Reduced Toxicity of Shiga Toxin (Stx) Type 2c in Mice Compared to Stx2d Is Associated with Instability of Stx2c Holotoxin

Abstract

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.

Epidemiology of Shiga Toxin-Producing Escherichia coli O157 in the Province of Alberta, Canada, 2009–2016

“…Isolates encoding stx 2a, the Shiga toxin subtype with the highest cytotoxic potential [14] and classically associated with the risk of HUS, were involved in approximately 85% of HUS cases. stx 2c, although of comparatively lower cytotoxicity and higher heat lability [15], was involved in over 30% of cases of HUS, not all of them justifiable by the concomitant presence of other Shiga toxin subtypes. No cases of HUS were documented among patients infected with the stx 1a stx 2a stx 2c genotype, despite the combination of three different toxins, and those infected with the stx 1a stx 2a genotype isolates were statistically less likely to develop HUS, regardless of the presence of stx 2a.…”

The Virulence of Escherichia coli O157:H7 Isolates in Mice Depends on Shiga Toxin Type 2a (Stx2a)-Induction and High Levels of Stx2a in Stool

“…However, the risk of developing HUS is increased for children under 10 years age and in patients of any age who develop leukocytosis, experience treatment delays, or receive antibiotics and/or anti-motility agents (Tarr et al, 2005). Although the presence of the locus of enterocyte effacement (LEE) in an STEC isolate, the production of specific Stx subtypes, and E. coli O157:H7 clade classification have been associated with STEC that cause severe human disease (Manning et al, 2008;Neupane et al, 2011;Amigo et al, 2015;Bunger et al, 2015), no other pathogen-specific factors have been identified that can be used to predict the severity of STEC related disease. The focus of this study was to determine whether factors, such as stx-subtype, Stx protein levels, stx-phage insertion sites, and virulence of O157:H7 clinical isolates in a mouse model correlated with disease presentation in patients.…”

Bimodal Response to Shiga Toxin 2 Subtypes Results from Relatively Weak Binding to the Target Cell

“…Stx2, on the other hand, is composed of a diverse and heterogeneous group of subtypes, from Stx2a to Stx2h (17)(18)(19). These subtypes differ in potency and species specificity, with Stx2a, Stx2c, and Stx2d linked to severe human illness (20)(21)(22)(23)(24). Stx2a is associated with more severe infections than the other Stx2 subtypes or Stx1a (6,21,(25)(26)(27).…”