Genomic Rearrangements and Functional Diversification of lecA and lecB Lectin-Coding Regions Impacting the Efficacy of Glycomimetics Directed against Pseudomonas aeruginosa

Abstract

LecA and LecB tetrameric lectins take part in oligosaccharide-mediated adhesion-processes of Pseudomonas aeruginosa. Glycomimetics have been designed to block these interactions. The great versatility of P. aeruginosa suggests that the range of application of these glycomimetics could be restricted to genotypes with particular lectin types. The likelihood of having genomic and genetic changes impacting LecA and LecB interactions with glycomimetics such as galactosylated and fucosylated calix[4]arene was investigated over a collection of strains from the main clades of P. aeruginosa. Lectin types were defined, and their ligand specificities were inferred. These analyses showed a loss of lecA among the PA7 clade. Genomic changes impacting lec loci were thus assessed using strains of this clade, and by making comparisons with the PAO1 genome. The lecA regions were found challenged by phage attacks and PAGI-2 (genomic island) integrations. A prophage was linked to the loss of lecA. The lecB regions were found less impacted by such rearrangements but greater lecB than lecA genetic divergences were recorded. Sixteen combinations of LecA and LecB types were observed. Amino acid variations were mapped on PAO1 crystal structures. Most significant changes were observed on LecBPA7, and found close to the fucose binding site. Glycan array analyses were performed with purified LecBPA7. LecBPA7 was found less specific for fucosylated oligosaccharides than LecBPAO1, with a preference for H type 2 rather than type 1, and Lewisa rather than Lewisx. Comparison of the crystal structures of LecBPA7 and LecBPAO1 in complex with Lewisa showed these changes in specificity to have resulted from a modification of the water network between the lectin, galactose and GlcNAc residues. Incidence of these modifications on the interactions with calix[4]arene glycomimetics at the cell level was investigated. An aggregation test was used to establish the efficacy of these ligands. Great variations in the responses were observed. Glycomimetics directed against LecB yielded the highest numbers of aggregates for strains from all clades. The use of a PAO1ΔlecB strain confirmed a role of LecB in this aggregation phenotype. Fucosylated calix[4]arene showed the greatest potential for a use in the prevention of P. aeruginosa infections.

The Pseudomonas aeruginosa Lectin LecB Causes Integrin Internalization and Inhibits Epithelial Wound Healing

“…In addition, LecB is a tetramer with four fucose-binding sites, but UEA-1 is a dimer offering only two binding sites (35). It is also worth noting that the sequence of LecB varies slightly between different strains of P. aeruginosa (51,52). Since ligand binding among LecB variants is conserved, we expect that these LecB variants are utilized in similar ways as we reported here for LecB in the P. aeruginosa strain PAO1.…”

Pathoblockers or antivirulence drugs as a new option for the treatment of bacterial infections

“…LecB has been studied in detail using multivalent and small molecule approaches. Interestingly, the sequence of LecB differs among clinical isolates of this highly variable pathogen, with some mutations in close proximity to the carbohydrate binding site, but carbohydrate-binding function is preserved across all lectins investigated [ 42 – 43 ]. The glycopeptide dendrimer 13 ( Fig.…”

Conformations of the type-1 lacto-N-biose I unit in protein complex structures

“…structures of glycan-binding proteins (for example, lectins, adhesins, haemagglutinins and viral capsid proteins) from various organisms including humans and pathogens have been extensively studied and represent the majority of the LNBcontaining structures in the PDB. The pathogens include a fungus (or yeast, Candida glabrata; Diderrich et al, 2015), a nematode (Toxascaris leonina; Hwang et al, 2016), a protist (Toxoplasma gondii; Garnett et al, 2009), bacteria (Helicobacter pylori, Shiga toxin-producing Escherichia coli and Pseudomonas aeruginosa; Moonens et al, 2012Moonens et al, , 2016Boukerb et al, 2016) and viruses (influenza virus, norovirus, rotavirus and polyomavirus; Vachieri et al, 2014;Choi et al, 2008;Liu et al, 2017;Stehle & Harrison, 1997). Eight PDB entries for enzymes contain the -LNB unit as part of their substrate (discussed below).…”

“…Complex crystal structures of plant lectin IV from the African shrub Griffonia simplicifolia with Le b tetrasaccharide (Delbaere et al, 1993), microneme protein 1 from a protozoan parasite (T. gondii) with 3-SLN (Garnett et al, 2009), galectin Tl-gal from a canine gastrointestinal nematode parasite (T. leonina) with LNT (Hwang et al, 2016) and the adhesin Epa6A domain from a fungal pathogen (C. glabrata) with LNB (Diderrich et al, 2015) have been reported. For bacterial glycan-binding proteins and lectins, several crystal structures, including those of the adhesin BabA from H. pylori, the fimbrial adhesin subunit FedF from Shiga toxin-producing E. coli and the LecB lectin from P. aeruginosa, complexed with Lewis and type-1 ABO(H) blood group antigens have been reported (Moonens et al, 2012(Moonens et al, , 2016Boukerb et al, 2016). PDB entry 5f9d for BabA contains the whole Le b heptasaccharide (Fig.…”