Interactions of Pseudomonas aeruginosa PA-IIL lectin with quail egg white glycoproteins

Pseudomonas aeruginosa produces several lectins, including the galactophilic PA-IL and the fucose- and mannose-binding PA-IIL. The great advantage of these two lectins is their stability in purified preparations. Following observations that pigeon egg white blocks Escherichia coli P-fimbriae and PA-IL, we examined the interactions of diverse avian egg white components with PA-IIL. This lectin may represent both mannose- and fucose-specific microbial adhesins. For comparison, Con A (which also binds mannose) and Ulex europaeus lectin (UEA-I, which binds fucose) were analyzed in parallel. The lectin interactions with chicken, quail, and pigeon egg whites and several purified chicken egg white glycoproteins were examined by a hemagglutination inhibition test and Western blotting. Both analyses showed that like Con A and unlike UEA-I, which was not sensitive to any of these three egg whites, PA-IIL most strongly reacted with the quail egg white. However, in contrast with Con A, its interactions with the chicken egg white components, excluding avidin, were very poor. The results of this study might indicate the possibility that some of the egg white components that interacted with the above two mannose-binding lectins (exhibiting individual heterogeneity) might be associated with the innate immunity against mannose-specific microbial or viral adhesion during the fowl embryonic period.     

The mink as an animal model for Pseudomonas aeruginosa adhesion: binding of the bacterial lectins (PA-IL and PA-IIL) to neoglycoproteins and to sections of pancreas and lung tissues from healthy mink

Lung infection with Pseudomonas aeruginosa, leading to chronic lung disease with impaired function, is the major course of morbidity and mortality among cystic fibrosis patients. The bacterium produces two lectins that bind to alpha-D-galactose (PA-IL) and L-fucose (PA-IIL), respectively, and lectin-carbohydrate interactions may be involved in microbial pathogenicity by creating bacterial adherence to epithelial and endothelial cells. An ideal animal model for P. aeruginosa infection has until now not been established, but the mink seems to be the only animal that has been reported to develop spontaneous P. aeruginosa infections in the airways. Since cystic fibrosis also severely may affect pancreatic function, we incubated sections from mink lungs and pancreas with a medium containing Pseudomonas lectins in order to detect in situ binding of the bacterial lectins. In the lungs, both lectins adhered to seromucinous glands located in the submucosa of the larger bronchi. Additionally, PA-IL reacted with the capillaries in the alveolar walls and with the small blood vessels forming the vasa vasorum around the larger vessels, while PA-IIL marked the goblet cells in the bronchial surface epithelium. In the pancreas, both lectins bound to the epithelium in the excretory ducts, and additionally, PA-IL strongly stained the pancreatic capillaries while PA-IIL staining was noticed in the apical part of acinar cells in the exocrine part of the gland while no lectin reaction could be recorded in the endocrine cells. Judging from the results in the present paper the mink should be considered a suitable model to study P. aeruginosa adherence.     

Structures of the lectins from Pseudomonas aeruginosa: insight into the molecular basis for host glycan recognition

The opportunistic human pathogen Psuedomonas aeruginosa produces two lectins in close association with virulence factors: PA-IL adn PA-IIL, which bind to galactose- and fucose/mannose-containing glycoconjugates, respectively. We review here the structural aspects of these lectins relative to their putative roles in host recognition, cell surface adhesion and biofilm formation.     

The hemagglutinating activities of Pseudomonas aeruginosa lectins PA-IL and PA-IIL exhibit opposite temperature profiles due to different receptor types

The two Pseudomonas aeruginosa lectins PA-IL and PA-IIL, which are very similar in subunit size, composition and properties, but differ in carbohydrate specificity, were shown to exhibit opposite temperature profiles in hemagglutination tests. The galactophilic PA-IL, which interacts with the erythrocyte I antigen (together with B or P system antigens), resembles Ii system-specific 'cold hemagglutinins' (including antibodies and lectins of animals and plants) in low (4 degrees C) temperature optimum, while the hemagglutination by the fucose- and mannose-binding PA-IIL (like that of antibodies and lectins which do not bind to these antigens) increases on raising the temperature from 4 to 37 degrees C and even to 42 degrees C. The preferential production of both P. aeruginosa lectins at 28 degrees C and their much stronger interaction with enzyme (protease or sialidase)-damaged cells, as well as the lower temperature optimum (4 degrees C) of PA-IL-binding to the host cells, may be associated with the saprophytic rather than parasitic designation of this bacterium.     

