Shiga Toxin Type 2dact Displays Increased Binding to Globotriaosylceramide in vitro and Increased Lethality in Mice after Activation by Elastase

Abstract

Shiga toxin type 2dact (Stx2dact), an Stx2 variant originally identified from Escherichia coli O91:H21 strain B2F1, displays increased cytotoxicity after activation by elastase present in intestinal mucus. Activation is a result of cleavage of two amino acids from the C-terminal tail of the A2 subunit. In this study, we hypothesized that activation leads to increased binding of toxin to its receptor on host cells both in vitro and in vivo. To test this theory, Stx2dact was treated with elastase or buffer alone and then each toxin was assessed for binding to purified globotriaosylceramide (Gb3) in an enzyme-linked immunosorbent assay, or cells in culture by immunofluorescence, or flow cytometry. Elastase- and buffer-treated Stx2dact were also evaluated for binding to mouse kidney tissue and for relative lethality in mice. We found that activated Stx2dact had a greater capacity to bind purified Gb3, cells in culture, and mouse kidney tissue and was more toxic for mice than was non-activated Stx2dact. Thus, one possible mechanism for the augmented cytotoxicity of Stx2dact after activation is its increased capacity to bind target cells, which, in turn, may cause greater lethality of elastase-treated toxin for mice and enhanced virulence for humans of E. coli strains that express Stx2dact.

Synchronous Disease Kinetics in a Murine Model for Enterohemorrhagic E. coli Infection Using Food-Borne Inoculation

“…Upon establishment of intestinal colonization, Stx promotes intestinal damage resulting in bloody diarrhea. The toxin subsequently translocates across the colonic epithelium into the bloodstream, where it targets distal tissues, including the microvasculature of the kidney, which expresses high levels of the Stx receptor, globotriaosylceramide (Gb3) (Obrig et al, 1993; Obata et al, 2008; Obrig, 2010; Melton-Celsa et al, 2012; Bunger et al, 2013). Hence, EHEC infection exhibits distinct clinical phases such as non-bloody diarrhea, followed by bloody diarrhea, and systemic disease, the latter manifested most commonly by hemolytic uremic syndrome (HUS), the triad of hemolytic anemia, thromobocytopenia and renal failure (Keepers et al, 2006; Obrig, 2010; Melton-Celsa et al, 2012; Davis et al, 2014; Melton-Celsa and O'Brien, 2014; Freedman et al, 2016).…”

“…The native murine AE pathogen C. rodentium (CR), which like EHEC forms attaching and effacing lesions, was lysogenized with phage Φ1720a-02, isolated from an Stx-producing E. coli strain (Mallick et al, 2012, 2014). This phage encodes and produces Stx variant Stx2dact, which is activated ~18-fold by intestinal mucus (Mallick et al, 2012; Bunger et al, 2013), resulting in high potency in mice (Teel et al, 2002; Bunger et al, 2013). The oral gavage of mice with a high (5 × 10 9 CFU) dose of an overnight culture of CR(Φ1720a-02), herein referred to as “CR(ΦStx 2dact )” for simplicity, recapitulates many of the features of human EHEC infection in an Stx 2dact -dependent manner, including colitis, renal damage, weight loss, and systemic injury.…”

Affinity-Based Screening of Tetravalent Peptides Identifies Subtype-Selective Neutralizers of Shiga Toxin 2d, a Highly Virulent Subtype, by Targeting a Unique Amino Acid Involved in Its Receptor Recognition

“…Stx2a (10,11) and Stx2d, which is activated by elastase derived from the intestinal mucosa (12)(13)(14), are highly virulent and have been linked with HUS, the most serious sequela of EHEC infection. The pathophysiologic importance of these subtypes was also confirmed by the finding that Stx2a and Stx2d are highly toxic when injected into mice (15,16) or primates (17)(18)(19). Therefore, Stx neutralizers, particularly those customized to specifically neutralize Stx2a and Stx2d, would be highly valuable therapeutic agents for treating infections caused by various EHEC strains.…”

Shiga Toxin 2 Subtypes of Enterohemorrhagic E. coli O157:H- E32511 Analyzed by RT-qPCR and Top-Down Proteomics Using MALDI-TOF-TOF-MS

“…In contrast, there are fewer AA substitutions across subtypes in the catalytically-active A1 fragment of the A-subunit. This suggests that differences in the toxicity of Stx1, Stx2, and their subtypes have less to do with differences in A1 toxicity, whose sequence is highly conserved across subtypes, and more to do with stability of the AB 5 holotoxin, its transport, its attachment to surface receptors on eukaryotic cells, and its intracellular translocation.…”

“…It is this quaternary complex that is responsible for transport of the catalytically-active part of the A-subunit (A1 fragment). The A2/B 5 complex is also responsible for recognizing and attaching to the glycolipid surface receptors on eukaryotic cells, specifically the glycolipid globotriaosylceramide (Gb3) receptor [5].…”