Vol. 62, No. 5
INFECTION AND IMMUNITY, May 1994, p. 1896-1900 0019-9567/94/$04.00+0 Copyright © 1994, American Society for Microbiology
Pseudomonas aeruginosa Outer Membrane Adhesins for Human Respiratory Mucus Glycoproteins CHRISTOPHE CARNOY,' ANDREE SCHARFMAN,' EDWIGE VAN BRUSSEL,'
GENEVIEVE LAMBLIN,' REUBEN RAMPHAL,2 AND PHILIPPE ROUSSEL'* Unite 377, Institut National de la Sante et de la Recherche Medicale, Place de Verdun, 59045 Lille Cedex, and Department of Medicine, University of Florida, Gainesville, Florida 326102
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Received 20 October 1993/Returned for modification 15 November 1993/Accepted 25 February 1994
The attachment of Pseudomonas aeruginosa to human respiratory mucus represents an important step in the development of lung infection, especially in cases of cystic fibrosis. For this purpose, microtiter plate adhesion assays have been developed and have suggested that nonpilus adhesins of P. aeruginosa are the most important ones for binding to human respiratory mucins. In order to characterize these mucin-binding adhesins, outer membrane proteins (OMP) from two adhesive strains, 1244-NP and PAK-NP, and their poorly adhesive rpoN mutants, 1244-N3 and PAK-Ni, were prepared by a mild extraction with Zwittergent 3-14. Mucin-binding adhesins were detected after polyacrylamide gel electrophoresis and blotting of the OMP on nitrocellulose replicas, using human bronchial mucins labeled with 1251. The binding properties of these OMP with lactotransferrin, another glycoprotein abundant in respiratory mucus, were also studied. Radiolabeled mucins detected four bands at 48, 46, 28, and 25 kDa with strain PAK-NP. With the nonmucoid strain 1244-NP, five bands were observed at 48, 46, 42, 28, and 25 kDa. The bands at 48 and 25 kDa were also visualized by radiolabeled lactotransferrin. These bands were partially or completely displaced by nonradiolabeled respiratory mucin glycopeptides but not by tetramethylurea, suggesting that they recognized carbohydrate sites. In contrast, the poorly adhesive strains showed weakly binding bands. These results demonstrate that outer membranes from two different nonpiliated P. aeruginosa strains express multiple adhesins with an affinity for human respiratory mucins and/or lactotransferrin. kindly provided by S. Lory from the University of Washington (Seattle). They had been used in a previous study of the binding of P. aeruginosa mutants to mucins (16). Bacteria were grown in tryptic soy broth (TSB medium) (Difco, Detroit, Mich.) for 16 and 24 h. After centrifugation of the cultures at 4,000 x g, the pellets were washed twice with phosphate-buffered saline (PBS) and suspended in 50 mM sodium citrate buffer containing 0.1% (wt/vol) Zwittergent 3-14 (Calbiochem, San Diego, Calif.), 1 mM phenylmethylsulfonyl fluoride (Sigma, St. Louis, Mo.), and 10 mM EDTA. This mixture was incubated for 25 min at 45°C with occasional mixing. The bacteria were then pelleted by centrifugation at 10,500 x g for 20 min, and the supernatant containing the extracted proteins was dialyzed extensively against deionized water containing 0.02% (wt/vol) sodium azide to remove the detergent. The lyophilized supernatant was used as a crude OMP preparation. The OMP content was measured by the bicinchoninic acid protein assay (Pierce, Rockford, Ill.). SDS-PAGE and Western blotting of the OMP. The crude OMP extracts were treated by 2-mercaptoethanol and were studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (5 to 15% gradient) according to the method of Laemmli (9); 40 ,ug of protein was loaded in each lane. The low-molecular-weight calibration kit (PharmaciaLKB, Uppsala, Sweden) was used as a protein standard. Proteins were stained with Coomassie blue R250. Part of the gel was submitted to semidry electrotransfer on a nitrocellulose sheet (Amersham, Arlington Heights, Ill.) with a Multiphor II NovaBlot unit (Pharmacia-LKB) according to the manufacturer's instructions. To check the efficiency of the transfer, some blots were stained with amido black (24). Preparation of human respiratory mucins and mucin glycopeptides. Sputum from a patient (blood group 0) suffering from chronic bronchitis was diluted with deionized water
