The 1.9 Å crystal structure of the extracellular matrix protein Bap1 from Vibrio cholerae provides insights into bacterial biofilm adhesion.

Growth of the cholera bacterium Vibrio cholerae in a biofilm community contributes to both its pathogenicity and survival in aquatic environmental niches. The major components of V. cholerae biofilms include Vibriopolysaccharide (VPS) and the extracellular matrix proteins RbmA, RbmC, and Bap1. To further elucidate the previously observed overlapping roles of Bap1 and RbmC in biofilm architecture and surface attachment, here we investigated the structural and functional properties of Bap1. Soluble expression of Bap1 was possible only after the removal of an internal 57-amino-acid-long hydrophobic insertion sequence. The crystal structure of Bap1 at 1.9 Å resolution revealed a two-domain assembly made up of an eight-bladed ß-propeller interrupted by a ß-prism domain. The structure also revealed metal-binding sites within canonical calcium blade motifs, which appear to have structural rather than functional roles. Contrary to results previously observed with RbmC, the Bap1 ß-prism domain did not exhibit affinity for complex N-glycans, suggesting an altered role of this domain in biofilm-surface adhesion. Native polyacrylamide gel shift analysis did suggest that Bap1 exhibits lectin activity with a preference for anionic or linear polysaccharides. Our results suggest a model for V. cholerae biofilms in which Bap1 and RbmC play dominant but differing adhesive roles in biofilms, allowing bacterial attachment to diverse environmental or host surfaces.

Structural basis of mammalian glycan targeting by Vibrio cholerae cytolysin and biofilm proteins.

Vibrio cholerae is an aquatic gram-negative microbe responsible for cholera, a pandemic disease causing life-threatening diarrheal outbreaks in populations with limited access to health care. Like most pathogenic bacteria, V. cholerae secretes virulence factors to assist colonization of human hosts, several of which bind carbohydrate receptors found on cell-surfaces. Understanding how pathogenic virulence proteins specifically target host cells is important for the development of treatment strategies to fight bacterial infections. Vibrio cholerae cytolysin (VCC) is a secreted pore-forming toxin with a carboxy-terminal ß-prism domain that targets complex N-glycans found on mammalian cell-surface proteins. To investigate glycan selectivity, we studied the VCC ß-prism domain and two additional ß-prism domains found within the V. cholerae biofilm matrix protein RbmC. We show that the two RbmC ß-prism domains target a similar repertoire of complex N-glycan receptors as VCC and find through binding and modeling studies that a branched pentasaccharide core (GlcNAc2-Man3) represents the likely footprint interacting with these domains. To understand the structural basis of V. cholerae ß-prism selectivity, we solved high-resolution crystal structures of fragments of the pentasaccharide core bound to one RbmC ß-prism domain and conducted mutagenesis experiments on the VCC toxin. Our results highlight a common strategy for cell-targeting utilized by both toxin and biofilm matrix proteins in Vibrio cholerae and provide a structural framework for understanding the specificity for individual receptors. Our results suggest that a common strategy for disrupting carbohydrate interactions could affect multiple virulence factors produced by V. cholerae, as well as similar ß-prism domains found in other vibrio pathogens.

NMR structure of the Bacillus cereus hemolysin II C-terminal domain reveals a novel fold.

In addition to multiple virulence factors, Bacillus cereus a pathogen that causes food poisoning and life-threatening wound infections, secretes the pore-forming toxin hemolysin II (HlyII). The HlyII toxin has a unique 94 amino acid C-terminal domain (HlyIIC). HlyIIC exhibits splitting of NMR resonances due to cis/trans isomerization of a single proline near the C-terminus. To overcome heterogeneity, we solved the structure of P405M-HlyIIC, a mutant that exclusively stabilizes the trans state. The NMR structure of HlyIIC reveals a novel fold, consisting of two subdomains αA-ß1-ß2 and ß3-ß4-αB-ß5, that come together in a barrel-like structure. The barrel core is fastened by three layers of hydrophobic residues. The barrel end opposite the HlyIIC-core has a positively charged surface, that by binding negatively charged moieties on cellular membranes, may play a role in target-cell surface recognition or stabilization of the heptameric pore complex. In the WT domain, dynamic flexibility occurs at the N-terminus and the first α-helix that connects the HlyIIC domain to the HlyII-core structure. In the destabilizing P405M mutant, increased flexibility is evident throughout the first subdomain, suggesting that the HlyIIC structure may have arisen through gene fusion.

Glycan specificity of the Vibrio vulnificus hemolysin lectin outlines evolutionary history of membrane targeting by a toxin family.

Pore-forming toxins (PFTs) are a class of pathogen-secreted molecules that oligomerize to form transmembrane channels in cellular membranes. Determining the mechanism for how PFTs bind membranes is important in understanding their role in disease and for developing possible ways to block their action. Vibrio vulnificus, an aquatic pathogen responsible for severe food poisoning and septicemia in humans, secretes a PFT called V. vulnificus hemolysin (VVH), which contains a single C-terminal targeting domain predicted to resemble a ß-trefoil lectin fold. In order to understand the selectivity of the lectin for glycan motifs, we expressed the isolated VVH ß-trefoil domain and used glycan-chip screening to identify that VVH displays a preference for terminal galactosyl groups including N-acetyl-d-galactosamine and N-acetyl-d-lactosamine. The X-ray crystal structure of the VVH lectin domain solved to 2.0Å resolution reveals a heptameric ring arrangement similar to the oligomeric form of the related, but inactive, lectin from Vibrio cholerae cytolysin. Structures bound to glycerol, N-acetyl-d-galactosamine, and N-acetyl-d-lactosamine outline a common and versatile mode of recognition allowing VVH to target a wide variety of cell-surface ligands. Sequence analysis in light of our structural and functional data suggests that VVH may represent an earlier step in the evolution of Vibrio PFTs.