Shiga Toxin Uptake and Sequestration in Extracellular Vesicles Is Mediated by Its B-Subunit

Annie Willysson (1) , Anne-Lie Ståhl (1) , Daniel Gillet (2) , Julien Barbier (2) , Jean-Christophe Cintrat (2) , Valérie Chambon (3) , Anne Billet (3) , Ludger Johannes (3, 4) , Diana Karpman (1)

1 Clinical Sciences - Department of Pediatrics [Lund, Sweden]
2 SIMoS - Service d'Ingénierie Moléculaire pour la Santé (ex SIMOPRO)
3 CBMCT - UMR 3666 / U1143 - Chimie biologique des membranes et ciblage thérapeutique
4 CBMCT - UMR 3666 / U1143 - Chimie biologique des membranes et ciblage thérapeutique

Shiga toxin (Stx)-stimulated blood cells shed extracellular vesicles (EVs) which can transfer the toxin to the kidneys and lead to hemolytic uremic syndrome. The toxin can be taken up by renal cells within EVs wherein the toxin is released, ultimately leading to cell death. The mechanism by which Stx is taken up, translocated, and sequestered in EVs was addressed in this study utilizing the B-subunit that binds to the globotriaosylceramide (Gb3) receptor. We found that Stx1B was released in EVs within minutes after stimulation of HeLa cells or red blood cells, detected by live cell imaging and flow cytometry. In the presence of Retro-2.1, an inhibitor of intracellular retrograde trafficking, a continuous release of Stx-positive EVs occurred. EVs from HeLa cells possess the Gb3 receptor on their membrane, and EVs from cells that were treated with a glycosylceramide synthase inhibitor, to reduce Gb3, bound significantly less Stx1B. Stx1B was detected both on the membrane and within the shed EVs. Stx1B was incubated with EVs derived from blood cells, in the absence of cells, and was shown to bind to, and be taken up by, these EVs, as demonstrated by electron microscopy. Using a membrane translocation assay we demonstrated that Stx1B was taken up by blood cell-and HeLa-derived EVs, an effect enhanced by chloropromazine or methyl-ß-cyclodextrin, suggesting toxin transfer within the membrane. This is a novel mechanism by which EVs derived from blood cells can sequester their toxic content, possibly to evade the host response.

Mots clés: Shiga toxin; extracellular vesicles; red blood cells; HeLa cells; globotriaosylceramide

Domaines: Bactériologie

Annie Willysson, Anne-Lie Ståhl, Daniel Gillet, Julien Barbier, Jean-Christophe Cintrat, et al.. Shiga Toxin Uptake and Sequestration in Extracellular Vesicles Is Mediated by Its B-Subunit. Toxins, 2020, 12 (7), ⟨10.3390/toxins12070449⟩. ⟨cea-03023143⟩