Vibrio cholerae Cytolysin Recognizes the Heptasaccharide Core of Complex N-Glycans with Nanomolar Affinity

Abstract

Pathogens selectively target host cells using adhesion molecules and secreted virulence factors that may utilize protein, lipid, or carbohydrate ligands on the cell surface. The human intestinal pathogen Vibrio cholerae secretes a pore-forming toxin, Vibrio cholerae cytolysin (VCC), which contains two domains that are structurally similar to known carbohydrate-binding proteins. These tandem domains are attached to the carboxy-terminus of the cytolytic domain and contain a β-trefoil fold and a β-prism fold. VCC has been shown to bind glycosylated proteins, and removal of the β-prism domain leads to a large decrease in lytic activity against rabbit erythrocytes. Despite these clues, the identity of the glycan receptors of VCC and the role of glycan binding in toxin activity remains unknown. To better understand this specificity, we used a combination of structural and functional approaches to characterize the carbohydrate-binding activity of the VCC toxin. We first probed the monosaccharide-binding activity of VCC and demonstrated that the toxin exhibits millimolar affinity for aldohexoses. To understand this specificity, we solved the crystal structure of the VCC β-prism domain bound to methyl-α-mannose. Next, we utilized a mammalian glycan screen to determine that the β-prism domain preferentially binds complex N-glycans with a heptasaccharide GlcNAc4 Man3 core (NGA2). Fluorescence anisotropy and surface plasmon resonance indicated an approximately 100-nanomolar affinity of the β-prism domain for the heptasaccharide core. Our results suggest that carbohydrate-binding domains on the VCC toxin facilitate high-affinity targeting of mammalian cell membranes, which may contribute to the ability of VCC to lyse cells at picomolar concentrations.

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

“…So unlike staphylococcal toxins, which bind proteins and lipids, the acquisition of lectin domains by Vibrio toxins may be an alternative mechanism for targeting cells that also leads to a broader efficacy against various human cell lines. The slight preference for rabbit erythrocytes, as we have observed before (18), may arise by a higher density of complex N-glycans on these cells.…”

“…The Interplay between Sugar Binding Mutants and Rim Domain Mutants-In addition to direct rim domain interactions, lectin-glycan interactions play an important role in membrane targeting by VCC (18,36). To ensure that rim domain mutants do not have unintended effects on the ␤-prism domain lectin activity, we measured binding of VCC WT and a selection of single alanine mutants to the glycoprotein asialofetuin by ITC.…”

“…1B). 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).…”

The β-Prism Lectin Domain of Vibrio cholerae Hemolysin Promotes Self-assembly of the β-Pore-forming Toxin by a Carbohydrate-independent Mechanism

“…Briefly, pro-VCC, which includes the 14-kDa prodomain (amino acid residues 26 -741 of 82-kDa prepro-VCC) (27), was cloned into the pET32a(ϩ) expression vector (Novagen) and transformed into SHuffle TM T7 competent Escherichia coli cells (New England Biolabs), which provided a nonreducing cytosol appropriate for proper folding of the cytolysin (21). To prepare VCC 50 , a stop codon was introduced after the cytolytic domain, and to obtain the mutant VCC D617A , a point mutation was introduced into the pro-VCC nucleotide sequence (24,25). The proteins were purified to homogeneity by hydrophobic interaction chromatography on phenyl-Sepharose CL-4B, followed by elution with ethylene glycol (22).…”

“…Deletion of the 15-kDa ␤-prism fold bearing sequence and structural homology to the carbohydrate-binding domain of the plant lectin jacalin reduces hemolytic activity by ϳ1000-fold (22) and profoundly influences apoptotic and immunoregulatory activities (23). Interestingly, the role of the ␤-prism lectin domain in asialofetuin-binding and pore-forming activities critically depends on a single Asp 617 residue (24,25).…”