Production,properties and specificity of a new bacterial L-fucose- and D-arabinose-binding lectin of the plant aggressive pathogen Ralstonia solanacearum,and its comparison to related plant and microbial lectins

The worldwide distributed plant aggressive pathogen Ralstonia solanacearum, which causes lethal wilt in many agricultural crops, produces a potent L-fucose-binding lectin (RSL) exhibiting sugar specificity similar to that of PA-IIL of the human aggressive opportunistic pathogen Pseudomonas aeruginosa. Both lectins show L-fucose > L-galactose > D-arabinose > D-mannose specificity, but the affinities of RSL to these sugars are substantially lower. Unlike Ulex europaeus anti-H lectin, but like PA-IIL and Aleuria aurantia lectin (AAL), RSL agglutinates H-positive human erythrocytes regardless of their type, O, A, B, or AB, and animal erythrocytes (papain-treated ones more strongly than untreated ones). It also interacts with H and Lewis chains in the saliva of "secretors" and "nonsecretors." RSL purification is easier than that of PA-IIL since R. solanacearum extracts do not contain a galactophilic PA-IL-like activity. Mass spectrometry and 35 N-terminal amino acid sequencing enabled identification of the RSL protein (subunit approximately 9.9 kDa, approximately 90 amino acids) in the complete genome sequence of this bacterium. Despite the greater phylogenetic proximity of R. solanacearum to P. aeruginosa, and the presence of a PA-IIL-like gene in its genome, the RSL structure is not related to that of PA-IIL, but to that of the fucose-binding lectin of the mushroom (fungus) Aleuria aurantia, which like the two bacteria is a soil inhabitant.     

Interaction of Pseudomonas aeruginosa galactophilic lectin PA-IL with pigeon egg white glycoproteins

Pseudomonas aeruginosa produces a galactophilic lectin, PA-IL, that resembles P-fimbrial adhesins of uropathogenic Escherichia coli strains in binding to human P blood group antigens. We examined, in the present study, its interaction with pigeon egg white glycoproteins carrying N-glycans with terminal Galalpha1-4Gal which inhibit the adhesion of P-fimbriae. For comparison, the lectin concanavalin A (Con A) and additional avian egg whites (of hen and quail) were also examined. The results obtained in both hemagglutination inhibition and Western blot analyses showed that PA-IL, unlike Con A, preferentially reacted with the pigeon egg white glycoproteins. These results, which confirmed PA-IL similarity in sugar specificity to E. coli P-fimbriae, demonstrated the advantage of this purified lectin for representing P-type and additional galactophilic microbial adhesins unavailable in purified stable form, in Western blot analyses.     

Structural basis for oligosaccharide-mediated adhesion of Pseudomonas aeruginosa in the lungs of cystic fibrosis patients

Pseudomonas aeruginosa galactose- and fucose-binding lectins (PA-IL and PA-IIL) contribute to the virulence of this pathogenic bacterium, which is a major cause of morbidity and mortality in cystic fibrosis patients. The crystal structure of PA-IIL in complex with fucose reveals a tetrameric structure. Each monomer displays a nine-stranded, antiparallel b-sandwich arrangement and contains two close calcium cations that mediate the binding of fucose in a recognition mode unique among carbohydrate-protein interactions. Experimental binding studies, together with theoretical docking of fucose-containing oligosaccharides, are consistent with the assumption that antigens of the Lewis a (Le(a)) series may be the preferred ligands of this lectin. Precise knowledge of the lectin-binding site should allow a better design of new antibacterial-adhesion prophylactics.     

