The cholesterol-dependent cytolysins, pneumolysin and streptolysin O, require binding to red blood cell glycans for hemolytic activity (#5)
Lucy K. Shewell
1
,
Richard M. Harvey
2
,
Melanie A. Higgins
2
,
Christopher J. Day
1
,
Lauren E. Hartley-Tassell
1
,
Austen Y. Chen
2
,
Christine M. Gillen
3
,
David B.A. James
4
,
Francis Alonzo III
4
,
Victor J. Torres
4
,
Mark J. Walker
3
,
Adrienne W. Paton
2
,
James C. Paton
2
,
Michael P. Jennings
1
- Institute for Glycomics, Griffith University, Gold Coast, QLD, Australia
- Research Centre for Infectious Diseases, University of Adelaide, Adelaide, SA, Australia
- School of Chemistry and Molecular Biosciences and the Australian Infectious Diseases Research Centre, University of Queensland, Brisbane, QLD, Australia
- Department of Microbiology, New York University School of Medicine, New York, NY, USA
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 actual 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 KD of 1.88 x 10-5 M. Ply haemolytic
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 haemolytic
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 prior to membrane insertion. A similar analysis conducted
on streptolysin O (SLO) from Streptococcus pyogenes revealed that this
CDC also has glycan binding properties and that haemolytic activity against
RBCs can be blocked with the glycan lacto-N-neotetraose (LNnT) 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.