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Infection and Immunity, March 2003, p. 1527-1537, Vol. 71, No. 3
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.3.1527-1537.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Mutant Escherichia coli Heat-Labile Toxin B Subunit That Separates Toxoid-Mediated Signaling and Immunomodulatory Action from Trafficking and Delivery Functions

Sylvia A. Fraser,1 Lolke de Haan,1 Arron R. Hearn,2 Heather K. Bone,1 Robert J. Salmond,1 A. Jennifer Rivett,2 Neil A. Williams,1 and Timothy R. Hirst1*

Departments of Pathology & Microbiology,1 Biochemistry, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, United Kingdom2

Received 23 September 2002/ Returned for modification 8 November 2002/ Accepted 18 November 2002

The homopentameric B-subunit components of Escherichia coli heat-labile enterotoxin (EtxB) and cholera toxin (CtxB) possess the capacity to enter mammalian cells and to activate cell-signaling events in leukocytes that modulate immune cell function. Both properties have been attributed to the ability of the B subunits to bind to GM1-ganglioside receptors, a ubiquitous glycosphingolipid found in the plasma membrane. Here we describe the properties of EtxB(H57S), a mutant B subunit with a His->Ser substitution at position 57. The mutant was found to be severely defective in inducing leukocyte signaling, as shown by failure to (i) trigger caspase 3-mediated CD8+-T-cell apoptosis, (ii) activate nuclear translocation of NF-{kappa}B in Jurkat T cells, (iii) induce a potent anti-B-subunit response in mice, or (iv) serve as a mucosal adjuvant. However, its GM1 binding, cellular uptake, and delivery functions remained intact. This was further validated by the finding that EtxB(H57S) was as effective as EtxB in delivering a conjugated model class I epitope into the major histocompatibility complex class I pathway of a dendritic cell line. These observations imply that GM1 binding alone is not sufficient to trigger the signaling events responsible for the potent immunomodulatory properties of EtxB. Moreover, they demonstrate that its signaling properties play no role in EtxB uptake and trafficking. Thus, EtxB(H57S) represents a novel tool for evaluating the complex cellular interactions and signaling events occurring after receptor interaction, as well as offering an alternative means of delivering attached peptides in the absence of the potent immunomodulatory signals induced by wild-type B subunits.

* Corresponding author. Mailing address: Department of Pathology & Microbiology, University of Bristol, School of Medical Sciences, University Walk, Bristol BS8 1TD, United Kingdom. Phone: 44-117-9287538. Fax: 44-117-9300543. E-mail: t.r.hirst{at}bristol.ac.uk.

Editor: J. T. Barbieri

Infection and Immunity, March 2003, p. 1527-1537, Vol. 71, No. 3
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.3.1527-1537.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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