Biochemical characterization and ligand identification of Pseudomonas aeruginosa lectins and their orthologs in other pathogenic bacteria

Carbohydrates and their binding proteins (Lectins) play important role in all living organisms and the development of new analytical technologies in the past few years allowed us to partially decipher the significance of carbohydrates in biology. Moreover, the role of lectins in pathogenicity was pointed out in the last decade, revealing that lectins play a crucial role in bacterial infection through, for example, virulence and adherence to the host as well as in biofilm formation. In this work, various biophysical and structural techniques were employed to identify anti-biofilm agents for human-opportunistic, Gram-negative bacterium pathogens, such as Pseudomonas aeruginosa and Burkholderia cenocepacia, by targeting their lectins and studying the binding properties of lectin blockers upon binding on their desired target. Furthermore, a new lectin (PllA) from the entomopathogenic bacterium Photorhabdus luminescens was characterized; this resulted in a new tool for lectinology and biomedical research, as a probe for detecting α-galactoside-terminating glycoconjugates.Kohlenhydrate und dessen Bindeproteine (Lektine) spielen eine wichtige Rolle in allen Lebewesen. Durch die Entwicklung von neuen analytischen Verfahren wird die Bedeutsamkeit von Kohlenhydraten in der Biologie ersichtlich. Es zeigt sich, dass Lektine bei der Pathogenität durch Bakterien in Bezug auf die Virulenz, Haftfestigkeit und deren Eigenschaft zur Biofilmbildung eine wesentliche Rolle spielen. In dieser Arbeit wurden verschiedene biophysikalische und strukturelle Techniken angewendet, um Biofilm verhindernde Wirkstoffe gegen opportunistische Erreger, wie den Gram-negativen Bakterien Pseudomonas aeruginosa und Burkholderia cenocepacia, zu erforschen. Dazu wurden die Bindeeigenschaften der Lektinblocker an entsprechenden Zielen untersucht. Außerdem wurde ein neues Lektin (PIIA) vom entomopathogenen Bakterium Photorhabdus luminescens charakterisiert. Es bindet an α-Galactoside und kann zur Erkennung von Glykokonjugaten in der Lektinology und in der biomedizinischen Forschung eingesetzt werden

Photorhabdus luminescens lectin A (PllA) : A new probe for detecting α-galactoside-terminating glycoconjugates

Lectins play important roles in infections by pathogenic bacteria, for example, in host colonization, persistence, and biofilm formation. The Gram-negative entomopathogenic bacterium Photorhabdus luminescens symbiotically lives in insect-infecting Heterorhabditis nematodes and kills the insect host upon invasion by the nematode. The P. luminescens genome harbors the gene plu2096, coding for a novel lectin that we named PllA. We analyzed the binding properties of purified PllA with a glycan array and a binding assay in solution. Both assays revealed a strict specificity of PllA for -galactoside–terminating glycoconjugates. The crystal structures of apo PllA and complexes with three different ligands revealed the molecular basis for the strict specificity of this lectin. Furthermore, we found that a 90° twist in subunit orientation leads to a peculiar quaternary structure compared with that of its ortholog LecA from Pseudomonas aeruginosa.We also investigated the utility of PllA as a probe for detecting -galactosides. The -Gal epitope is present on wildtype pig cells and is the main reason for hyperacute organ rejection in pig to primate xenotransplantation. We noted that PllA specifically recognizes this epitope on the glycan array and demonstrated that PllA can be used as a fluorescent probe to detect this epitope on primary porcine cells in vitro. In summary, our biochemical and structural analyses of the P. luminescens lectin PllA have disclosed the structural basis for PllA’s high specificity for -galactoside–containing ligands, and we show that PllA can be used to visualize the -Gal epitope on porcine tissues

Photoswitchable Janus glycodendrimer micelles as multivalent inhibitors of LecA and LecB from Pseudomonas aeruginosa

The first example of the self-assembly and lectin binding properties of photoswitchable glycodendrimer micelles is reported. Light-addressable micelles were assembled from a library of 12 amphiphilic Janus glycodendrimers composed of variable carbohydrate head groups and hydrophobic tail groups linked to an azobenzene core. Spontaneous association in water gave cylindrical micelles with uniform size distribution as determined by dynamic light scattering (DLS) and small angle neutron scattering (SANS). Trans-cis photoisomerization of the azobenzene dendrimer core was used to probe the self-assembly behaviour and lectin binding properties of cylindrical micelles, revealing moderate-to-potent inhibition of lectins LecA and LecB from Pseudomonas aeruginosa

Development of a competitive binding assay for the Burkholderia cenocepacia lectin BC2L-A and structure activity relationship of natural and synthetic inhibitors

Burkholderia cenocepacia is an opportunistic Gram-negative pathogen and especially hazardous for cystic fibrosis patients. In analogy to its relative Pseudomonas aeruginosa, B. cenocepacia possess numerous lectins with roles in adhesion and biofilm formation. The LecB homolog BC2L-A is important for biofilm structure and morphology. Inhibitors of this D-mannose specific C-type lectin could be useful as tools in B. cenocepacia biofilm research and potentially as anti-biofilm compounds against chronic infections. Here, we report the development of a fluorescence polarization-based competitive binding assay and its application in an extensive structure–activity relationship study of inhibitors of BC2L-A. In contrast to its homolog LecB, BC2L-A is highly selective for D-mannose-based ligands with an absolute requirement of its hydroxyl group at C6. A strict diastereoselectivity was observed for (6S)-mannoheptose-derived ligands. Intriguingly, bioisosteric substitution or methylation of hydroxyl groups directly involved in the calcium-coordination resulted in loss of inhibition for the two homologous lectins BC2L-A and LecB.publishe

Photorhabdus luminescens lectin A (PllA) - a new probe for detecting α-galactoside-terminating glycoconjugates.

Lectins play important roles in infections by pathogenic bacteria, for example, in host colonization, persistence and biofilm formation. The Gram-negative entomopathogenic bacterium Photorhabdus luminescens symbiotically lives in insect-infecting Heterorhabditis nematodes and kills the insect host upon invasion by the nematode. The P. luminescens genome harbors the gene plu2096 coding for a novel lectin that we named PllA. We analyzed the binding properties of purified PllA with a glycan array and a binding assay in solution. Both assays revealed a strict specificity of PllA for alpha-galactoside-terminating glycoconjugates. The crystal structures of apo PllA and complexes with three different ligands revealed the molecular basis for the strict specificity of this lectin. Furthermore, we found that a 90 degree twist in subunit orientation leads to a peculiar quaternary structure compared with that of its ortholog LecA from Pseudomonas aeruginosa. We also investigated the utility of PllA as a probe for detecting alpha-galactosides. The alpha-Gal epitope is present on wild-type pig cells and the main reason for hyperacute organ rejection in pig to primate xenotransplantation. We noted that PllA specifically recognizes this epitope on the glycan array and demonstrated that PllA can be used as a fluorescent probe to detect this epitope on primary porcine cells in vitro. In summary, our biochemical and structural analyses of the P. luminescens lectin PllA have disclosed the structural basis for PllAs high specificity for alpha-galactoside-containing ligands, and we show that PllA can be used to visualize alpha-Gal epitope on porcine tissues