title: The Relationship between Glycan Binding and Direct Membrane Interactions in Vibrio cholerae Cytolysin, a Channel-forming Toxin.
abstract: Bacterial pore-forming toxins (PFTs) are structurally diverse pathogen-secreted proteins that form cell-damaging channels in the membranes of host cells. Most PFTs are released as water-soluble monomers that first oligomerize on the membrane before inserting a transmembrane channel. To modulate specificity and increase potency, many PFTs recognize specific cell surface receptors that increase the local toxin concentration on cell membranes, thereby facilitating channel formation. Vibrio cholerae cytolysin (VCC) is a toxin secreted by the human pathogen responsible for pandemic cholera disease and acts as a defensive agent against the host immune system. Although it has been shown that VCC utilizes specific glycan receptors on the cell surface, additional direct contacts with the membrane must also play a role in toxin binding. To better understand the nature of these interactions, we conducted a systematic investigation of the membrane-binding surface of VCC to identify additional membrane interactions important in cell targeting. Through cell-based assays on several human-derived cell lines, we show that VCC is unlikely to utilize high affinity protein receptors as do structurally similar toxins from Staphylococcus aureus. Next, we identified a number of specific amino acid residues that greatly diminish the VCC potency against cells and investigated the interplay between glycan binding and these direct lipid contacts. Finally, we used model membranes to parse the importance of these key residues in lipid and cholesterol binding. Our study provides a complete functional map of the VCC membrane-binding surface and insights into the integration of sugar, lipid, and cholesterol binding interactions.
authors.affiliation: Department of Molecular Biology and Biochemistry, Molecular Biophysics Program, Wesleyan University, Middletown, Connecticut 06459.
authors.forename: Swastik
authors.initials: S
authors.lastname: De
authors.affiliation: Department of Molecular Biology and Biochemistry, Molecular Biophysics Program, Wesleyan University, Middletown, Connecticut 06459.
authors.forename: Adele
authors.initials: A
authors.lastname: Bubnys
authors.affiliation: Department of Microbiology, New York University School of Medicine, New York, New York 10016.
authors.forename: Francis
authors.initials: F
authors.lastname: Alonzo
authors.affiliation: Department of Molecular Biology and Biochemistry, Molecular Biophysics Program, Wesleyan University, Middletown, Connecticut 06459.
authors.forename: Jinsol
authors.initials: J
authors.lastname: Hyun
authors.affiliation: Biotechnology-Bioservices Center, University of Connecticut, Storrs, Connecticut 06269.
authors.forename: Jeffrey W
authors.initials: JW
authors.lastname: Lary
authors.affiliation: Biotechnology-Bioservices Center, University of Connecticut, Storrs, Connecticut 06269; Department of Molecular and Cell Biology and Department of Chemistry, University of Connecticut, Storrs, Connecticut 06269.
authors.forename: James L
authors.initials: JL
authors.lastname: Cole
authors.affiliation: Department of Microbiology, New York University School of Medicine, New York, New York 10016.
authors.forename: Victor J
authors.initials: VJ
authors.lastname: Torres
authors.affiliation: Department of Molecular Biology and Biochemistry, Molecular Biophysics Program, Wesleyan University, Middletown, Connecticut 06459. Electronic address: rolson@wesleyan.edu.
authors.forename: Rich
authors.initials: R
authors.lastname: Olson
mesh_terms: D000595:Amino Acid Sequence; D002460:Cell Line; D003603:Cytotoxins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007473:Ion Channels / Q000378:metabolism*; D009504:Neutrophils / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism*; D011485:Protein Binding; D020349:Surface Plasmon Resonance; D014734:Vibrio cholerae / Q000378:metabolism*
chemical_list: D003603:Cytotoxins; D007473:Ion Channels; D011134:Polysaccharides
keywords: bacterial pathogenesis; bacterial toxin; cholesterol; membrane; structure-function