title: The cholesterol-dependent cytolysins pneumolysin and streptolysin O require binding to red blood cell glycans for hemolytic activity.
abstract: The cholesterol-dependent cytolysin (CDC) pneumolysin (Ply) is a key virulence factor of Streptococcus pneumoniae. Membrane cholesterol is required for the cytolytic activity of this toxin, but it is not clear whether cholesterol is the only cellular receptor. Analysis of Ply binding to a glycan microarray revealed that Ply has lectin activity and binds glycans, including the Lewis histo-blood group antigens. Surface plasmon resonance analysis showed that Ply has the highest affinity for the sialyl LewisX (sLeX) structure, with a K(d) of 1.88 × 10(-5) M. Ply hemolytic activity against human RBCs showed dose-dependent inhibition by sLeX. Flow cytometric analysis and Western blots showed that blocking binding of Ply to the sLeX glycolipid on RBCs prevents deposition of the toxin in the membrane. The lectin domain responsible for sLeX binding is in domain 4 of Ply, which contains candidate carbohydrate-binding sites. Mutagenesis of these predicted carbohydrate-binding residues of Ply resulted in a decrease in hemolytic activity and a reduced affinity for sLeX. This study reveals that this archetypal CDC requires interaction with the sLeX glycolipid cellular receptor as an essential step before membrane insertion. A similar analysis conducted on streptolysin O from Streptococcus pyogenes revealed that this CDC also has glycan-binding properties and that hemolytic activity against RBCs can be blocked with the glycan lacto-N-neotetraose by inhibiting binding to the cell surface. Together, these data support the emerging paradigm shift that pore-forming toxins, including CDCs, have cellular receptors other than cholesterol that define target cell tropism.
authors.affiliation: Institute for Glycomics, Griffith University, Gold Coast, QLD 4222, Australia;
authors.forename: Lucy K
authors.initials: LK
authors.lastname: Shewell
authors.affiliation: Research Centre for Infectious Diseases, School of Molecular and Biomedical Science, University of Adelaide, Adelaide 5005, Australia;
authors.forename: Richard M
authors.initials: RM
authors.lastname: Harvey
authors.affiliation: Research Centre for Infectious Diseases, School of Molecular and Biomedical Science, University of Adelaide, Adelaide 5005, Australia;
authors.forename: Melanie A
authors.initials: MA
authors.lastname: Higgins
authors.affiliation: Institute for Glycomics, Griffith University, Gold Coast, QLD 4222, Australia;
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Day
authors.affiliation: Institute for Glycomics, Griffith University, Gold Coast, QLD 4222, Australia;
authors.forename: Lauren E
authors.initials: LE
authors.lastname: Hartley-Tassell
authors.affiliation: Research Centre for Infectious Diseases, School of Molecular and Biomedical Science, University of Adelaide, Adelaide 5005, Australia;
authors.forename: Austen Y
authors.initials: AY
authors.lastname: Chen
authors.affiliation: School of Chemistry and Molecular Biosciences and the Australian Infectious Diseases Research Centre, University of Queensland, Brisbane, QLD 4072, Australia; and.
authors.forename: Christine M
authors.initials: CM
authors.lastname: Gillen
authors.affiliation: Department of Microbiology, New York University School of Medicine, New York, NY 10016.
authors.forename: David B A
authors.initials: DB
authors.lastname: James
authors.affiliation: Department of Microbiology, New York University School of Medicine, New York, NY 10016.
authors.forename: Francis
authors.initials: F
authors.lastname: Alonzo
authors.affiliation: Department of Microbiology, New York University School of Medicine, New York, NY 10016.
authors.forename: Victor J
authors.initials: VJ
authors.lastname: Torres
authors.affiliation: School of Chemistry and Molecular Biosciences and the Australian Infectious Diseases Research Centre, University of Queensland, Brisbane, QLD 4072, Australia; and.
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Walker
authors.affiliation: Research Centre for Infectious Diseases, School of Molecular and Biomedical Science, University of Adelaide, Adelaide 5005, Australia;
authors.forename: Adrienne W
authors.initials: AW
authors.lastname: Paton
authors.affiliation: Research Centre for Infectious Diseases, School of Molecular and Biomedical Science, University of Adelaide, Adelaide 5005, Australia; james.paton@adelaide.edu.au m.jennings@griffith.edu.au.
authors.forename: James C
authors.initials: JC
authors.lastname: Paton
authors.affiliation: Institute for Glycomics, Griffith University, Gold Coast, QLD 4222, Australia; james.paton@adelaide.edu.au m.jennings@griffith.edu.au.
authors.forename: Michael P
authors.initials: MP
authors.lastname: Jennings
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry; D001665:Binding Sites; D002241:Carbohydrates / Q000737:chemistry; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D004912:Erythrocytes / Q000378:metabolism*; D005434:Flow Cytometry; D006017:Glycolipids / Q000737:chemistry; D006461:Hemolysis; D006801:Humans; D016256:Lewis X Antigen / Q000737:chemistry; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009844:Oligosaccharides / Q000737:chemistry; D011134:Polysaccharides / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D013301:Streptolysins / Q000737:chemistry*; D020349:Surface Plasmon Resonance
chemical_list: D001426:Bacterial Proteins; D002241:Carbohydrates; D006017:Glycolipids; D016256:Lewis X Antigen; D009844:Oligosaccharides; D011134:Polysaccharides; D013301:Streptolysins; C032290:plY protein, Streptococcus pneumoniae; C025964:streptolysin O; C013084:lacto-N-neotetraose
keywords: Streptococcus pneumoniae; cholesterol-dependent cytolysin; glycan binding; pneumolysin; streptolysin O