Shiga toxin (Stx) is an AB5 ribotoxin made by Stx-producing Escherichia coli (STEC). These organisms cause diarrhea, hemorrhagic colitis and the hemolytic uremic syndrome. STEC make two types of Stxs, Stx1 and/or Stx2. Stx2 has one prototype (a) and six subtypes (b-g), but only STEC that make Stx2a, and/or Stx2c, or Stx2d are associated with severe disease. However, Stx2c is about 10-fold less toxic than Stx2d in vivo despite only two amino acid differences in the A subunit at positions 291 and 297. We made mutations at these two sites to create intermediate toxins between Stx2c and Stx2d, and determined the 50% cytotoxic dose on Vero cells before and after heat treatment, and the 50% lethal dose in mice of the toxins. We found that serine 291 was associated with increased toxicity in vivo and that either amino acid change from that in Stx2c to that in Stx2d increased heat stability. We also assessed the secondary structure of Stx2c and Stx2d by circular dichroism (CD) spectroscopy. The CD studies suggest that Stx2c has a less-ordered secondary structure than Stx2d. We conclude that both amino acids at positions 291 and 297 in Stx2c contribute to its decreased stability and in vivo toxicity compared to Stx2d.
Keywords: STEC; Shiga toxin; Stx2; Stx2c; Stx2d.