fibre; bar, 1 µm. (e) Bacteria bound to a collagen fibre in a longitudinal orientation; bar, 200 nm. (f, g) Bacteria bound to a collagen network; bar, 1 µm. (h, i) Interaction between bacteria and collagen; bars, 500 and 200 nm, respectively.

 
Effect of different treatments on A. pleuropneumoniae adhesion to swine collagen III
To determine the chemical nature of the A. pleuropneumoniae serotype 1 adhesion factor recognizing pig collagen III, several treatments were done to bacteria prior to the interaction with collagen (Table 1). Vortexing the bacteria did not significantly affect their adhesion to swine collagen, but growth at 18 °C partially decreased it (30 % inhibition). EDTA and EGTA inhibited the adherence by 60 %, and Ca2+ increased it by 20 %; however, Mg2+ did not have an effect. Carbohydrates like glucose, galactose, mannose, N-acetylglucosamine and N-acetylgalactosamine did not decrease the adherence significantly (5–10 % inhibition, P>0·05, data not shown); accordingly, metaperiodate did not affect it either. Porcine haemoglobin, fibrinogen and gelatin inhibited the adhesion by 60 %. Inhibition of bacterial adherence to collagen was also obtained when bacteria were heated at 56 °C or incubated with proteinase K and trypsin (45, 84 and 92 %, respectively); thus, bacterial surface proteins seem to be involved in the interaction between A. pleuropneumoniae and collagen. Finally, pig convalescent-phase serum decreased the bacterial adhesion to collagen films by 40 %; the possibility that acute-phase proteins inhibited the adhesion was discarded, since serum depleted of IgG did not inhibit the adherence of bacteria to collagen (data not shown).

Collagen-binding protein of A. pleuropneumoniae
To test if A. pleuropneumoniae serotype 1 OMPs are involved in the binding to pig-lung collagen, they were purified by using sarcosyl. In Fig. 4 a typical electrophoretic profile of OMPs showing several enriched proteins (Fig. 4a, lane 4) is compared with total bacterial extract and internal membrane proteins (Fig. 4a, lanes 2 and 3). OMPs were incubated with biotinylated pig-lung collagen types I, III, IV and V, biotinylated swine fibrinogen, and also with fibronectin and laminin, which were reacted with mAbs. Only a 60 kDa band reacted with all the collagens (Fig. 4b, lanes 1–4, respectively); BSA was the negative control (Fig. 4b, lane 5). Fibrinogen also recognized the 60 kDa band; however, fibronectin and laminin recognized other OMPs (data not shown). All A. pleuropneumoniae serotypes except 6 and 11 showed the 60 kDa collagen adhesin (Fig. 4c). To check the lack of reaction of serotypes 6 and 11, other strains (Fe/710 of serotype 6 and 56153 of serotype 11) were tested and the results were similar (data not shown). Serotypes 6 and 11 were also tested with the anti-OMP60 antibodies; only serotype 11 was recognized by this Ab (data not shown).



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Fig. 4. Collagen-binding protein from A. pleuropneumoniae. (a) SDS-PAGE (10 %) pattern of A. pleuropneumoniae serotype 1 proteins stained with Coomassie blue. Lanes: 1, molecular mass markers; 2, bacterial total extract; 3, cytoplasmic membrane extract; 4, OMPs. (b, c) Overlay assays of OMPs electroblotted onto a nitrocellulose membrane. (b) A protein of 60 kDa from A. pleuropneumoniae serotype 1 was recognized by biotinylated pig-lung collagen I, III, IV and V (lanes 1, 2, 3 and 4, respectively); no reaction was found with BSA (lane 5). (c) The 60 kDa adhesin of A. pleuropneumoniae was recognized by pig collagen I, III, IV and V in all serotypes except 6 and 11. E. coli (E.c.) was the negative control.

