Mechanisms of Shiga toxin-mediated signaling and toxicity

Sammanfattning: AbstractShiga toxin (Stx) is the main virulence factor of enterohemorrhagic Escherichia coli (EHEC). EHEC strains cause gastrointestinal infection and release Stx that can gain access to the circulation. Patients may develop hemolytic uremic syndrome with extensive kidney damage. In the first paper we investigated if blood cell-derived microvesicles released during EHEC infection contain Stx. Toxin-containing microvesicles were found in plasma from EHEC-infected patients and mice, and in kidney cells. Toxin transferred within microvesicles retained its toxic potential. This study demonstrated a novel mechanism of Stx delivery to the kidney. In the second paper the role of the Stx receptor, globotriaosylceramide (Gb3) in toxicity and intracellular transport of Stx delivered via microvesicles was investigated. Stx-positive microvesicles were taken up by both Gb3-positive and Gb3–negative cells but the toxin only exhibited toxicity when the cells expressed endogenous Gb3.Stx induces calcium influx, cytotoxicity and microvesicle release in cells. In the third paper we found that ATP is released from HeLa cells after Stx stimulation and that ATP functions as a secondary messenger to promote all the above-mentioned toxin effects. Blocking purinergic P2X signaling with NF449 inhibited Stx-induced calcium influx, microvesicle release and protected cells from the cytotoxic effects.In the fourth paper the role of extracellular ATP was investigated in a murine model of EHEC infection. Infected mice were injected with apyrase to cleave extracellular ATP. Apyrase protected the mice form EHEC-induced intestinal damage and delayed symptoms.The fifth paper is a review that summarizes the role of extracellular vesicles in normal physiology and kidney diseases.In summary this thesis defined a novel mechanism of Stx transfer to the kidneys, an ATP-mediated effect of Stx-signaling and the role of Gb3 on the cellular effect of Stx-positive microvesicles.