Preventing Ralstonia solanacearum adhesion with glycans from cashew, cocoa, coffee, pumpkin, and tomato seed extract

Ralstonia solanacearum wilts many plants, causing heavy agricultural losses. Its pathogenic strain ATCC 11696 produces 2 hemagglutinating lectins: RSL and RS-IIL. These lectins may bind to terminal l-fucose-, d-arabinose-, and d-mannose-bearing seedling xylem cell wall glycans, thus enabling pathogen adhesion to them, with devastating infection establishment. Blocking the active sites of these lectins with seed embryo-surrounding oligo- and poly-saccharides hampers binding of the lectins to the embryos. The current study shows that seeds of cashew, cocoa, coffee, pumpkin, and tomato contain low and high molecular mass glycans that block RSL and RS-IIL (like its homologous Pseudomonas aeruginosa PA-IIL lectin). The blocking of the pathogen lectins, which is attributable to the documented composition of the oligo- and poly-saccharides of these seeds, is similar to that observed with animal glycoproteins of avian egg whites (protecting their embryos from infections) and of milk and royal jelly, which likewise protect mammal and bee neonates, respectively. RSL was most strongly inhibited by cashew seed glycans, and RS-IIL by coffee seed glycans. Western blot analyses with these lectins instead of antibodies revealed the hitherto undescribed presence of lectin-binding glycoproteins in the coffee, pumpkin, tomato, and cashew (but not cocoa) seeds. The use of these lectins for unveiling potent embryo-protecting seed glycans might be helpful for seedling-bioprotection projects similar to those planned for animal protection against antibiotic-resistant infections.     

Interactions of the fucose-specific Pseudomonas aeruginosa lectin, PA-IIL, with mammalian glycoconjugates bearing polyvalent Lewis(a) and ABH blood group glycotopes

Pseudomonas aeruginosa Fuc > Man specific lectin, PA-IIL, is an important microbial agglutinin that might be involved in P. aeruginosa infections in humans. In order to delineate the structures of these lectin receptors, its detailed carbohydrate recognition profile was studied both by microtiter plate biotin/avidin-mediated enzyme-lectin-glycan binding assay (ELLSA) and by inhibition of the lectin-glycan interaction. Among 40 glycans tested for binding, PA-IIL reacted well with all human blood group ABH and Le(a)/Le(b) active glycoproteins (gps), but weakly or not at all with their precursor gps and N-linked gps. Among the sugar ligands tested by the inhibition assay, the Le(a) pentasaccharide lacto-N-fucopentaose II (LNFP II, Galbeta1-3[Fucalpha1-4]GlcNAcbeta1-3Galbeta1-4Glc) was the most potent one, being 10 and 38 times more active than the Le(x) pentasaccharide (LNFP III, Galbeta1-4 [Fucalpha1-3]GlcNAcbeta1-3Galbeta1-4Glc) and sialyl Le(x) (Neu5Acalpha2-3Galbeta1-4[Fucalpha1-3] GlcNAc), respectively. It was 120 times more active than Man, while Gal and GalNAc were inactive. The decreasing order of PA-IIL affinity for the oligosaccharides tested was: Le(a) pentaose > or = sialyl Le(a) tetraose > methyl alphaFuc > Fuc and Fucalpha1-2Gal (H disaccharide)>2'-fucosyllactose (H trisaccharide), Le(x) pentaose, Le(b) hexaose (LNDFH I) and gluco-analogue of Le(y) tetraose (LDFT)>H type I determinant (LNFP I)>Le(x) trisaccharide (Galbeta1-4[Fucalpha1-3]GlcNAc) > sialyl Le(x) trisaccharide > Man > Gal, GalNAc, and Glc (inactive). The results presented here, in accordance with the crystal 3D structural data, imply that the combining site of PA-IIL is a small cavity-type best fitting Fucalpha1- with a specific shallow groove subsite for the remainder part of the Le(a) saccharides, and that polyvalent glycotopes enhance the reactivity. The Fuc > Man Ralstonia solanacearum lectin RSL, which resembles PA-IIL in sugar specificity, differs from it in it's better fit to the B and A followed by H oligosaccharides vs. Fuc, whereas, the second R. solanacearum lectin RS-IIL (the structural homologue of PA-IIL) binds Man > Fuc. These results provide a valuable information on PA-IIL interactions with mammalian glycoforms and the possible spectrum of attachment sites for the homing of this aggressive bacterium onto the target molecules. Such information might be useful for the antiadhesive therapy of P. aeruginosa infections.     

