Membrane interaction of Pasteurella multocida toxin involves sphingomyelin

Michael C. Brothers, Mengfei Ho, Ram Maharjan, Nathan C. Clemons, Yuka Bannai, Mark A. Waites, Melinda J. Faulkner, Theresa B. Kuhlenschmidt, Mark S. Kuhlenschmidt, Steven R. Blanke, Chad Rienstra, Brenda A. Wilson

Research output: Contribution to journalArticlepeer-review


Pasteurella multocida toxin (PMT) is an AB toxin that causes pleiotropic effects in targeted host cells. The N-terminus of PMT (PMT-N) is considered to harbor the membrane receptor binding and translocation domains responsible for mediating cellular entry and delivery of the C-terminal catalytic domain into the host cytosol. Previous studies have implicated gangliosides as the host receptors for PMT binding. To gain further insight into the binding interactions involved in PMT binding to cell membranes, we explored the role of various membrane components in PMT binding, utilizing four different approaches: (a) TLC-overlay binding experiments with 125I-labeled PMT, PMT-N or the C-terminus of PMT; (b) pull-down experiments using reconstituted membrane liposomes with full-length PMT; (c) surface plasmon resonance analysis of PMT-N binding to reconstituted membrane liposomes; (d) and surface plasmon resonance analysis of PMT-N binding to HEK-293T cell membranes without or with sphingomyelinase, phospholipase D or trypsin treatment. The results obtained revealed that, in our experimental system, full-length PMT and PMT-N did not bind to gangliosides, including monoasialogangliosides GM 1, GM 2 or GM 3, but instead bound to membrane phospholipids, primarily the abundant sphingophospholipid sphingomyelin or phosphatidylcholine with other lipid components. Collectively, these studies demonstrate the importance of sphingomyelin for PMT binding to membranes and suggest the involvement of a protein co-receptor. Pasteurella multocida toxin (PMT) binds preferentially to phosphatidylcholine and sphingomyelin developed on TLC plates, incorporated into liposomes, or loaded onto BiaCore L1 chips. Surface plasmon resonance analysis of PMT binding to ghosts from HEK-293T cells treated with phospholipase D, sphingomyelinase, or trypsin suggests interplay among phosphatidylcholine, sphingomyelin and protein coreceptors in the cellular binding of PMT

Original languageEnglish (US)
Pages (from-to)4633-4648
Number of pages16
JournalFEBS Journal
Issue number23
StatePublished - Dec 2011


  • dermonecrotic toxin
  • ganglioside
  • phosphatidylcholine
  • surface plasmon resonance
  • thin layer chromatography

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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