Pseudomonas aeruginosa lectin LecB is located in the outer membrane and is involved in biofilm formation Denis Tielker, 1 Stephanie Hacker, 2 Remy Loris, 3 Martin Strathmann, 4 Jost Wingender, 4 Susanne Wilhelm, 1 Frank Rosenau 1 and Karl-Erich Jaeger 1 Correspondence Karl-Erich Jaeger karl-erich.jaeger@fz-juelich.de 1 Institut fu ¨ r Molekulare Enzymtechnologie, Heinrich-Heine-Universita ¨t Du ¨ sseldorf, Forschungszentrum Juelich, D-52426 Juelich, Germany 2 Lehrstuhl fu ¨ r Biologie der Mikroorganismen, Ruhr-Universita ¨ t Bochum, D-44801 Bochum, Germany 3 Laboratorium voor Ultrastructuur, Vlaams Interuniversitair Instituut voor Biotechnologie and Vrije Universiteit Brussel, B-1050 Brussel, Belgium 4 Biofilm Centre, Abteilung Aquatische Mikrobiologie, Universita ¨ t Duisburg-Essen, D-47057 Duisburg, Germany Received 13 October 2004 Revised 1 February 2005 Accepted 3 February 2005 Pseudomonas aeruginosa is an opportunistic pathogen which causes a variety of diseases, including respiratory tract infections in patients suffering from cystic fibrosis. Therapeutic treatment of P. aeruginosa infections is still very difficult because the bacteria exhibit high intrinsic resistance against a variety of different antibiotics and, in addition, form stable biofilms, e.g. in the human lung. Several virulence factors are produced by P. aeruginosa, among them the two lectins LecA and LecB, which exert different cytotoxic effects on respiratory epithelial cells and presumably facilitate bacterial adhesion to the airway mucosa. Here, the physiology has been studied of the lectin LecB, which binds specifically to L-fucose. A LecB-deficient P. aeruginosa mutant was shown to be impaired in biofilm formation when compared with the wild-type strain, suggesting an important role for LecB in this process. This result prompted an investigation of the subcellular localization of LecB by cell fractionation and subsequent immunoblotting. The results show that LecB is abundantly present in the bacterial outer-membrane fraction. It is further demonstrated that LecB could be released specifically by treatment of the outer-membrane fraction with p-nitrophenyl a-L-fucose, whereas treatment with D-galactose had no effect. In contrast, a LecB protein carrying the mutation D104A, which results in a defective sugar-binding site, was no longer detectable in the membrane fraction, suggesting that LecB binds to specific carbohydrate ligands located at the bacterial cell surface. Staining of biofilm cells using fluorescently labelled LecB confirmed the presence of these ligands. INTRODUCTION Lectins represent a specific class of carbohydrate-binding proteins different from enzymes or antibodies (Barondes et al., 1988). They are found in a wide range of organisms including viruses, bacteria, plants and animals, and are believed to play an important role in cell–cell interactions (Gabius et al., 2002). Specific recognition of or attachment to target cells was demonstrated for the mannose-specific lectin FimH from Escherichia coli, which mediates adhesion between the bacterium and the urothelium (Beachey, 1981). Furthermore, lectins may have significant biotechnological and medical potential, as exemplified by the galactoside- specific mistletoe lectin, which is used on a large scale to support anti-cancer therapy (Beuth et al., 1995). Pseudomonas aeruginosa, an important opportunistic pathogen associated with chronic airway infections, synthe- sizes two lectins, LecA and LecB (formerly respectively named PA-IL and PA-IIL) (Gilboa-Garber, 1982). Strains of P. aeruginosa that produce high levels of these virulence factors exhibit an increased virulence potential (Gilboa- Garber & Garber, 1992). The lectins play a prominent role in human infections, as it was demonstrated that a P. Abbreviations: CF, cystic fibrosis; CLSM, confocal laser scanning microscopy; pNPC, p-nitrophenyl caproate; pNPF, p-nitrophenyl a-L- fucose. 0002-7701 G 2005 SGM Printed in Great Britain 1313 Microbiology (2005), 151, 1313–1323 DOI 10.1099/mic.0.27701-0