Glycan Specificity of the Vibrio vulnificus Hemolysin Lectin Outlines Evolutionary History of Membrane Targeting by a Toxin Family

Abstract

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 key to understanding their role in disease and possible ways to block their action. Vibrio vulnificus, an aquatic pathogen responsible for severe food poisoning and septicemia in humans, secretes a PFT called Vibrio vulnificus hemolysin (VVH), which contains a single C-terminal targeting domain predicted to resemble a β-trefoil lectin fold. In order to understand the selectivity of the lectin for glycan motifs, we expressed the isolated VVH β-trefoil domain and used glycan-chip screening to identify that VVH displays a preference for terminal galactosyl groups including N-acetyl-D-galactosamine (GalNAc) and N-acetyl-D-lactosamine (LacNAc). The X-ray crystal structure of the VVH lectin domain solved to 2.0 Å 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, GalNAc, and LacNAc 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.

Phobalysin, a Small β-Pore-Forming Toxin of Photobacterium damselae subsp. damselae

“…In support of this, an amino acid substitution in the ␤-trefoil domain renders PhlyP substantially less hemolytic, but the activity is fully recovered by replacing the defective domain with the wild-type sequence (13). Notably, PhlyP's overall structure resembles that of Vibrio vulnificus cytolysin (VVC) in that it too lacks a C-terminal lectin domain, but the primary sequences of PhlyP and VCC are more closely related (57), underscoring the remarkable diversification of small ␤-PFTs. Loss of membrane integrity after attack by PhlyP was followed by a broad range of toxic effects in HaCaT cells, all of which might contribute to the severe consequences of infection by P. damselae subsp.…”

“…VCC contains two contiguous lectin domains: the so-called ␤-trefoil domain and the more C-terminal ␤-prism domain (55). Deletion of the ␤-prism domain virtually eliminated hemolytic activity (56), which led to the conclusion that the ␤-trefoil domain in VCC is inactive (57). Although devoid of the ␤-prism domain, PhlyP applied at nanomolar concentrations kills mammalian cells, suggesting that its trefoil domain is active.…”

SEDPHAT – A platform for global ITC analysis and global multi-method analysis of molecular interactions

“…Third, manual user-dependent interference in what is considered the experimental data should be avoided as much as possible to rule out bias, and this can be accomplished automatically and reliably with the peak shape analysis and truncated singular value decomposition algorithms in NITPIC [18]. First examples for the application of the NITPIC-SEDPHAT analysis strategy can be found in [35,38,48,53–62,66,78–80]. By letting the thermogram integration software NITPIC feed data into SEDPHAT, errors from the individual injections are honored and carried through to yield rigorous final parameter uncertainties in gITC.…”

The Relationship between Glycan Binding and Direct Membrane Interactions in Vibrio cholerae Cytolysin, a Channel-forming Toxin

“…The VCC ␤-prism domain targets complex N-glycans on host cell membranes with nanomolar affinity (18) and increases the affinity of the toxin for cells. The ␤-trefoil domain in VCC is inactive, but in homologous toxins, it recognizes terminal galactosyl glycan moieties with micromolar affinity (19). In addition to binding glycans, there is evidence that VCC activity is enhanced by cholesterol, sphingolipids, and ceramides in the cell membrane (20), although the mechanism by which this occurs is still not well understood.…”