The cholesterol-dependent cytolysins pneumolysin and streptolysin O require binding to red blood cell glycans for hemolytic activity

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; Michael P. Jennings

doi:10.1073/pnas.1412703111

Edited by Emil C. Gotschlich, The Rockefeller University, New York, NY, and approved November 3, 2014 (received for review July 9, 2014)

Significance

The pneumococcus accounts for 25% of deaths in children under 5 y of age in developing countries. One of the most important virulence factors expressed by this pathogen is the pore-forming toxin, pneumolysin (Ply), an example of a Gram-positive cholesterol-dependent cytolysin (CDC). We show that Ply interacts with the Lewis histo-blood group antigen sialyl LewisX and that blocking this interaction can protect RBCs from lysis. We also identify glycan receptors on RBCs for the CDC streptolysin O from group A streptococcus. Our study supports the emerging paradigm shift that CDCs have cellular receptors other than cholesterol that define target cell tropism.

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.

Footnotes

↵¹L.K.S. and R.M.H. contributed equally to this work.
↵²To whom correspondence may be addressed. Email: james.paton{at}adelaide.edu.au or m.jennings{at}griffith.edu.au.

Author contributions: L.K.S., R.M.H., V.J.T., M.J.W., A.W.P., J.C.P., and M.P.J. designed research; L.K.S., R.M.H., M.A.H., C.J.D., L.E.H.-T., D.B.A.J., F.A., and A.W.P. performed research; R.M.H., M.A.H., A.Y.C., and C.M.G. contributed new reagents/analytic tools; L.K.S., R.M.H., M.A.H., C.J.D., L.E.H.-T., and D.B.A.J. analyzed data; and L.K.S., R.M.H., M.A.H., J.C.P., and M.P.J. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1412703111/-/DCSupplemental.

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