Key Residues in Vibrio Cholerae Cytolysin Involved in Membrane Binding

Vibrio cholerae cytolysin (VCC) is a pore-forming toxin (PFT) secreted by the human pathogen Vibrio cholerae that has a predominant role in lethality in ΔCT (classical cholera toxin null) strains. VCC is a potent toxin with the ability to lyse cells in vitro at picomolar concentrations. In order to form heptameric lytic pores, VCC makes high-affinity interactions with cell membranes utilizing a combination of glycan receptors and lipid/cholesterol interactions. While previous research in our lab has illuminated aspects of glycan interactions made by one of the two lectin domains attached to VCC, knowledge regarding non-glycan mediated interactions between the host cell membrane and VCC's membrane-contacting rim domain is still lacking. To better understand direct membrane interactions, we performed systematic alanine scanning mutagenesis to over 30 amino acid residues predicted to interact with the membrane based on the crystal structure of the heptameric pore. We found several residues that when mutated to alanine, display drastically decreased protein activity; in some cases more than a 1000-fold loss. To ensure that the observed loss in activity is not related to a destabilization of the protein, we confirmed that our most drastic mutants are well behaved in solution and are not prone to aggregation. We also showed that the decrease in activity is not due to any loss of glycan interactions between the protein and cell-surface receptors. Our results identify residues important in membrane binding and give us a starting point to understand the nature of these interactions. Our work provides a template for investigating mechanisms by which PFTs target cell membranes with high-affinity.