Please use this identifier to cite or link to this item: http://hdl.handle.net/2440/89666
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Type: Journal article
Title: The cholesterol-dependent cytolysins pneumolysin and streptolysin O require binding to red blood cell glycans for hemolytic activity
Author: Shewell, L.
Harvey, R.
Higgins, M.
Day, C.
Hartley-Tassell, L.
Chen, A.
Gillen, C.
James, D.
Alonzo, F.
Torres, V.
Walker, M.
Paton, A.
Paton, J.
Jennings, M.
Citation: Proceedings of the National Academy of Sciences of the United States of America, 2014; 111(49):E5312-E5320
Publisher: National Academy of Sciences
Issue Date: 2014
ISSN: 0027-8424
1091-6490
Statement of
Responsibility: 
Lucy K. Shewell, Richard M. Harvey, Melanie A. Higgins, Christopher J. Day, Lauren E. Hartley-Tassell, Austen Y. Chen, Christine M. Gillen, David B. A. James, Francis Alonzo, Victor J. Torres, Mark J. Walker, Adrienne W. Paton, James C. Paton, and Michael P. Jennings
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.
Keywords: Streptococcus pneumoniae; cholesterol-dependent cytolysin; glycan binding; pneumolysin; streptolysin O
Rights: © 2014 National Academy of Sciences. Freely available online through the PNAS open access option.
RMID: 0030016528
DOI: 10.1073/pnas.1412703111
Grant ID: http://purl.org/au-research/grants/nhmrc/565526
Appears in Collections:Molecular and Biomedical Science publications

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