Proteolytic cleavage of the A subunit is essential for maximal cytotoxicity of Escherichia coli O157 : H7 Shiga-like toxin-1

Members of the bacterial Shiga toxin family consist of a single A subunit t hat is non-covalently associated with a pentamer of B subunits. These toxin s bind to receptors on susceptible mammalian cells and enter the cells by e ndocytic uptake. During cell entry, the 32 kDa A subunit is cleaved by the membrane-anchored protease furin to generate a catalytically active, 27.5 k Da A, fragment and a 4.5 kDa A, fragment. Previous studies have shown that mutating the furin site to prevent cleavage did not significantly affect to xin potency, suggesting that cleavage is not required for toxin activity. H ere it is confirmed that preventing cleavage at the usual processing site d oes not prevent proteolytic processing of the Escherichia coli Shiga-like t oxin-l A subunit. However, simultaneous mutation of both the primary furin- recognition site and a nearby putative furin cleavage site did prevent intr acellular processing of the A subunit. Comparison of the cytotoxicities of purified recombinant toxins to cultured mammalian cells demonstrated that e ven on prolonged incubation with toxin, the unprocessed mutant was 60-fold less toxic than the wild-type protein or other mutants still capable of bei ng proteolytically processed during cell entry.