title: Mutant Escherichia coli heat-labile toxin B subunit that separates toxoid-mediated signaling and immunomodulatory action from trafficking and delivery functions.
abstract: 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-kappaB 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.
authors.affiliation: Department of Pathology & Microbiology, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, United Kingdom.
authors.forename: Sylvia A
authors.initials: SA
authors.lastname: Fraser
authors.forename: Lolke
authors.initials: L
authors.lastname: de Haan
authors.forename: Arron R
authors.initials: AR
authors.lastname: Hearn
authors.forename: Heather K
authors.initials: HK
authors.lastname: Bone
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Salmond
authors.forename: A Jennifer
authors.initials: AJ
authors.lastname: Rivett
authors.forename: Neil A
authors.initials: NA
authors.lastname: Williams
authors.forename: Timothy R
authors.initials: TR
authors.lastname: Hirst
mesh_terms: D000276:Adjuvants, Immunologic / Q000494:pharmacology*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D017209:Apoptosis; D001427:Bacterial Toxins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D015496:CD4-Positive T-Lymphocytes / Q000502:physiology; D018414:CD8-Positive T-Lymphocytes / Q000502:physiology; D004768:Enterotoxins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D029968:Escherichia coli Proteins; D005260:Female; D005677:G(M1) Ganglioside / Q000378:metabolism; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D016328:NF-kappa B / Q000378:metabolism; D021381:Protein Transport; D013329:Structure-Activity Relationship; D014121:Toxoids / Q000494:pharmacology*
chemical_list: D000276:Adjuvants, Immunologic; D001427:Bacterial Toxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D016328:NF-kappa B; D014121:Toxoids; D005677:G(M1) Ganglioside; C027538:heat-labile enterotoxin, E coli