 
Purification, specificity and localization of the 60 kDa adhesin
The 60 kDa OMP of A. pleuropneumoniae serotype 1 that recognized pig-lung collagen was purified by affinity chromatography, and its purity was confirmed by SDS-PAGE. Only this polypeptide was eluted from the collagen column (Fig. 5a, lane 2). Fig. 5b, lane 2, shows the 60 kDa protein purified by electroelution. The purity of this protein was also checked by isoelectric focusing; only one protein was observed (data not shown). To test the importance and specificity of the 60 kDa OMP in A. pleuropneumoniae adherence to collagen, an inhibition assay with the anti-OMP60 was done. Antibodies blocked the cell binding to collagen with a concentration-dependent effect (Fig. 5c). The putative collagen-binding protein was detected on the bacterial surface and surrounding material by using the anti-OMP60 and colloidal gold–protein A, in electron microscopy (Fig. 5d, 1 and 2); flagella were observed in some preparations as was recently described (Negrete et al., 2003). Cells did not react with protein A (Fig. 5d, 3) or rabbit preimmune serum (Fig. 5d, 4).



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Fig. 5. Purification of the A. pleuropneumoniae 60 kDa OMP, and inhibition of adhesion to pig-lung collagen. (a) Purification by collagen affinity column. (b) Purification by electroelution. (c) Inhibition of A. pleuropneumoniae adherence to pig-lung collagen III films, by different dilutions of a rabbit anti-OMP60 antiserum (1, without Ab; 2, 1 : 1000; 3, 1 : 500; 4, 1 : 200; 5, 1 : 100; 6, 1 : 50; 7, 1 : 10); data represent the means of three independent experiments in triplicate. (d1 and 2) The A. pleuropneumoniae 60 kDa OMP is detected around the bacterial surface with the anti-OMP60, and protein A-colloidal gold. (d3) Negative control showing bacterial cells with protein A. (d4) Negative control of bacterial cells treated with preimmune serum and protein A. Bars, 500 nm.

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial adhesion is defined as the irreversible attachment of bacteria to tissues, and is considered as an early step for infection establishment (Ofek & Doyle, 1994; Patti et al., 1994; Westerlund & Korhonen, 1993). Collagen adhesion is crucial for pathogens, such as meningitis-associated E. coli, which penetrates the epithelium, reaching sites of secondary infection (Pouttu et al., 1999). As A. pleuropneumoniae showed affinity for pig respiratory-tract mucus (Bélanger et al., 1992), and it was able to secrete proteolytic enzymes (Negrete et al., 1994), it probably adheres to and degrades the mucus and underlying epithelia, leading to contact with collagen. This process was found in Helicobacter pylori, which colonized the mucus and adhered to gastric cells, leading to binding to collagen IV (Trust et al., 1991). A. actinomycetemcomitans was able to bind to collagen I; this feature is a decisive colonization factor of the human oral cavity (Mintz & Fives, 1999). A similar mechanism has been described in Streptococcus pyogenes, where cysteine protease damaged the skin surface and penetrated the tissue (Lukomski et al., 1999).

A. pleuropneumoniae is a swine-specific pathogen with tropism for the respiratory tract, capable of reaching the lung (Fenwick & Henry, 1994); thus it is important to know its adhesion ability to this organ, where 60 % of the connective tissue is collagen (Van-Kuppevelt et al., 1995). Here, the four types of pig-lung collagen were dissolved in acetic acid, and protein contaminants digested with pepsin at 4 °C, a temperature at which collagen is resistant; collagen types were then separated by differential precipitation with NaCl (Duance, 1990). The electrophoretic profile of pig-lung collagens was consistent with that of collagens I and IV of human placenta (Miller & Rhodes, 1982), confirming that collagen is a highly conserved protein. Our preparations of swine collagens I and IV were not apparently mixed with the other collagens tested; however, the preparations of collagens III and V could have contained some type I. Neither pig fibrillar collagens nor basal-membrane collagen were contaminated with fibronectin or laminin.

