Heptabladed ß-propeller lectins PLL2 and PHL from Photorhabdus spp. recognize O-methylated sugars and influence the host immune system.

O-methylation is an unusual sugar modification with a function that is not fully understood. Given its occurrence and recognition by lectins involved in the immune response, methylated sugars were proposed to represent a conserved pathogen-associated molecular pattern. We describe the interaction of O-methylated saccharides with two ß-propeller lectins, the newly described PLL2 from the entomopathogenic bacterium Photorhabdus laumondii, and its homologue PHL from the related human pathogen Photorhabdus asymbiotica. The crystal structures of PLL2 and PHL revealed up to 10 out of 14 potential binding sites per protein subunit to be occupied with O-methylated structures. The avidity effect strengthens the interaction by 4 orders of magnitude. PLL2 and PHL also interfere with the early immune response by modulating the production of reactive oxygen species and phenoloxidase activity. Since bacteria from Photorhabdus spp. have a complex life cycle involving pathogenicity towards different hosts, the involvement of PLL2 and PHL might contribute to the pathogen overcoming insect and human immune system defences in the early stages of infection. DATABASES: Structural data are available in PDB database under the accession numbers 6RG2, 6RGG, 6RFZ, 6RG1, 6RGU, 6RGW, 6RGJ, and 6RGR.

Characterization of novel lectins from Burkholderia pseudomallei and Chromobacterium violaceum with seven-bladed ß-propeller fold.

Burkholderia pseudomallei and Chromobacterium violaceum are bacteria of tropical and subtropical soil and water that occasionally cause fatal infections in humans and animals. Microbial lectins mediate the adhesion of organisms to host cells, which is the first phase in the development of infection. Here we report the discovery of two novel lectins from the above-mentioned bacteria - BP39L and CV39L. The crystal structures revealed that the lectins possess a seven-bladed ß-propeller fold. Functional studies conducted on a series of oligo- and polysaccharides confirmed the preference of BP39L for mannosylated saccharides and CV39L for rather more complex polysaccharides with a monosaccharide preference for ß-l-fucose. The presented data indicate that the proteins belong to a currently unknown family of lectins.

Lectin PLL3, a Novel Monomeric Member of the Seven-Bladed ß-Propeller Lectin Family.

The Photorhabdus species is a Gram-negative bacteria of the family Morganellaceae that is known for its mutualistic relationship with Heterorhabditis nematodes and pathogenicity toward insects. This study is focused on the characterization of the recombinant lectin PLL3 with an origin in P. laumondii subsp. laumondii. PLL3 belongs to the PLL family of lectins with a seven-bladed ß-propeller fold. The binding properties of PLL3 were tested by hemagglutination assay, glycan array, isothermal titration calorimetry, and surface plasmon resonance, and its structure was determined by X-ray crystallography. Obtained data revealed that PLL3 binds similar carbohydrates to those that the other PLL family members bind, with some differences in the binding properties. PLL3 exhibited the highest affinity toward l-fucose and its derivatives but was also able to interact with O-methylated glycans and other ligands. Unlike the other members of this family, PLL3 was discovered to be a monomer, which might correspond to a weaker avidity effect compared to homologous lectins. Based on the similarity to the related lectins and their proposed biological function, PLL3 might accompany them during the interaction of P. laumondii with both the nematode partner and the insect host.

Synthesis of ß-d-galactopyranoside-Presenting Glycoclusters, Investigation of Their Interactions with Pseudomonas aeruginosa Lectin A (PA-IL) and Evaluation of Their Anti-Adhesion Potential.

