Elastase in intestinal mucus enhances the cytotoxicity of Shiga toxin type 2d

Shiga toxin variant type 2d (Stx2d) produced by some strains of Shiga toxin-producing Escherichia coli is composed of an enzymatically active A subunit and a B (binding) pentamer. The cytotoxicity of Stx2d is increased (activated) 10-1000-fold for Vero cells when the toxin is incubated with mucus obtained from the small intestine of mice. In this study we isolated an Stx2d activator and identified it as a mouse elastase with strong homology to human elastase IIIB. Moreover, commercially available porcine pancreatic elastase preparations also activated Stx2d cytotoxicity although with a lower specific activity than isolated mouse elastase. Elastase directly nicked the Stx2d A subunit to A₁ and A₂, an event that did not correlate with activation. However, elastase also reduced the size and changed the isoelectric point of the A₂ peptide, as determined by SDS-polyacrylamide gel electrophoresis and two-dimensional electrophoresis followed by Western immunoblot analysis. This elastase-mediated size and charge shift in the A₂ peptide of Stx2d occurred concurrently with activation of the toxin. Both the reduction in size of the Stx2d A₂ peptide by incubation with elastase as well as the associated activation of Stx2d cytotoxicity were fully inhibited by elastatinal, an elastase-specific inhibitor.