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Microvesicles are shed from cell surfaces during infectious or inflammatory conditions and may contribute to the pathogenesis of disease. During Shiga toxin-producing Escherichia coli (STEC) infection, microvesicles are released from blood cells. These microvesicles play a part in inflammation, thrombosis, hemolysis, and the transfer of the main virulence factor of STEC strains, Shiga toxin, to target organ cells. This chapter describes how to isolate blood cell- and cell culture-derived microvesicles from plasma or cell culture medium, respectively, and how to characterize these microvesicles by various methods, with special focus on Shiga toxin-associated microvesicles.

Shiga toxin is the main virulence factor of non-invasive enterohemorrhagic Escherichia coli strains capable of causing hemolytic uremic syndrome. Our group has previously shown that the toxin can reach the kidney within microvesicles where it is taken up by renal cells and the vesicles release their cargo intracellularly, leading to toxin-mediated inhibition of protein synthesis and cell death. The aim of this study was to examine if recipient cells must express the globotriaosylceramide (Gb3) toxin receptor for this to occur, or if Gb3-negative cells are also susceptible after uptake of Gb3-positive and toxin-positive microvesicles. To this end we generated Gb3-positive A4GALT-transfected CHO cells, and a vector control lacking Gb3 (CHO-control cells), and decreased Gb3 synthesis in native HeLa cells by exposing them to the glycosylceramide synthase inhibitor PPMP. We used these cells, and human intestinal DLD-1 cells lacking Gb3, and exposed them to Shiga toxin 2-bearing Gb3-positive microvesicles derived from human blood cells. Results showed that only recipient cells that possessed endogenous Gb3 (CHO-Gb3 transfected and native HeLa cells) exhibited cellular injury, reduced cell metabolism and protein synthesis, after uptake of toxin-positive microvesicles. In Gb3-positive cells the toxin introduced via vesicles followed the retrograde pathway and was inhibited by the retrograde transport blocker Retro-2.1. CHO-control cells, HeLa cells treated with PPMP and DLD-1 cells remained unaffected by toxin-positive microvesicles. We conclude that Shiga toxin-containing microvesicles can be taken up by Gb3-negative cells but the recipient cell must express endogenous Gb3 for the cell to be susceptible to the toxin., (Copyright © 2020 Johansson, Willysson, Kristoffersson, Tontanahal, Gillet, Ståhl and Karpman.)

Shiga toxin (Stx) is the main virulence factor of enterohemorrhagic Escherichia coli (EHEC), that cause gastrointestinal infection leading to hemolytic uremic syndrome. The aim of this study was to investigate if Stx signals via ATP and if blockade of purinergic receptors could be protective. Stx induced ATP release from HeLa cells and in a mouse model. Toxin induced rapid calcium influx into HeLa cells, as well as platelets, and a P2X1 receptor antagonist, NF449, abolished this effect. Likewise, the P2X antagonist suramin blocked calcium influx in Hela cells. NF449 did not affect toxin intracellular retrograde transport, however, cells pre-treated with NF449 exhibited significantly higher viability after exposure to Stx for 24 hours, compared to untreated cells. NF449 protected HeLa cells from protein synthesis inhibition and from Stx-induced apoptosis, assayed by caspase 3/7 activity. The latter effect was confirmed by P2X1 receptor silencing. Stx induced the release of toxin-positive HeLa cell- and platelet-derived microvesicles, detected by flow cytometry, an effect significantly reduced by NF449 or suramin. Suramin decreased microvesicle levels in mice injected with Stx or inoculated with Stx-producing EHEC. Taken together, we describe a novel mechanism of Stx-mediated cellular injury associated with ATP signaling and inhibited by P2X receptor blockade.

Shiga toxin (Stx)-producing Escherichia coli (STEC) cause hemolytic uremic syndrome (HUS). This study investigated whether Stx2 induces hemolysis and whether complement is involved in the hemolytic process. RBCs and/or RBC-derived microvesicles from patients with STEC-HUS (n = 25) were investigated for the presence of C3 and C9 by flow cytometry. Patients exhibited increased C3 deposition on RBCs compared with controls (p < 0.001), as well as high levels of C3- and C9-bearing RBC-derived microvesicles during the acute phase, which decreased after recovery. Stx2 bound to P1 (k) and P2 (k) phenotype RBCs, expressing high levels of the P(k) Ag (globotriaosylceramide), the known Stx receptor. Stx2 induced the release of hemoglobin and lactate dehydrogenase in whole blood, indicating hemolysis. Stx2-induced hemolysis was not demonstrated in the absence of plasma and was inhibited by heat inactivation, as well as by the terminal complement pathway Ab eculizumab, the purinergic P2 receptor antagonist suramin, and EDTA. In the presence of whole blood or plasma/serum, Stx2 induced the release of RBC-derived microvesicles coated with C5b-9, a process that was inhibited by EDTA, in the absence of factor B, and by purinergic P2 receptor antagonists. Thus, complement-coated RBC-derived microvesicles are elevated in HUS patients and induced in vitro by incubation of RBCs with Stx2, which also induced hemolysis. The role of complement in Stx2-mediated hemolysis was demonstrated by its occurrence only in the presence of plasma and its abrogation by heat inactivation, EDTA, and eculizumab. Complement activation on RBCs could play a role in the hemolytic process occurring during STEC-HUS., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Background: Aggregates formed between leukocytes and platelets in the circulation lead to release of tissue factor (TF)-bearing microparticles contributing to a prothrombotic state. As enterohemorrhagic Escherichia coli (EHEC) may cause hemolytic uremic syndrome (HUS), in which microthrombi cause tissue damage, this study investigated whether the interaction between blood cells and EHEC virulence factors Shiga toxin (Stx) and lipopolysaccharide (LPS) led to release of TF., Methodology/principal Findings: The interaction between Stx or LPS and blood cells induced platelet-leukocyte aggregate formation and tissue factor (TF) release, as detected by flow cytometry in whole blood. O157LPS was more potent than other LPS serotypes. Aggregates formed mainly between monocytes and platelets and less so between neutrophils and platelets. Stimulated blood cells in complex expressed activation markers, and microparticles were released. Microparticles originated mainly from platelets and monocytes and expressed TF. TF-expressing microparticles, and functional TF in plasma, increased when blood cells were simultaneously exposed to the EHEC virulence factors and high shear stress. Stx and LPS in combination had a more pronounced effect on platelet-monocyte aggregate formation, and TF expression on these aggregates, than each virulence factor alone. Whole blood and plasma from HUS patients (n = 4) were analyzed. All patients had an increase in leukocyte-platelet aggregates, mainly between monocytes and platelets, on which TF was expressed during the acute phase of disease. Patients also exhibited an increase in microparticles, mainly originating from platelets and monocytes, bearing surface-bound TF, and functional TF was detected in their plasma. Blood cell aggregates, microparticles, and TF decreased upon recovery., Conclusions/significance: By triggering TF release in the circulation, Stx and LPS can induce a prothrombotic state contributing to the pathogenesis of HUS.