Identification and Characterization of the Glycan Binding Site of Vibrio Cholerae Cytolysin

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Vibrio cholerae cytolysin (VCC) is a pore-forming toxin secreted by the Vibrio cholera O1 El Tor strain. VCC is hypothesized to perform an important function by lysing immune cells and thereby protecting the bacteria from host defenses. VCC consists of multiple domains including two possible carbohydrate-binding lectin domains (β-prism and β-trefoil). Recently it has been found that the β-prism domain binds to both mono and oligosaccharides, but interacts with highest affinity to glycans found on eukaryotic cell membranes. Crystal structures exist for VCC in water-soluble and membrane assembled forms, but the precise molecular mechanism by which VCC recognizes carbohydrates is still not well understood. Here we show that the Asp 617 residue in the β-prism domain plays an important role in both monosaccharide and cell surface glycan binding. Site directed mutagenesis of the Asp 617 to alanine resulted in a ∼80-fold decrease in monosaccharide binding activity and a ∼280-fold decrease in cell-surface glycan binding activity. Furthermore, the Asp 617 mutant displayed a decline in hemolytic activity compared to wild type VCC when incubated with rabbit red blood cells. We also show that both monosaccharides and purified cell surface glycans can compete with VCC and cause a dose dependent lag in the half-life of rabbit blood hemolysis. These results indicate that the VCC interacts with glycans on target cell membranes and Asp 617 is involved in this binding process. Having more insight into the molecular mechanism and residues involved in sugar recognition may enable the development of sugar-based therapies against VCC and other pore-forming toxins.