Dvora Sudakevitz

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 (Lea) 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.

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 Å resolution, and the native protein was solved at 2.4 Å resolution. Each monomer adopts a β‐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.

PA-I and PA-II lectin interactions with the ABO-(H) and P blood group glycosphingolipid antigens may contribute to the broad spectrum adherence of Pseudomonas aeruginosa to human tissues in secondary infections

Pseudomonas aeruginosa may cause serious infections in most human tissues/organs. Its adherence to them is mediated by a battery of adhesins including the PA-I and PA-II lectins, which are produced in this bacterium in high quantities. PA-I binds to thed-galactose of the erythrocyte glycosphingolipids exhibiting highest affinities for B and Pk (followed by P1) antigens, while PA-II preferentially binds to thel-fucose of H, A and B antigens. IntactP. aeruginosa cells also exhibit a clear Pk and P1 over p preference. Such affinities for the most common human ABH and P system antigens may underlie the widespread tissue infectivity and pathogenicity of this bacterium.

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 Mr 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 Mr 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’.

Purified Pseudomonas aeruginosa PA-I lectin induces gut growth when orally ingested by rats

The effects of PA-I lectin isolated from the human pathogen Pseudomonas aeruginosa upon cellular metabolism in vivo have been studied using the rat gut as a model system. Orally ingested PA-I lectin stimulated metabolic activity and induced polyamine accumulation and growth in the small intestine, caecum and colon. The nature and extent of the changes induced by PA-I lectin were similar to those caused by dietary kidney bean lectin and were likely to lead to impaired epithelial cell function and integrity. This finding contributes to our understanding of the possible roles of these lectins in Pseudomonas aeruginosa infection.

Blocking of Pseudomonas aeruginosa and Chromobacterium violaceum lectins by diverse mammalian milks.

Pseudomonas aeruginosa and Chromobacterium violaceum morbid and mortal infections are initiated by bacterial adherence to host-cell receptors via their adhesins, including lectins (which also contribute to bacterial biofilm formation). Pseudomonas aeruginosa produces a galactophilic lectin, PA-IL (LecA), and a fucophilic (Lewis-specific) lectin, PA-IIL (LecB), and C. violaceum produces a fucophilic (H-specific) lectin, CV-IIL. The antibiotic resistance of these bacteria prompted the search for glycosylated receptor-mimicking compounds that would function as glycodecoys for blocking lectin attachment to human cell receptors. Lectins PA-IL and PA-IIL have been shown to be useful for such glycodecoy probing, clearly differentiating between human and cow milks. This article describes their usage, together with CV-IIL and the plant lectin concanavalin A, for comparing the anti-lectin-dependent adhesion potential of diverse mammalian milks. The results show that the diverse milks differ in blocking (hemagglutination inhibition) and differential binding (Western blots) of these lectins. Human milk most strongly inhibited the 3 bacterial lectins (with PA-IIL superiority), followed by alpaca, giraffe, and monkey milks, whereas cow milk was a weak inhibitor. Lectin PA-IL was inhibited strongly by human, followed by alpaca, mare, giraffe, buffalo, and monkey milks, weakly by camel milk, and not at all by rabbit milk. Lectins PA-IIL and CV-IIL were also most sensitive to human milk, followed by alpaca, monkey, giraffe, rabbit, and camel milks but negligibly sensitive to buffalo and mare milks. Plant lectin concanavalinA, which was used as the reference, differed from them in that it was much less sensitive to human milk and was equally as sensitive to cow milk. These results have provided important information on the anti-lectin-dependent adhesion potential of the diverse milks examined. They showed that human followed by alpaca, giraffe, and Rhesus monkey milks efficiently blocked the binding of both the galactophilic and fucophilic (>mannophilic) pathogen lectins. The results also proved the advantage of isolated pathogenic bacterial lectins as superb probes for unveiling bacterial adhesion-blocking glycodecoys. The chosen milks or their polymeric glycans might be implicated in blocking lectin-dependent adhesion of antibiotic-resistant pathogens leading to skin, eye, ear, and gastrointestinal infections.