This work has shown the ability of A. pleuropneumoniae to bind strongly to pig-lung collagen. With the experiments performed we cannot differentiate whether this bacterium recognizes conserved regions among collagen types, or distinct sites for affinity to them. YadA protein from Yersinia is able to recognize different collagen chains on several types (Schulze-Koops et al., 1992). Bacterial attachment to collagen III, the predominant protein in pulmonary tissue of adult and suckling pigs (Mills & Haaworrth, 1987), was accomplished rapidly and was stable and on the increase in a slower process over several hours. We also have evidence that A. pleuropneumoniae can cleave azocoll, a synthetic collagen (unpublished results). Therefore, A. pleuropneumoniae adherence to and further degradation of collagen could be contributing to the tissue damage, and this adherence could play a key role in the extensive lung colonization by this pathogen in vivo.

Electron microscopy observations showed that pig-lung collagen III is a striated fibre structurally similar to that of chicken embryo cornea, dermis and tendon (Birk et al., 1996). Bacterial attachment to collagen fibres was strong, since it was not affected by intensive washes or Triton X-100. Initially, a polar binding of most bacteria was found; perhaps some polar structure is involved in the initial recognition of collagen. Tip-oriented adhesion has also been observed in the oral spirochaete Treponema denticola, though in an inverse process compared with A. pleuropneumoniae, because T. denticola contacts fibronectin-coated surfaces first along its length and with time in a polar orientation (Ellen et al., 1994). More studies must be done to understand the significance of the initial A. pleuropneumoniae polar contact to pig collagen.

Pre-incubation of cells with pig convalescent-phase sera against A. pleuropneumoniae serotype 1 partially reduced their interaction with collagen; thus, collagen adhesins are antigens expressed in pleuropneumonia, suggesting that animals produce Abs blocking the adhesion to collagen, as found in infection by Prevotella intermedia (Grenier, 1996). Inhibition of collagen adherence by fibrinogen could be explained by the shared regions with collagen, or related to steric hindrance due to close proximity of both adhesins, or even both proteins may share the A. pleuropneumoniae binding component. Adhesion of P. intermedia to human collagen I also was inhibited by fibrinogen, though it was not demonstrated if it is the same adhesin (Grenier, 1996). Haemoglobin binds to the surface of A. pleuropneumoniae whole cells (Bélanger et al., 1995), and then it could obstruct the further binding to collagen. Treatment of cells with EDTA or EGTA diminished the adhesion to collagen, indicating that interaction is dependent on divalent ions; this was confirmed for CaCl2, which increased the adhesion, although MgCl2 did not affect it. In contrast, carbohydrates and metaperiodate did not affect the adherence; thus the collagen adhesin could not be a glycoconjugate, LPS, or capsular polysaccharide.

A. pleuropneumoniae cells treated with proteinase K or trypsin, or heated at 56 °C, which eliminate some OMPs or alter the surface protein pattern (Chiang et al., 1991; Mintz & Fives, 1999), notably diminished the adhesion of bacteria to collagen III, indicating that mainly surface proteins are involved in the binding. Similarly, A. actinomycetemcomitans treated with trypsin (Mintz & Fives, 1999), or P. intermedia with proteinase K (Grenier, 1996), showed a drastic decrease in binding to collagen I. A. pleuropneumoniae grown at 18 °C, a temperature at which other bacteria do not synthesize or assemble fimbriae (Strom & Lory, 1993), slightly decreased the adhesion to collagen. Therefore, A. pleuropneumoniae adhesion to collagen could be via OMPs and fimbriae. In other bacteria, these collagen adhesins are crucial for colonization of the host, as occurs with Yersinia enterocolitica YadA OMP, which binds to several collagen types (Schulze-Koops et al., 1992), E. coli fimbriae Dr, which recognize collagen V (Westerlund et al., 1989), and the Staphylococcus aureus Cna cytoplasmic membrane protein, which shows high affinity to collagen II and is considered necessary for bacteria to bind to cartilage (Switalski et al., 1993).