Comparison of the Antimicrobial Adhesion Potential of Human Body Fluid Glycoconjugates Using Fucose-Binding Lectin (PA-IIL) of Pseudomonas aeruginosa and Ulex europaeus Lectin (UEA-I)

Pseudomonas aeruginosa produces a fucose-binding lectin (PA-IIL) which strongly binds to human cells. This lectin was shown to be highly sensitive to inhibition by fucose-bearing human milk glycoproteins. Since the glycans of these glycoproteins mimic human cell receptors, they may function as decoys in blocking lectin-dependent pathogen adhesion to the host cells. Human saliva and seminal fluid also contain such compounds, and body fluids of individuals who are “secretors” express additional fucosylated (alpha 1,2) residues. The latter are selectively detected by Ulex europaeus lectin UEA-I. The aim of the present research was to compare the PA-IIL and UEA-I interactions with human salivas and seminal fluids of “secretors” and “nonsecretors” with those obtained with the respective milks. Using hemagglutination inhibition and Western blot analyses, we showed that PA-IIL interactions with the saliva and seminal fluid glycoproteins were somewhat weaker than those obtained with the milk and that “nonsecretor” body fluids were not less efficient than those of “secretors” in PA-IIL blocking. UEA-I, which interacted only with the “secretors” glycoproteins, was most sensitive to those of the seminal fluids.     

A new Ralstonia solanacearum high-affinity mannose-binding lectin RS-IIL structurally resembling the Pseudomonas aeruginosa fucose-specific lectin PA-IIL

The plant pathogen Ralstonia solanacearum produces two lectins, each with different affinity to fucose. We described previously the properties and sequence of the first lectin, RSL (subunit M(r) 9.9 kDa), which is related to fungal lectins (Sudakevitz, D., Imberty, A., and Gilboa-Garber, N., 2002, J Biochem 132: 353-358). The present communication reports the discovery of the second one, RS-IIL (subunit M(r) 11.6 kDa), a tetrameric lectin, with high sequence similarity to the fucose-binding lectin PA-IIL of Pseudomonas aeruginosa. RS-IIL recognizes fucose but displays much higher affinity to mannose and fructose, which is opposite to the preference spectrum of PA-IIL. Determination of the crystal structure of RS-IIL complexed with a mannose derivative demonstrates a tetrameric structure very similar to the recently solved PA-IIL structure (Mitchell, E., et al., 2002, Nature Struct Biol 9: 918-921). Each monomer contains two close calcium cations that mediate the binding of the monosaccharide and explain the outstandingly high affinity to the monosaccharide ligand. The binding loop of the cations is fully conserved in RS-IIL and PA-IIL, whereas the preference for mannose versus fucose can be attributed to the change of a three-amino-acid sequence in the 'specificity loop'.     

The galactophilic lectin, LecA, contributes to biofilm development in Pseudomonas aeruginosa

