Shiga toxin delivery in extracellular vesicles induces high mortality and severe kidney injury in a Gb3-dependent manner

Escherichia coli O157:H7 is a non-invasive Shiga toxin (Stx)-producing pathogen that causes gastroenteritis and hemolytic uremic syndrome characterized by acute kidney injury. Stx binding to cells induces release of extracellular vesicles (EVs) containing the toxin. This study aimed to demonstrate the lethal effects of toxin-positive vesicles, and the importance of the Stx receptor, globotriaosylceramide (Gb3), for these effects. HeLa cells were stimulated with Stx2 to induce vesicle release. EVs < 450 nm were isolated and cytotoxicity confirmed. BALB/c mice were injected intravenously with Stx2 126 ng/kg, the same toxin concentration in Stx2-EVs or EVs without toxin. Mortality was significantly increased in mice challenged with Stx2-EVs (n = 8/8) compared to mice challenged with free Stx2 (n = 4/7). Urea levels were high, and kidney tubulointerstitial pathology and fibrinogen staining prominent in mice challenged with Stx2-EVs. Mice challenged with EVs alone (n = 7) remained unaffected. C57BL/6 mice and Gb3-negative littermates were injected with Stx2-EVs at toxin concentrations of 126 or 200 ng/kg. Gb3-negative mice (n = 9) injected with Stx2-EVs at both toxin concentrations were totally protected whereas wild-type mice developed disease, n = 2/4 at the lower and 4/5 at the higher toxin concentration. These mice remained unaffected by the same concentrations of free toxin (n = 10) suggesting that toxin delivered in vesicles was lethal. This study shows that Stx2 delivered systemically within EVs is lethal, causing severe kidney damage, and the Gb3 toxin receptor is crucial for disease induction by circulating Stx2-EVs.