Structural basis for mannose recognition by a lectin from opportunistic bacteria Burkholderia cenocepacia

Abstract

Chronic colonization of the lungs by opportunist bacteria such as Pseudomonas aeruginosa and members of the Bcc (Burkholderia cepacia complex) is the major cause of morbidity and mortality among CF (cystic fibrosis) patients. PA-IIL (lecB gene), a soluble lectin from Ps. aeruginosa, has been the subject of much interest because of its very strong affinity for fucose. Orthologues have been identified in the opportunist bacteria Ralstonia solanacearum, Chromobacterium violaceum and Burkholderia of Bcc. The genome of the J2315 strain of B. cenocepacia, responsible for epidemia in CF centres, contains three genes that code for proteins with PA-IIL domains. The shortest gene was cloned in Escherichia coli and pure recombinant protein, BclA (B. cenocepacia lectin A), was obtained. The presence of native BclA in B. cenocepacia extracts was checked using a proteomic approach. The specificity of recombinant BclA was characterized using surface plasmon resonance showing a preference for mannosides and supported with glycan array experiments demonstrating a strict specificity for oligomannose-type N-glycan structures. The interaction thermodynamics of BclA with methyl alpha-D-mannoside demonstrates a dissociation constant (K(d)) of 2.75 x 10(-6) M. The X-ray crystal structure of the complex with methyl alpha-D-mannoside was determined at 1.7 A (1 A=0.1 nm) resolution. The lectin forms homodimers with one binding site per monomer, acting co-operatively with the second dimer site. Each monomer contains two Ca2+ ions and one sugar ligand. Despite strong sequence similarity, the differences between BclA and PA-IIL in their specificity, binding site and oligomerization mode indicate that the proteins should have different roles in the bacteria.

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

“…These results are consistent with the known dissociation constant of methyl α-D-mannoside and BC2L-A of Kd = 2.7 µM and the poor binding of L-fucose. 10 The assay was then evaluated by titration of BC2L-A (1.5 µM) in presence of the reporter ligand 3 (10 nM) with a dilution series of D-mannose (1, Figure 1B) After establishing the assay, we tested various derivatives of L-fucose and D-mannose for inhibition of BC2L-A ( Table 1). The common structural motif contained the 6-membered tetrahydropyran ring structure and all three calcium-coordinating secondary hydroxy groups were present.…”

“…10,19,20 In contrast to the tetramer forming LecB, BC2L-A forms a dimeric assembly of its 13.8 kDa monomers. 10 The carbohydrate specificity of BC2L-A was determined by glycan array analysis, which revealed a high specificity of this lectin for mannose-containing ligands. In contrast to the high affinity of LecB to L-fucose, BC2L-A shows a low affinity to L-fucose (IC50 = 2.3 mM).…”

“…8,9 lecB-like genes were also identified in several other Gram-negative bacteria such as Chromobacterium violaceum, Ralstonia solanacearum, as well as in B. cenocepacia. [10][11][12] B. cenocepacia has three lectins homologous to P. aeruginosa LecB: BclA (BCAM0186), BclB (BCAM0184) or BclC (BCAM0185), also called BC2L-A, BC2L-B and BC2L-C. BC2L-A contains only a LecB domain, whereas BC2L-B and BC2L-C have additional N-terminal domains. This additional domain is without homology to known domains in BC2L-B, and in BC2L-C it contains a tumor necrosis factor-fold domain.…”

A Burkholderia cenocepacia Orphan LuxR Homolog Is Involved in Quorum-Sensing Regulation

“…Lectins play a role in adhesion, biofilm formation, and host recognition (37). Of three recently identified lecB-like genes in B. cenocepacia (46), CepR2 regulates only the BclA gene. The members of the AraC family of regulators generally act as positive transcriptional regulators and have been shown to influence pathogenesis in a number of species, including Pseudomonas, Salmonella, Yersinia, and Vibrio species (27).…”

Glycodendrimers and Their Derivatives as Potential Therapeutic Agents

“…It is suggested, that LecB undergoes transient N-glycosylation that plays a role in the secretion mechanism. [15] The search for a putative binding partner led to the proporsal of another membrane protein OprF, that is non-specific, weakly cationselective porin protein. LecB may mediate the adhesion of P. aeruginosa cells to receptors, which are located on its surface and facilitate biofilm formation, promoting colonization of host tissues.…”