Pseudomonas aeruginosa is the major pathogen in chronic bacterial lung colonization in cases of cystic fibrosis (6). Respiratory infection by P. aeruginosa is considered to be the main cause of the poor prognosis in cases of cystic fibrosis. The reasons for rapid colonization have not been elucidated, but several studies have shown alterations in the carbohydrate composition of respiratory mucins (1, 3-5, 11) and the greater affinity of P. aeruginosa for mucins from patients with cystic fibrosis, suggesting that these glycoproteins might be involved in the colonization process (2). It has been shown previously that the adhesion of P. aeruginosa to mucin might be mediated by pilus and alginate (15, 17), but recently, the observation of the binding of nonpiliated mutants to mucins (16) and neoglycolipids (14, 20) suggested the existence of a class of nonpilus adhesins. In order to characterize these nonpilus adhesins, we examined the interactions of outer membrane proteins (OMP) from two adhesive strains with two species of glycoproteins secreted in human respiratory mucus, mucins and lactotransferrin. Mucins are high-molecular-weight 0-linked glycoproteins secreted by goblet cells and mucous glands of the mucosa (10), whereas lactotransferrin is an N-linked glycoprotein (75 to 80 kDa) secreted by the serous glands of the mucosa (8, 23). Several adhesins in both strains were identified. MATERIALS AND METHODS Bacteria. The P. aeruginosa strains used in this study, the nonpiliated, nonmucoid strains 1244-NP and PAK-NP and the poorly adhesive rpoN mutants 1244-N3 and PAK-Ni, were * Corresponding author. Mailing address: Unite 377, INSERM, Place de Verdun, F-59045 Lille Cedex, France. Phone: (33) 20 52 94 84 or (33) 20 53 85 62.
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FIG. 1. PAGE and blotting of OMP obtained from PAK-NP and 1244-NP. OMP from PAK-NP (lanes 1 and 2) and 1244-NP (lanes 3 and 4) were stained by Coomassie blue directly on the gel (lanes 1 and 3) or by amido black after blotting (lanes 2 and 4). Each lane contained 40 pLg of protein. In all figures, the unlabeled lane contained molecular mass markers.
(1:12) and stirred overnight. Then the suspension was centrifuged; the supernatant was dialyzed against deionized water and lyophilized. The preparation of high-molecular-mass mucins was obtained by CsBr density gradient centrifugation (7). This preparation was further purified by a second step of CsBr density gradient centrifugation. Mucin glycopeptides were obtained by pronase (Calbiochem) digestion of mucins (13) and prepared by gel chromatography on Sepharose CL-4B (Pharmacia-LKB) (13). Labeling of human secretory glycoproteins. Radioiodination was performed with lodo-beads (Pierce) according to the manufacturer's instructions. Ten milligrams of human tracheobronchial mucins dissolved in 1 ml of phosphate buffer (75 mM Na2HPO4-KH2PO4, 75 mM NaCl, 0.02Cc [wt/vol] NaN3 [pH 7.2]) was iodinated with 750 ,uCi of 1251 (Amersham) by the addition of two lodo-beads. The iodination mixture was maintained for 15 min at room temperature with stirring. The reaction was terminated by transferring the solution to a tube containing sodium iodide (final concentration, 0.5 M). Radiolabeled mucins were separated from residual iodide by gel filtration on a Sepharose CL-4B column (40 by 1 cm) equilibrated with PBS. Fractions containing the iodinated samples were pooled and stored at 4°C. An average specific activity of 16 x 10" cpm/mg of mucins was obtained. In some experiments, desialylated radiolabeled mucins were used. For this purpose, radiolabeled mucins were treated twice by neuraminidase from Clostridium perfringens (Sigma) (0.03 U per 10 mg of mucin for 24 h). The radioiodination of human milk lactotransferrin (a gift from G. Spik, Villeneuve d'Ascq, France), 4 mg in I ml of phosphate buffer, was performed according to the same procedure. Detection of mucin-binding OMP. For the detection of OMP binding to mucin or lactotransferrin, nitrocellulose replicas were first maintained in PBS containing 2% bovine serum albumin (BSA) (Sepracor, Villeneuve la Garenne, France) (wt/vol) for 2 h at room temperature under mild agitation. The BSA solution was replaced by radiolabeled mucins at a con-
1 2 3 4 FIG. 2. OMP adhesins from P. aeniginosa PAK-NP and PAK-NI. OMP from PAK-NP (lanes 1 and 2) and PAK-NI (lanes 3 and 4) were separated by PAGE and stained with Coomassie blue (lanes 1 and 3) or blotted and revealed with '25I-labeled mucins after blocking with BSA (lanes 2 and 4). Each lane contained 40 ,ug of protein. The arrows indicate the major bands at 48, 46, 28, and 25 kDa.