Multivalent human blood group ABH and Lewis glycotopes are key recognition factors for a LFuc>Man binding lectin from phytopathogenic Ralstonia solanacearum.

Ralstonia solanacearum lectin (RSL), that might be involved in phytopathogenicity, has been defined as LFuc>>Man specific. However, the effects of polyvalency of glycotopes and mammalian structural units on binding have not been established. In this study, recognition factors of RSL were comprehensively examined with natural multivalent glycotopes and monomeric ligands using enzyme linked lectin-sorbent and inhibition assays. Among the glycans tested, RSL reacted strongly with multivalent blood group A(h) (GalNAcalpha1-3[Fucalpha1-2]Gal) and H (Fucalpha1-2Gal) active glycotopes, followed by B(h) (Galalpha1-3[Fucalpha1-2]Gal), Le(a) (Galbeta1-3[Fucalpha1-4]GlcNAc) and Le(b) (Fucalpha1-2Galbeta1-3[Fucalpha1-4]GlcNAc) active glycotopes. But weak or negligible binding was observed for blood group precursors having Galbeta1-3/4GlcNAcbeta1- (Ibeta/IIbeta) residues or Galbeta1-3GalNAcalpha1- (Talpha), GalNAcalpha1-Ser/Thr (Tn) bearing glycoproteins. These results indicate that the density and degree of exposure of multivalent ligands of alpha1-2 linked LFuc to Gal at the non-reducing end is the most critical factor for binding. An inhibition study with monomeric ligands revealed that the combining site of RSL should be of a groove type to fit trisaccharide binding with highest complementarity to blood group H trisaccharide (H(L); Fucalpha1-2Galbeta1-4Glc). The outstandingly broad RSL saccharide-binding profile might be related to the unusually wide spectrum of plants that suffer from R. solanacearum pathogenicity and provide ideas for protective antiadhesion strategies.

The Five Bacterial Lectins (PA-IL, PA-IIL, RSL, RS-IIL, and CV-IIL): Interactions with Diverse Animal Cells and Glycoproteins

Among the ten different lectins discovered in the old biochemistry laboratory at Bar-Ilan University during the years 1972–2006 (Fig. 9.1), five were isolated from three soil bacteria: Pseudomonas aeruginosa (PA) [1–3], Ralstonia solanacearum (RS) [4, ], and Chromobacterium violaceum (CV) [6].

Relative intensities of recognition factors at two combining sites of Ralstonia solanacearum lectin (RSL) for accommodating LFucα1-->, DManα1--> and Galβ1-->3/4GlcNAc glycotopes.

Owing to the weak reactivities of monomeric dManα1 and Galβ1→3/4GlcNAcβ (I β/II β) glycotopes with Ralstonia solanacearum lectin (RSL), their recognition roles were previously ignored. In this study, the interaction intensities of RSL toward four monomeric glycotopes lFucα1→, dManα1→ and I β/II β within two combining sites were established by both enzyme‐linked lectinosorbent and inhibition assays. It was found that high density of lFucα1→ complex enhanced the recognition intensities at lFucα1→ site, polyvalent dManα1→ was essential for binding at the dManα1→ site and polyvalent I β/II β was required at lFucα1→ site. The peculiar recognition systems of RSL are very different from other well known microbial lectins.

Regulation of Lectin Production by the Human Pathogens Pseudomonas aeruginosa and Chromobacterium violaceum : Effects of Choline, Trehalose, and Ethanol

The worldwide-distributed Pseudomonas aeruginosa (PA) and the geographically restricted (confined to tropical and subtropical zones) Ralstonia solanacearum and Chromobacterium violaceum are Gram-negative proteobacteria that dwell in soil and water. They are essentially beneficial saprophytes that vigorously decompose plant and animal remnants and organic debris, contributing to world carbon and nitrogen cycling (Fig. 11.1). In accordance with their distinguished role in nature, these bacteria are endowed with very prosperous arsenals of cell-binding adhesins, toxicating proteinaceous and nonproteinaceous factors, and hydrolytic enzymes as virulence factors (VIFs), enabling them to home in on dead or damaged cells and molecules and attack them.