LecA (PA-IL) is a cytotoxic lectin and adhesin produced by Pseudomonas aeruginosa which binds hydrophobic galactosides with high specificity and affinity. By using a lecA-egfp translation fusion and immunoblot analysis of the biofilm extracellular matrix, we show that lecA is expressed in biofilm-grown cells. In static biofilm assays on both polystyrene and stainless steel, biofilm depth and surface coverage was reduced by mutation of lecA and enhanced in the LecA-overproducing strain PAO-P47. Biofilm surface coverage by the parent strain, PAO-P47 but not the lecA mutant on steel coupons was also inhibited by growth in the presence of either isopropyl-beta-D-thiogalactoside (IPTG) or p-nitrophenyl-alpha-D-galactoside (NPG). Furthermore, mature wild-type biofilms formed in the absence of these hydrophobic galactosides could be dispersed by the addition of IPTG. In contrast, addition of p-nitrophenyl-alpha-L-fucose (NPF) which has a high affinity for the P. aeruginosa LecB (PA-IIL) lectin had no effect on biofilm formation or dispersal. Planktonic growth of P. aeruginosa PAO1 was unaffected by the presence of IPTG, NPG or NPF, nor was the strain able to utilize these sugars as carbon sources, suggesting that the observed effects on biofilm formation were due to the competitive inhibition of LecA-ligand binding. Similar results were also obtained for biofilms grown under dynamic flow conditions on steel coupons, suggesting that LecA contributes to P. aeruginosa biofilm architecture under different environmental conditions.     

PA-I lectin from Pseudomonas aeruginosa binds acyl homoserine lactones

The study analyses the binding affinities of Pseudomonas aeruginosa PA-I lectin (PA-IL) to three N-acyl homoserine lactones (AHSL), quorum sensing signal molecules responsible for cell-cell communication in bacteria. It shows that like some plant lectins, PA-IL has a dual function and, besides its carbohydrate-binding capacity, can accommodate AHLS. Formation of complexes between PA-IL and AHSL with acyl side chains composed of 4, 6 or 12 methyl groups is characterized by changes in the emissions of two incorporated fluorescent markers, TNS and IAEDANS, both derivatives of naphthalene sulfonic acid. PA-IL shows increasing affinities to lactones with longer aliphatic side chains. The values of the apparent dissociation constants (K(d)), which are similar to the previously determined K(d) for the adenine high affinity binding, and the similar effects of lactones and adenine on the TNS emission indicate one identical binding site for these ligands, which is suggested to represent the central cavity of the oligomeric molecule formed after the association of the four identical subunits of PA-IL. Intramolecular distances between the fluorescent markers and protein Trp residues are determined by fluorescence resonance energy transfer (FRET).     

Mitogenic activity of edible mushroom lectins

A special group of lectins were isolated from three popular Asian edible mushrooms: Volvariella volvacea, Pleurotus flabellatus and Hericium erinacium, and their mitogenic activities towards mouse T cells were compared to the extensively investigated Agaricus bisporus lectin (ABL) and the Jack bean lectin, Concanavalin A (Con A). Among the four mushroom lectins tested, V. volvacea lectin (VVL) exhibited strong mitogenic activity as demonstrated by 3H-thymidine incorporation, which was at least 10-fold more effective than that of Con A, and the other mushroom lectins did not exhibit any proliferative activity. Treatment with VVL and ABL resulted in activation of the protein tyrosine kinase, p56lck, and expression of early activation markers, CD69 and CD25, but only VVL induced intracellular calcium influx while ABL triggered cell death. The calcium influx was sensitive to calcium channel antagonists such as nifedipine and verapamil. The P. flabellatus lectin (PFL) and H. erinacium lectin (HEL) did not stimulate p56lck expression and cell proliferation. Neither of these lectins interfered with Con A-mediated lymphocyte proliferation, which further indicated that both PFL and HEL were non-mitogenic. Taken all results together, VVL induced mitogenesis through T cell receptors and the subsequent calcium signaling pathway.     

PA-II,the L-fucose and D-mannose binding lectin of Pseudomonas aeruginosa stimulates human peripheral lymphocytes and murine splenocytes

Pseudomonas aeruginosa lectin PA-II agglutinates human peripheral lymphocytes and stimulates mitogenesis (predominantly in T cells), like the plant lectins PHA and Con A. Murine splenocytes are also agglutinated and stimulated by PA-II as by Con A. Sialidase treatment of the human and murine cells enhances their agglutination and augments the stimulation of human lymphocytes at low PA-II concentrations. The PA-II agglutinating and mitogenic effects are specifically inhibited by L-fucose. The bacterial source and the specificity of PA-II for L-fucose are both rare features among the hitherto described mitogenic lectins. However, since this lectin also binds mannose, a mannose-bearing receptor might be involved in its mitogenicity.     

