Extracellular vesicles in Shiga toxin-mediated disease

Abstract: Extracellular vesicles are released during infectious, inflammatory and prothrombotic conditions. These are membrane-derived vesicles shed by cells during activation, senescence and/or apoptosis. Extracellular vesicles contain and transfer components of the parent cell from which they are shed. During gastrointestinal infection with enterohemorrhagic Escherichia coli (EHEC) extracellular vesicles are released from blood cells into the circulation and contain Shiga toxin, the main EHEC virulence factor with potent cytotoxic effects. EHEC can cause hemorrhagic colitis or hemolytic uremic syndrome, characterized by acute kidney injury, thrombocytopenia, and non-immune hemolytic anemia. This PhD thesis addressed mechanisms of release and uptake of Shiga toxin-positive extracellular vesicles and characterized prothrombotic and proinflammatory properties related to vesicles released after Shiga toxin stimulation of blood cells. The thesis also provides methodology for the of these extracellular vesicles.Paper I shows that once Shiga toxin is taken up by cells it is released within vesicles within minutes and does not undergo retrograde transport. Toxin could bind directly to extracellular vesicles and could transfer from the surface to the inside of the vesicle suggesting sequestration, possibly to evade the host response. This is a novel mechanism by which vesicles could protect their content while in the circulation.Paper II investigated the importance of the toxin receptor, globotriaosylceramide (Gb3), in recipient cells for the uptake of Shiga toxin-containing extracellular vesicles by the cells and exertion of a cytotoxic effect. Various Gb3-negative and Gb3-positive cells were used showing that the recipient cell must possess endogenous Gb3 for the toxin, taken up in extracellular vesicles, to exert a toxic effect on cell viability.Paper III investigated the pro-thrombotic and proinflammatory properties of Shiga toxin-induced blood cell-derived extracellular vesicles. These vesicles possessed more tissue factor activity and thrombin- generating activity and induced platelet aggregation. The vesicles contained pro-inflammatory cytokines and induced the release of IL-8 from endothelial cells co-incubated with monocytes.Paper IV describes isolation procedures of blood cell- and cell culture-derived microvesicles and characterization of the vesicles by various methods including flow cytometry, electron microscopy, nanoparticle tracking, ELISA, proteomics and live cell imaging.In summary, this thesis characterized important aspects of Shiga toxin-induced extracellular vesicles including their uptake, intracellular effects, release, and properties that can contribute to the development of hemolytic uremic syndrome.