centration of 100 p.g/ml in PBS containing 1% BSA (wt/vol), and the replicas were left under mild agitation for another 2 h at room temperature. The blots were washed with PBS (four times for 15 min each) and then 0.05% (vol/vol) Tween 20 (Merck, Munich, Germany) in PBS (four times for 15 min each), dried on filter paper, and then exposed at - 70°C to X-ray film (Hyperfilm-MP; Amersham). In some experiments, blots were treated with 0.5 M tetramethylurea (Merck) after incubation with labeled mucins. Displacement of radiolabeled glycoproteins bound to OMP. After incubation with radiolabeled mucins or lactotransferrin, some blots were incubated with a 100-fold excess of unlabeled respiratory mucin glycopeptides (10 mg/ml) for 16 h at 4°C and then they were treated as described previously. Controls were incubated for the same period with PBS. RESULTS Kinetics of production of OMP by nonpiliated strains of P. aeruginosa. P. aeruginosa (strains PAK-NP and 1244-NP) was grown in TSB medium at 37°C for 24 h. Bacteria were sampled at 16 and 24 h, and then OMP were prepared and subsequently analyzed by PAGE. With time, there was a progressive increase of recovered OMP. The major bands were mainly localized in two areas, at 40 to 50 kDa and at about 15 to 30 kDa. A large number of bands was already present at 16 h, and in order to avoid possible OMP degradation, bacteria were cultured for 16 h in most experiments (Fig. 1). Binding assay. OMP from PAK-NP and 1244-NP were separated by PAGE before being transferred to nitrocellulose sheets. As shown in Fig. 1, the OMP were homogeneously transferred to nitrocellulose sheets since the protein profiles of the blots stained with amido black were identical to the profiles observed with polyacrylamide gels after staining with Coomassie blue. There was no difference in the number or intensity of the different bands, indicating that the transfer was quantitative. The blocking of nonspecific binding sites on the nitrocellulose with Tween was also studied by treating different blots
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with increasing concentrations of Tween 20 (0.05, 0.2, and 0.5%). It appeared that Tween 20, even at a low concentration (0.05%), could elute low-molecular-mass proteins from the nitrocellulose sheet. We therefore decided to replace Tween 20 by 2% BSA in the blocking process. Mucin-binding properties of OMP from the nonpiliated strains PAK-NP and 1244-NP. Four main bands were detected at 48, 46, 28, and 25 kDa by radiolabeled mucins with OMP from strain PAK-NP (Fig. 2). Minor bands were also observed in the background at 42 and 35 kDa and below 25 kDa. Five bands were observed at 48, 46, 42, 28, and 25 kDa with the OMP from 1244-NP. Minor bands were also observed in the background below 25 kDa (Fig. 3). The bands at 25 and 28 kDa disappeared after blocking of the nitrocellulose with Tween 20 (data not shown). Mucin-binding properties of OMP from poorly adhesive rpoN mutants of strains PAK-NP and 1244-NP. OMP from the poorly adhesive mutant PAK-Nl were compared with OMP from PAK-NP for their mucin-binding properties (Fig. 2): there were only very faint bands at about 48 kDa and 25 to 28 kDa. Equally, OMP from the poorly adhesive mutant 1244-N3 were compared with OMP from 1244-NP for their binding properties (Fig. 3): there were only very faint bands at 25 to 28 kDa and below 25 kDa. Specificity of the adhesins. In order to check the specificity of the labeling, different experiments were performed. After contact with radiolabeled mucins, some blots were treated for 16 h with solutions of cold mucin glycopeptides. Compared with those of controls, the bands were largely attenuated after contact with nonradiolabeled mucin glycopeptides (Fig. 4). The bands in the region of 40 to 50 kDa almost disappeared. Tetramethylurea has been suggested to inhibit a hydrophobic nonspecific protein-mucin interaction (21); therefore, some blots corresponding to OMP from strains 1244-NP and PAK-NP were treated with 0.5 M tetramethylurea after incubation with radiolabeled mucins. No modification of the labeling could be observed (data not shown). Since sialic acid has been suggested to be involved in the binding of piliated strains of P. aeruginosa to mucins (18), it
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