Structural basis of calcium and galactose recognition by the lectin PA-IL of Pseudomonas aeruginosa

The structure of the tetrameric Pseudomonas aeruginosa lectin I (PA-IL) in complex with galactose and calcium was determined at 1.6 A resolution, and the native protein was solved at 2.4 A resolution. Each monomer adopts a beta-sandwich fold with ligand binding site at the apex. All galactose hydroxyl groups, except O1, are involved in a hydrogen bond network with the protein and O3 and O4 also participate in the co-ordination of the calcium ion. The stereochemistry of calcium galactose binding is reminiscent of that observed in some animal C-type lectins. The structure of the complex provides a framework for future design of anti-bacterial compounds.     

Structural basis of the preferential binding for globo-series glycosphingolipids displayed by Pseudomonas aeruginosa lectin I

The opportunistic pathogen Pseudomonas aeruginosa contains several carbohydrate-binding proteins, among which is the P. aeruginosa lectin I (PA-IL), which displays affinity for alpha-galactosylated glycans. Glycan arrays were screened and demonstrated stronger binding of PA-IL toward alphaGal1-4betaGal-terminating structures and weaker binding to alphaGal1-3betaGal ones in order to determine which human glycoconjugates could play a role in the carbohydrate-mediated adhesion of the bacteria. This was confirmed in vivo by testing the binding of the lectin to Burkitt lymphoma cells that present large amounts of globotriaosylceramide antigen Gb3/CD77/P(k). Trisaccharide moieties of Gb3 (alphaGal1-4betaGal1-4Glc) and isoglobotriaosylceramide (alphaGal1-3betaGal1-4Glc) were tested by titration microcalorimetry, and both displayed similar affinity to PA-IL in solution. The crystal structure of PA-IL complexed to alphaGal1-3betaGal1-4Glc trisaccharide has been solved at 1.9-A resolution and revealed how the second galactose residue makes specific contacts with the protein surface. Molecular modeling studies were performed in order to compare the binding mode of PA-IL toward alphaGal1-3Gal with that toward alphaGal1-4Gal. Docking studies demonstrated that alphaGal1-4Gal creates another network of contacts for achieving a very similar affinity, and 10-ns molecular dynamics in explicit water allowed for analyzing the flexibility of each disaccharide ligand in the protein binding site. The higher affinity observed for binding to Gb3 epitope, both in vivo and on glycan array, is likely related to the presentation effect of the oligosaccharide on a surface, since only the Gb3 glycosphingolipid geometry is fully compatible with parallel insertion of neighboring trisaccharide heads in two binding sites of the same tetramer of PA-IL.     

Analyses of Pseudomonas aeruginosa Lectin Binding to α-Galactosylated Glycans

The specificity and binding capacity of the galactophilic lectin from the Gram negative bacterium Pseudomonas aeruginosa (PA-IL) was determined by solid phase measurements using galactosylated neoglycoproteins immobilized on microtiter plates. The bacterial lectin reacted with both short chain (monosaccharide) and long chain (pentasaccharide) glycoconjugates. Among the Galα1-XGal disaccharides, the highest affinity was observed towards the Galα1-3Gal structure. Raising the incubation temperature enhanced the lectin-polysaccharide agglutination, and it is suggested that binding to certain conformations of polysaccharides could vary between lectins with the same monocarbohydrate specificity and that this activity may, in part, be temperature dependent. Histochemical examination of lectin binding to different porcine tissues suggests a differential glycosylation of the carbohydrate antigens on endothelial cells in various parts of the vascular system. In the pancreas, PA-IL also adhered to the excretory ducts. These observations on PA-IL binding could be of importance both to determine infection foci in P. aeruginosa-mediated vacuities and to determine its role for pancreatic involvement in cystic fibrosis.