INFECTION AND IMMUNITY, May 1994, p. 1896-1900 Vol. 62, No. 50019-9567/94/$04.00+0Copyright © 1994, American Society for Microbiology

Pseudomonas aeruginosa Outer Membrane Adhesins for HumanRespiratory 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, France,"and Department of Medicine, University of Florida, Gainesville, Florida 326102

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 thedevelopment of lung infection, especially in cases of cystic fibrosis. For this purpose, microtiter plate adhesionassays have been developed and have suggested that nonpilus adhesins ofP. aeruginosa are the most importantones for binding to human respiratory mucins. In order to characterize these mucin-binding adhesins, outermembrane proteins (OMP) from two adhesive strains, 1244-NP and PAK-NP, and their poorly adhesive rpoNmutants, 1244-N3 and PAK-Ni, were prepared by a mild extraction with Zwittergent 3-14. Mucin-bindingadhesins were detected after polyacrylamide gel electrophoresis and blotting of the OMP on nitrocellulosereplicas, using human bronchial mucins labeled with 1251. The binding properties of these OMP withlactotransferrin, another glycoprotein abundant in respiratory mucus, were also studied. Radiolabeled mucinsdetected four bands at 48, 46, 28, and 25 kDa with strain PAK-NP. With the nonmucoid strain 1244-NP, fivebands were observed at 48, 46, 42, 28, and 25 kDa. The bands at 48 and 25 kDa were also visualized byradiolabeled lactotransferrin. These bands were partially or completely displaced by nonradiolabeledrespiratory mucin glycopeptides but not by tetramethylurea, suggesting that they recognized carbohydratesites. In contrast, the poorly adhesive strains showed weakly binding bands. These results demonstrate thatouter membranes from two different nonpiliated P. aeruginosa strains express multiple adhesins with an affinityfor human respiratory mucins and/or lactotransferrin.

Pseudomonas aeruginosa is the major pathogen in chronicbacterial lung colonization in cases of cystic fibrosis (6).Respiratory infection by P. aeruginosa is considered to be themain cause of the poor prognosis in cases of cystic fibrosis. Thereasons for rapid colonization have not been elucidated, butseveral studies have shown alterations in the carbohydratecomposition of respiratory mucins (1, 3-5, 11) and the greateraffinity of P. aeruginosa for mucins from patients with cysticfibrosis, suggesting that these glycoproteins might be involvedin 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 ofnonpiliated 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 exam-ined the interactions of outer membrane proteins (OMP) fromtwo adhesive strains with two species of glycoproteins secretedin human respiratory mucus, mucins and lactotransferrin.Mucins are high-molecular-weight 0-linked glycoproteins se-creted by goblet cells and mucous glands of the mucosa (10),whereas lactotransferrin is an N-linked glycoprotein (75 to 80kDa) 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, thenonpiliated, nonmucoid strains 1244-NP and PAK-NP and thepoorly 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 84or (33) 20 53 85 62.

kindly provided by S. Lory from the University of Washington(Seattle). They had been used in a previous study of thebinding 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 ofthe cultures at 4,000 x g, the pellets were washed twice withphosphate-buffered saline (PBS) and suspended in 50 mMsodium citrate buffer containing 0.1% (wt/vol) Zwittergent3-14 (Calbiochem, San Diego, Calif.), 1 mM phenylmethylsul-fonyl fluoride (Sigma, St. Louis, Mo.), and 10 mM EDTA. Thismixture was incubated for 25 min at 45°C with occasionalmixing. The bacteria were then pelleted by centrifugation at10,500 x g for 20 min, and the supernatant containing theextracted proteins was dialyzed extensively against deionizedwater containing 0.02% (wt/vol) sodium azide to remove thedetergent. The lyophilized supernatant was used as a crudeOMP preparation. The OMP content was measured by thebicinchoninic acid protein assay (Pierce, Rockford, Ill.).SDS-PAGE and Western blotting of the OMP. The crude

OMP extracts were treated by 2-mercaptoethanol and werestudied by sodium dodecyl sulfate-polyacrylamide gel electro-phoresis (SDS-PAGE) (5 to 15% gradient) according to themethod of Laemmli (9); 40 ,ug of protein was loaded in eachlane. The low-molecular-weight calibration kit (Pharmacia-LKB, Uppsala, Sweden) was used as a protein standard.Proteins were stained with Coomassie blue R250. Part of thegel was submitted to semidry electrotransfer on a nitrocellu-lose sheet (Amersham, Arlington Heights, Ill.) with a Mul-tiphor II NovaBlot unit (Pharmacia-LKB) according to themanufacturer's instructions. To check the efficiency of thetransfer, some blots were stained with amido black (24).

