Elastase in Intestinal Mucus Enhances the Cytotoxicity of Shiga Toxin Type 2d

Abstract

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 A 1 and A 2 , an event that did not correlate with activation. However, elastase also reduced the size and changed the isoelectric point of the A 2 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 A 2 peptide of Stx2d occurred concurrently with activation of the toxin. Both the reduction in size of the Stx2d A 2 peptide by incubation with elastase as well as the associated activation of Stx2d cytotoxicity were fully inhibited by elastatinal, an elastase-specific inhibitor.

Characterization of Shiga Toxin-Producing Escherichia coli Strains Isolated from Human Patients in Germany over a 3-Year Period

“…A mucus elastase-activatable variant of Stx2 called stx 2d (stx 2vha and stx 2vhb ) was previously described for an STEC strain of serotype O91:H21 (40,49). Toxin type 2d-positive Table 2).…”

Structure of Shiga Toxin Type 2 (Stx2) from Escherichia coli O157:H7

“…Second, the carboxyl terminus of the Stx2 A-subunit forms a short 2-turn ␣-helix after threading through the pore of the B-pentamer, in contrast to the tail of the Stx A-subunit, which in the crystals shows disorder in the same region. The carboxyl-terminal tail of the Stx2d-activable A2 has been shown to be involved in the enhanced toxicity of Stx2d-activable in the presence of elastase from mucus (33,34). Perhaps the more ordered and extended structure found in the terminus of the Stx2 A2, could, by analogy, also be found in Stx2d-activable, and contribute to the accessibility of that peptide to elastase, which cleaves the terminal two amino acids from the A2 carboxyl terminus (34).…”

Oral Intoxication of Mice with Shiga Toxin Type 2a (Stx2a) and Protection by Anti-Stx2a Monoclonal Antibody 11E10

“…The slides were then rinsed in de-ionized water and blocked overnight at 4°C in 1% goat serum diluted in antibody (Ab) diluent reagent solution (Invitrogen). Slides were incubated with primary Ab (polyclonal rabbit anti-Stx2a [34] diluted 1:500 in Ab diluent solution) for 1 h at room temperature. Slides were washed 3 times with PBS before the secondary Ab, goat anti-rabbit-Alexa-fluor 488 (Invitrogen) diluted 1:500 in Ab diluent, was applied for 1 h at room temperature.…”