Three biologically significant globotriasylceramide binding sites on the Verotoxin 1 B subunit, implications in toxin action, pathogenesis of disease and vaccine design

grantor: University of TorontoGastrointestinal infection with Verotoxigenic 'Escherichia coli ' (VTEC) is a cause of hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS). VTEC produce two AB5 toxin types, verotoxin (VT) 1 and VT2, comprised of a catalytic A subunit and a pentamer of globotriaosylceramide (Gb3) receptor binding B subunits. Using oligonucleotide-directed site-specific mutagenesis, the significance of three B subunit binding sites (I, II and III) was examined. Mutant binding data revealed that the amino acid substitutions predicted to affect Sites I or II, caused reductions in VT1 binding affinity, binding capacity and cytotoxicity. A Site III mutation, despite markedly reducing the binding capacity, increased binding affinity and had only minimal effects on cytotoxicity. Our data suggest that the high affinity receptor binding most relevant for cytotoxicity occurs at Site I and/or Site II, while Site III, despite recognizing additional receptors with lower affinity has limited relevance to cytotoxicity. To study receptor binding and disease production, a Site I mutant, Phe 30 Ala, was intravenously administered to rabbits. When given at a dose equivalent to ten times the 50% lethal dose of VT1, Phe 30 Ala had a significantly longer serum half-life, failed to localize preferentially in target organs and did not produce clinical disease despite the observation of mild VT-related histologic changes in target tissues. Thus, receptor binding and more specifically, Site I binding, is important in VT1 mediated disease. As Phe 30 Ala may serve as a natural toxoid we studied its immunogenicity in three inbred mouse strains. While our results demonstrate that an antibody response to the VT1 B subunit is dependent on the strain's ' H-2' haplotype, they also suggest that a B-cell immunodominant epitope is formed partly by Phe 30. A potential role for the A subunit in providing the T-cell help necessary to overcome a deficient B subunit antibody response in 'H-2b' mice was also supported.Ph.D