Preparation of human respiratory mucins and mucin glyco-peptides. Sputum from a patient (blood group 0) sufferingfrom chronic bronchitis was diluted with deionized water

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NONPILUS ADHESINS OF PSEUDOMONAS AERUGINOSA 1897

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FIG. 1. PAGE and blotting of OMP obtained from PAK-NP and1244-NP. OMP from PAK-NP (lanes 1 and 2) and 1244-NP (lanes 3and 4) were stained by Coomassie blue directly on the gel (lanes 1 and3) or by amido black after blotting (lanes 2 and 4). Each lane contained40 pLg of protein. In all figures, the unlabeled lane contained molecularmass markers.

(1:12) and stirred overnight. Then the suspension was centri-fuged; the supernatant was dialyzed against deionized waterand lyophilized. The preparation of high-molecular-mass mu-

cins was obtained by CsBr density gradient centrifugation (7).This preparation was further purified by a second step of CsBrdensity 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. Radioiodinationwas performed with lodo-beads (Pierce) according to themanufacturer's instructions. Ten milligrams of human tracheo-bronchial mucins dissolved in 1 ml of phosphate buffer (75 mMNa2HPO4-KH2PO4, 75 mM NaCl, 0.02Cc [wt/vol] NaN3 [pH7.2]) was iodinated with 750 ,uCi of 1251 (Amersham) by theaddition of two lodo-beads. The iodination mixture was main-tained for 15 min at room temperature with stirring. Thereaction was terminated by transferring the solution to a tubecontaining sodium iodide (final concentration, 0.5 M). Radio-labeled mucins were separated from residual iodide by gelfiltration on a Sepharose CL-4B column (40 by 1 cm) equili-brated with PBS. Fractions containing the iodinated sampleswere pooled and stored at 4°C. An average specific activity of16 x 10" cpm/mg of mucins was obtained. In some experi-ments, desialylated radiolabeled mucins were used. For thispurpose, radiolabeled mucins were treated twice by neuramin-idase from Clostridium perfringens (Sigma) (0.03 U per 10 mgof mucin for 24 h).The radioiodination of human milk lactotransferrin (a gift

from G. Spik, Villeneuve d'Ascq, France), 4 mg in I ml ofphosphate buffer, was performed according to the same pro-

cedure.Detection of mucin-binding OMP. For the detection of

OMP binding to mucin or lactotransferrin, nitrocellulose rep-licas 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. TheBSA solution was replaced by radiolabeled mucins at a con-

1 2 3 4FIG. 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) wereseparated by PAGE and stained with Coomassie blue (lanes 1 and 3)or blotted and revealed with '25I-labeled mucins after blocking withBSA (lanes 2 and 4). Each lane contained 40 ,ug of protein. The arrowsindicate 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 hat room temperature. The blots were washed with PBS (fourtimes for 15 min each) and then 0.05% (vol/vol) Tween 20(Merck, Munich, Germany) in PBS (four times for 15 mineach), dried on filter paper, and then exposed at - 70°C toX-ray film (Hyperfilm-MP; Amersham).

In some experiments, blots were treated with 0.5 M tetra-methylurea (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 unlabeledrespiratory mucin glycopeptides (10 mg/ml) for 16 h at 4°C andthen they were treated as described previously. Controls wereincubated 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) wasgrown in TSB medium at 37°C for 24 h. Bacteria were sampledat 16 and 24 h, and then OMP were prepared and subsequentlyanalyzed by PAGE. With time, there was a progressive in-crease of recovered OMP. The major bands were mainlylocalized in two areas, at 40 to 50 kDa and at about 15 to 30kDa. A large number of bands was already present at 16 h, andin order to avoid possible OMP degradation, bacteria werecultured for 16 h in most experiments (Fig. 1).

Binding assay. OMP from PAK-NP and 1244-NP wereseparated by PAGE before being transferred to nitrocellulosesheets. As shown in Fig. 1, the OMP were homogeneouslytransferred to nitrocellulose sheets since the protein profiles ofthe blots stained with amido black were identical to the profilesobserved with polyacrylamide gels after staining with Coo-massie blue. There was no difference in the number or intensityof the different bands, indicating that the transfer was quanti-tative.The blocking of nonspecific binding sites on the nitrocellu-

lose with Tween was also studied by treating different blots

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kDa

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FIG. 3. OMP adhesins from P. aeruginosa 1244-NP and 1244-N3.OMP from 1244-NP (lanes 1 and 2) and 1244-N3 (lanes 3 and 4) wereseparated by PAGE and stained with Coomassie blue (lanes 1 and 3)or blotted and revealed with '25I-labeled mucins after blocking withBSA (lanes 2 and 4). Each lane contained 40 Lg of protein. The arrowsindicate the bands at 48, 46, 42, 28, and 25 kDa.

with increasing concentrations of Tween 20 (0.05, 0.2, and0.5%). It appeared that Tween 20, even at a low concentration(0.05%), could elute low-molecular-mass proteins from thenitrocellulose sheet. We therefore decided to replace Tween20 by 2% BSA in the blocking process.