The bacterial outer membrane is an excellent surface compartment for anchoring and positioning adhesins, which have been implicated in pathogenesis (Debroy et al., 1995). The avidity of binding of A. pleuropneumoniae to pig-lung collagen suggests that it should possess several adhesins involved in this interaction. In this study, a 60 kDa OMP of serotype 1 strongly bound to all pig-lung collagens; thus this OMP could play a role in the early phase of pleuropneumonia. Apparently, this OMP is in a very low quantity since it is only seen when it reacts with biotinylated collagen. The 60 kDa OMP is perhaps a collagen adhesin, since Abs raised against this protein strongly inhibited the binding between whole bacteria and collagen. In A. pleuropneumoniae, a 55 kDa OMP was reported as binding to alveolar epithelial cells of serotypes 2 and 5 (Van Overbeke et al., 2002). However, the collagen adhesin described here has different features from the 55 kDa OMP adhesin, since it is expressed in cultures with NAD and not affected by periodate; also, the 60 kDa OMP collagen adhesin, but not the 55 kDa adhesin, had a blocked N-terminus (not shown).

A. pleuropneumoniae biotype 1 strains requiring NAD for growing have been differentiated into 14 serotypes (1a, 1b, 2–4, 5a, 5b, 6–12), based on the capsular antigenic structure. Rapp et al. (1986) reported a correlation between antigenic cross-reactivity among certain serotypes and all or some of their OMPs, observed as protein profiles in SDS-PAGE. In Mexico, serotypes 1, 3, 5 and 7 are the most frequently found in outbreaks, serotype 1 being the most pathogenic to pigs and the most adherent to swine buccoepithelial cells (Hamer et al., 2004). We found serotypes 1 and 7 to be the most adherent to swine collagen; thus, their adhesion value was taken as 100 %. However, no relationship was found between collagen adherence and virulence: serotype 11 has been reported as exceptionally virulent, together with serotypes 1, 5 and 9, but it only adhered 60 % compared with serotypes 1 and 7; serotypes 5 and 9 showed 70 and 80 % adherence, respectively, values similar to the majority of serotypes. Serotype 6, which is also virulent, showed 50 % adherence to pig collagen. As serotypes 6 and 11 showed low but not null adherence, perhaps they do not possess some of the collagen adhesins. In fact, these serotypes did not recognize biotinylated collagen by overlay. Since only serotype 11 showed the 60 kDa protein when treated with Abs against this adhesin, perhaps this serotype has a non-functional 60 kDa collagen adhesin, and serotype 6 does not possess this protein. Thus, fimbriae or other OMP(s) could be responsible for the low collagen adhesion in those serotypes. E. coli HB101 whole cells showed a very low adherence value to pig collagen (28 %), as reported (Virkola et al., 1993). Accordingly, in this bacterium the 60 kDa adhesin was not detected with biotinylated collagen, nor did it react with the anti-OMP60 Ab.

A. pleuropneumoniae releases LPS, exotoxins and proteases to the culture medium; Apx and proteases are also secreted inside membrane vesicles (Negrete et al., 2000). With this battery of products, the pathogen causes tissue damage leading to exposure of ECM components recognized by bacterial adhesins, and to the successful infection of the respiratory tract. Accordingly, A. pleuropneumoniae binds to pig tonsils, where it associates with the stratified epithelium, causing vacuolization and desquamation (Chiers et al., 1999). We are now identifying the gene encoding the 60 kDa OMP adhesin. Future studies should eventually identify the exact domains of contact and the global interaction between collagen or other ECM proteins and this bacterium.


   ACKNOWLEDGEMENTS
 
Project supported by CONACyT, México, grants 28755B, G38590B and 38823-N. The first author was a scholarship recipient from CONACyT, no. 112861. We thank Esteban Molina and Biol. Magda Reyes for their excellent technical assistance, and the Unit of Electron Microscopy of CINVESTAV-IPN.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 23 January 2004; revised 26 March 2004; accepted 26 March 2004.



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