By P. aeruginosa via interaction with specific sugar 
molecules on the MedChemExpress (��)-Hexaconazole surface of blood cells [52]. Our results described above suggested that the deletion of the oprF gene may affect the hemagglutination activity of the respective P. aeruginosa mutant. To test this assumption, erythrocyte cells were incubated for 1 h with P. aeruginosa PAO1 wild type, DlecB and DoprF mutants and agglutination was optically examined (Fig. 4). As expected, P. aeruginosa wild-type cells showed strong hemagglutination activity, whereas mutant strains P. aeruginosa lecB as well as oprF caused a significantly decreased agglutination. Purified LecB served as a positive and PBS buffer as a negative control. The agglutination caused by LecB as well as by wild-type P. aeruginosa cells could be inhibited by the addition of 20 mM L-fucose to the sample buffer. These results indicated that an interaction of the porin OprF andLecB Binds OprF on the Cell Surface of P. aeruginosaApparently, LecB is cell surface exposed and interacts with OprF via carbohydrate ligands. We further investigated this interaction by growing the oprF-deficient P. aeruginosa mutant H636 which harboured plasmid pBBC2 containing the lecB gene as anLectin LecB Interacts with Porin OprFFigure 3. SubGSK -3203591 custom synthesis Cellular localization of LecB in biofilm cells of the oprF-deficient mutant P. aeruginosa H636 grown 
for 48 h at 37uC. A. The same amount of culture supernatant, periplasm, cytoplasm and total membrane were subjected to SDS-PAGE analysis followed by immunoblotting using LecB antiserum. Fractions obtained from P. aeruginosa wild-type (wt) served as a positive control. Fractionation controls: B. Cellular fractions were analyzed using antisera directed against EstA (an outer membrane protein) and DsbA-(a periplasmic protein) and C. by determination of relative glucose-6phosphate dehydrogenase (cytoplasmic protein) activities. The percentages of relative enzyme activities present in the cytoplasm (CP), the periplasm (PP), the membrane fraction (MF) and the culture supernatant (SN) are shown. doi:10.1371/journal.pone.0046857.gthe lectin LecB in the outer membrane is required to mediate agglutination of red blood cells caused by P. aeruginosa.DiscussionP. aeruginosa is the major pathogen in the respiratory tract of patients suffering from cystic fibrosis. The treatment of these chronic P. aeruginosa airway infections is difficult due to the innate and adaptive antibiotic resistance of P. aeruginosa and the formation of biofilms on the respiratory epithelium [44,53]. The lectin LecB and the major outer membrane porin OprF have both been shown to be involved in adhesion to lung epithelial cells [30,54]. Ourstudy now demonstrates that LecB is bound to the bacterial outer membrane and interacts with OprF. In an earlier study, we already showed that LecB is localized in the bacterial outer membrane of P. aeruginosa biofilm cells [23], but it remained unclear whether it faces the periplasm 12926553 or the exterior. In this study, we washed biofilm cells with L-fucose which binds LecB with a high affinity (KD = 1.56106 M21) [14] and we observed the release of LecB from the cell surface. Further analysis of membrane fractions by Far-Western-blotting using purified LecB detected several putative LecB ligands. These results already indicated that LecB specifically interacts with glycoproteins present in the bacterial membrane. The major outer membrane porin OprF was then co-purified from wild-type P. aeruginosa using affinity.By P. aeruginosa via interaction with specific sugar molecules on the surface of blood cells [52]. Our results described above suggested that the deletion of the oprF gene may affect the hemagglutination activity of the respective P. aeruginosa mutant. To test this assumption, erythrocyte cells were incubated for 1 h with P. aeruginosa PAO1 wild type, DlecB and DoprF mutants and agglutination was optically examined (Fig. 4). As expected, P. aeruginosa wild-type cells showed strong hemagglutination activity, whereas mutant strains P. aeruginosa lecB as well as oprF caused a significantly decreased agglutination. Purified LecB served as a positive and PBS buffer as a negative control. The agglutination caused by LecB as well as by wild-type P. aeruginosa cells could be inhibited by the addition of 20 mM L-fucose to the sample buffer. These results indicated that an interaction of the porin OprF andLecB Binds OprF on the Cell Surface of P. aeruginosaApparently, LecB is cell surface exposed and interacts with OprF via carbohydrate ligands. We further investigated this interaction by growing the oprF-deficient P. aeruginosa mutant H636 which harboured plasmid pBBC2 containing the lecB gene as anLectin LecB Interacts with Porin OprFFigure 3. Subcellular localization of LecB in biofilm cells of the oprF-deficient mutant P. aeruginosa H636 grown for 48 h at 37uC. A. The same amount of culture supernatant, periplasm, cytoplasm and total membrane were subjected to SDS-PAGE analysis followed by immunoblotting using LecB antiserum. Fractions obtained from P. aeruginosa wild-type (wt) served as a positive control. Fractionation controls: B. Cellular fractions were analyzed using antisera directed against EstA (an outer membrane protein) and DsbA-(a periplasmic protein) and C. by determination of relative glucose-6phosphate dehydrogenase (cytoplasmic protein) activities. The percentages of relative enzyme activities present in the cytoplasm (CP), the periplasm (PP), the membrane fraction (MF) and the culture supernatant (SN) are shown. doi:10.1371/journal.pone.0046857.gthe lectin LecB in the outer membrane is required to mediate agglutination of red blood cells caused by P. aeruginosa.DiscussionP. aeruginosa is the major pathogen in the respiratory tract of patients suffering from cystic fibrosis. The treatment of these chronic P. aeruginosa airway infections is difficult due to the innate and adaptive antibiotic resistance of P. aeruginosa and the formation of biofilms on the respiratory epithelium [44,53]. The lectin LecB and the major outer membrane porin OprF have both been shown to be involved in adhesion to lung epithelial cells [30,54]. Ourstudy now demonstrates that LecB is bound to the bacterial outer membrane and interacts with OprF. In an earlier study, we already showed that LecB is localized in the bacterial outer membrane of P. aeruginosa biofilm cells [23], but it remained unclear whether it faces the periplasm 12926553 or the exterior. In this study, we washed biofilm cells with L-fucose which binds LecB with a high affinity (KD = 1.56106 M21) [14] and we observed the release of LecB from the cell surface. Further analysis of membrane fractions by Far-Western-blotting using purified LecB detected several putative LecB ligands. These results already indicated that LecB specifically interacts with glycoproteins present in the bacterial membrane. The major outer membrane porin OprF was then co-purified from wild-type P. aeruginosa using affinity.