Mucin-binding properties of OMP from the nonpiliatedstrains PAK-NP and 1244-NP. Four main bands were detectedat 48, 46, 28, and 25 kDa by radiolabeled mucins with OMPfrom strain PAK-NP (Fig. 2). Minor bands were also observedin the background at 42 and 35 kDa and below 25 kDa. Fivebands were observed at 48, 46, 42, 28, and 25 kDa with theOMP from 1244-NP. Minor bands were also observed in thebackground below 25 kDa (Fig. 3). The bands at 25 and 28 kDadisappeared after blocking of the nitrocellulose with Tween 20(data not shown).

Mucin-binding properties of OMP from poorly adhesiverpoN mutants of strains PAK-NP and 1244-NP. OMP from thepoorly adhesive mutant PAK-Nl were compared with OMPfrom PAK-NP for their mucin-binding properties (Fig. 2):there were only very faint bands at about 48 kDa and 25 to 28kDa. Equally, OMP from the poorly adhesive mutant 1244-N3were compared with OMP from 1244-NP for their bindingproperties (Fig. 3): there were only very faint bands at 25 to 28kDa and below 25 kDa.

Specificity of the adhesins. In order to check the specificityof the labeling, different experiments were performed. Aftercontact with radiolabeled mucins, some blots were treated for16 h with solutions of cold mucin glycopeptides. Comparedwith those of controls, the bands were largely attenuated aftercontact 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 hydropho-bic nonspecific protein-mucin interaction (21); therefore, someblots corresponding to OMP from strains 1244-NP andPAK-NP were treated with 0.5 M tetramethylurea after incu-bation with radiolabeled mucins. No modification of thelabeling could be observed (data not shown).

Since sialic acid has been suggested to be involved in thebinding of piliated strains of P. aeruginosa to mucins (18), it

ae . > 4; i S..0:ftj,AF * t'C'"'S

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1 2 3 4FIG. 4. Binding of radiolabeled human respiratory mucin to OMP

from P. aeruginosa PAK-NP and 1244-NP with and without subsequentdisplacement by unlabeled mucins. Blots of OMP from P. aeruginosaPAK-NP (lanes 1 and 3) and 1244-NP (lanes 2 and 4) were treated withradiolabeled respiratory mucins with (lanes 3 and 4) and without (lanes1 and 2) subsequent displacement by unlabeled respiratory mucinglycopeptides. Each lane contained 40 pLg of protein.

was interesting to check whether this was also the case fornonpiliated strains. Therefore, the binding to OMP was ana-lyzed after desialylation of radiolabeled human respiratorymucins with the neuraminidase of C. perfringens. The bandsobserved in the OMP preparations were not decreased butprobably enhanced after desialylation of human respiratorymucins (data not shown).

Lactotransferrin-binding properties of OMP from the non-piliated strains PAK-NP and 1244-NP. OMP from the PAKand 1244 mutants were also studied for lactotransferrin bind-ing since this glycoprotein is found in respiratory mucus. OMP

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aeruginosa PAK-NP and 1244-NP. Blots were obtained after PAGE ofOMP extracted from PAK-NP (lane 1) and from 1244-NP (lane 2).Autoradiography was performed after incubation with radiolabeledlactotransferrin.

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NONPILUS ADHESINS OF PSEUDOMONAS AERUGINOSA 1899

from strain PAK-NP contained three bands, at 48, 25, and 22kDa, with an affinity for radiolabeled lactotransferrin (Fig. 5).Bands at 48 and 25 kDa were also visualized with radiolabeledmucins (Fig. 2). With strain 1244-NP, OMP contained twobands, at 48 and 25 kDa, with an affinity for both radiolabeledlactotransferrin (Fig. 5) and radiolabeled mucin (Fig. 3) andone band at 22 kDa, only recognized by radiolabeled lacto-transferrin (Fig. 5). After contact with radiolabeled lactotrans-ferrin, some blots were treated for 16 h with solutions of coldmucin glycopeptides: the bands with molecular masses -25kDa were weakly attenuated (data not shown).