Pseudomonas aeruginosa is an opportunistic human pathogen associated with cystic fibrosis. This bacterium produces, among other virulence factors, a soluble d-galactose-specific lectin PA-IL (LecA). PA-IL plays an important role in the adhesion to the host cells and is also cytotoxic. Therefore, this protein is an interesting therapeutic target, suitable for inhibition by carbohydrate-based compounds. In the current study, ß-d-galactopyranoside-containing tri- and tetravalent glycoclusters were synthesized. Methyl gallate and pentaerythritol equipped with propargyl groups were chosen as multivalent scaffolds and the galactoclusters were built from the above-mentioned cores by coupling ethylene or tetraethylene glycol-bridges and peracetylated propargyl ß-d-galactosides using 1,3-dipolar azide-alkyne cycloaddition. The interaction between galactoside derivatives and PA-IL was investigated by several biophysical methods, including hemagglutination inhibition assay, isothermal titration calorimetry, analytical ultracentrifugation, and surface plasmon resonance. Their ability to inhibit the adhesion of P. aeruginosa to bronchial cells was determined by ex vivo assay. The newly synthesized multivalent galactoclusters proved to be significantly better ligands than simple d-galactose for lectin PA-IL and as a result, two representatives of the dendrimers were able to decrease adhesion of P. aeruginosa to bronchial cells to approximately 32% and 42%, respectively. The results may provide an opportunity to develop anti-adhesion therapy for the treatment of P. aeruginosa infection.

Selectivity of original C-hexopyranosyl calix[4]arene conjugates towards lectins of different origin.

As a part of ongoing activities towards the design of ligands against pathogenic lectins, a synthesis of original α-C-galacto/α-C-manno/α-C-fucopyranosyl glycomimetics based on a calix[4]arene scaffold and their binding evaluation is described. The interactions of the glycomimetics with seven lectins of various origins were carried out using agglutination inhibition assays. The 1,3-alternate tetra-C-fucosylated ligand and its derivative having a tertBu group at the upper rim of the calix[4]arene scaffold were the most potent towards the AAL lectin family (RSL, AFL, AAL, AOL) and BC2L-C. As AFL and RSL originate from important human (Aspergillus fumigatus) and plant (Ralstonia solanacearum) pathogens, the inhibition potency of both leading structures was assessed by surface plasmon resonance. With AFL, both structures exhibited an approximately three orders of magnitude increase in affinity compared to the reference l-fucose. The role of tertBu groups as "aglycon-assisted" events was illustrated by NMR. Furthermore, both compounds showed significantly increased ability to inhibit BC2L-C (from human pathogen Burkholderia cenocepacia) cell agglutination and were able to cross-link whole B. cenocepacia cells. Although the ligands failed to significantly inhibit the agglutination activity of LecA and LecB from Pseudomonas aeruginosa, tetra-C-galactosylated calix[4]arene with tertBu groups at the upper rim of the 1,3-alternate conformation inhibited P. aeruginosa biofilm formation efficiently. This systematic and comprehensive study highlights the fact that hydrolytically stable polyvalent C-glycomimetics should be regarded as potent and selective ligands capable of acting as antiadhesive agents.

Synthesis of α-l-Fucopyranoside-Presenting Glycoclusters and Investigation of Their Interaction with Photorhabdus asymbiotica Lectin (PHL).

Photorhabdus asymbiotica is a gram-negative bacterium that is not only as effective an insect pathogen as other members of the genus, but it also causes serious diseases in humans. The recently identified lectin PHL from P. asymbiotica verifiably modulates an immune response of humans and insects, which supports the idea that the lectin might play an important role in the host-pathogen interaction. Dimeric PHL contains up to seven l-fucose-specific binding sites per monomer, and in order to target multiple binding sites of PHL, α-l-fucoside-containing di-, tri- and tetravalent glycoclusters were synthesized. Methyl gallate and pentaerythritol were chosen as multivalent scaffolds, and the fucoclusters were built from the above-mentioned cores by coupling with different oligoethylene bridges and propargyl α-l-fucosides using 1,3-dipolar azide-alkyne cycloaddition. The interaction between fucoside derivates and PHL was investigated by several biophysical and biological methods, ITC and SPR measurements, hemagglutination inhibition assay, and an investigation of bacterial aggregation properties were carried out. Moreover, details of the interaction between PHL and propargyl α-l-fucoside as a monomer unit were revealed using X-ray crystallography. Besides this, the interaction with multivalent compounds was studied by NMR techniques. The newly synthesized multivalent fucoclusters proved to be up to several orders of magnitude better ligands than the natural ligand, l-fucose.