An A-B Subunit Chimera of the E.Coli Verotoxin 1 Provides A Receptor Probe and Potential Vaccine Approach.

ABSTRACT

We have generated a chimera between the enzymatically active A subunit of the E coli derived AB5 verotoxin and a single receptor-binding B subunit. The construct was made by in frame fusion of the 3’ terminus of the A subunit gene with the 5’ end of the B subunit gene via the deletion of the intervening bases of the verotoxin operon such that the C terminus of the A subunit is continuous with the N terminus of a single B subunit. The gene product is a single fusion protein of 38kDa molecular weight, reactive with polyclonal and monoclonal antibodies against either the A or B subunits of the wild type toxin. The chimera showed a 104-105 fold reduction in cell vero cell cytotoxicity but no toxicity for the globotriaosyl ceramide (Gb3) deficient VRP subclone. No Gb3 binding by TLC overlay was detected. Polyclonal rabbit anti-VT1A-B chimera serum neutralizes VT1 cytotoxicity in vitro but reacts only with the A subunit of the wildtype holotoxin by western blot. This A-B chimera illustrates the importance of the pentameric B subunit in receptor binding and potentially identifies a novel attenuation vaccination strategy.