DISCUSSION

The present data demonstrate that two nonpiliated P. aerugi-nosa strains express slightly different OMP that bind to humanrespiratory mucins and/or lactotransferrin. Previous studieshave demonstrated the adhesion of various strains of P.aeruginosa to human respiratory mucins (16, 18, 19) and theadhesion of nonpiliated P. aeruginosa strains to human salivarymucins (2). Sajjan et al. have suggested that the interactionbetween P. aeruginosa and human intestinal and respiratorymucins is nonspecific and is mediated by a hydrophobicinteraction (21). The present data clearly indicate that severalproteins of the outer membrane from P. aeruginosa interactwith mucins specifically and may be identified as adhesins: theyare competitively inhibited by glycopeptides and not blockedby tetramethylurea or albumin. Moreover, these data indicatethat the expression of these adhesins is correlated with theadhesive properties of the bacteria. OMP of the poorly adhe-sive strains PAK-Nl and 1244 N-3 react very weakly withradiolabeled mucins.Some protein bands recognize only mucins; this is the case

for the bands at 46 and 28 kDa obtained from PAK-NP (Fig.2). This is also the case for the bands at 46, 42, and 28 kDafrom 1244-NP (Fig. 3). The binding of radiolabeled mucins tothese different proteins is displaced, almost completely, byunlabeled mucin glycopeptides (Fig. 4), suggesting that carbo-hydrate epitopes are involved in these interactions. Since thecarbohydrate diversity of human respiratory mucins is verylarge (for a review, see reference 12), one may wonder whetherthese classes of proteins recognize identical carbohydrate sitesor, more probably, different sites. Some protein bands appearto recognize both mucins and lactotransferrin; this is the casefor the bands at 48 and 25 kDa from PAK-NP and 1244-NP(Fig. 2, 3, and 5). Although it is impossible to completelyeliminate the hypothesis of a superimposition of differentadhesins, there is a good possibility that some of these pro-teins, especially the band at 25 kDa, have an affinity for bothmucins and lactotransferrin. This raises the question of thesites that would be shared by mucins and lactotransferrin.Lactotransferrin is an N-linked glycoprotein (8, 23). Mucinsare mainly 0-linked glycoproteins (10). They contain a smallamount of mannose, and DNA sequencing of a few mucingenes has revealed very few possible sites of attachment forN-linked carbohydrate chains (for a review, see reference 10).In theory, therefore, the only possible structures that respira-tory mucins and lactotransferrin might have in common cor-respond to the periphery of the carbohydrate chains, especiallythe type 2 disaccharide units (Gal1-4GlcNAc) which havebeen shown to be possible sites of attachment for nonpiliatedP. aeruginosa strains (14, 20).

Reddy has recently described a P. aernginosa adhesin formucins with a molecular mass of 16 kDa (19). This adhesin wasnot observed in the present study. One of the reasons for thisapparent discrepancy is that the methods used to prepare

adhesins are different. The extraction protocol used in thepresent study is probably milder than that used by Reddy, whoperformed a sonication of the bacteria and may have releasedan internal lectin (19).Taken together, these data clearly demonstrate that differ-

ent strains of P. aenrginosa can express several proteins withdifferent binding properties to human mucus glycoproteins.OMP have been prepared in the presence of protease inhibi-tors, but we do not know if some of these mucin-binding bandsresult from posttranslational modifications or in vitro proteo-lytic degradation of a unique protein. Simpson et al. havesuggested that there are at least two classes of nonpilusadhesins that recognize mucins (22). It will therefore benecessary to purify and sequence these bands in order to findout whether they represent different forms of the same ad-hesin(s) or totally different proteins.We do not know if all these proteins are located on the

surfaces of the bacteria or if they are able to interact withglycoproteins in the lung environment in vivo. We also do notknow if such a diversity in the size of adhesins is also expressedin piliated and/or mucoid strains of P. aerlginosa or if, in suchstrains, these proteins are accessible for interactions withmucus glycoproteins. Nevertheless, the pathological implica-tions of such interactions between P. aerulginosa and mucusglycoproteins are potentially important in lung colonization,especially with regard to cystic fibrosis. Moreover, consideringthe importance of iron for the growth of P. aeniginiosa, thesignificance of an interaction between this bacterium andlactotransferrin will have to be elucidated.

ACKNOWLEDGMENTS

This work has been supported by the Association Francaise de Luttecontre la Mucoviscidose (France), the R6seau Regional d'etude desInteractions H6tes-Microorganismes (Centre Hospitalier et Universit6de Lille. Lille, France). and the Cystic Fibrosis Foundation (UnitedStates).

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