A Novel Mechanism of Bacterial Toxin Transfer within Host Blood Cell-Derived Microvesicles.

Shiga toxin (Stx) is the main virulence factor of enterohemorrhagic Escherichia coli, which are non-invasive strains that can lead to hemolytic uremic syndrome (HUS), associated with renal failure and death. Although bacteremia does not occur, bacterial virulence factors gain access to the circulation and are thereafter presumed to cause target organ damage. Stx was previously shown to circulate bound to blood cells but the mechanism by which it would potentially transfer to target organ cells has not been elucidated. Here we show that blood cell-derived microvesicles, shed during HUS, contain Stx and are found within patient renal cortical cells. The finding was reproduced in mice infected with Stx-producing Escherichia coli exhibiting Stx-containing blood cell-derived microvesicles in the circulation that reached the kidney where they were transferred into glomerular and peritubular capillary endothelial cells and further through their basement membranes followed by podocytes and tubular epithelial cells, respectively. In vitro studies demonstrated that blood cell-derived microvesicles containing Stx undergo endocytosis in glomerular endothelial cells leading to cell death secondary to inhibited protein synthesis. This study demonstrates a novel virulence mechanism whereby bacterial toxin is transferred within host blood cell-derived microvesicles in which it may evade the host immune system

Shiga Toxin and Lipopolysaccharide Induce Platelet-Leukocyte Aggregates and Tissue Factor Release, a Thrombotic Mechanism in Hemolytic Uremic Syndrome

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

Phenotypic Expression of ADAMTS13 in Glomerular Endothelial Cells

Background: ADAMTS13 is the physiological von Willebrand factor (VWF)-cleaving protease. The aim of this study was to examine ADAMTS13 expression in kidneys from ADAMTS13 wild-type (Adamts13+/+) and deficient (Adamts13-/-) mice and to investigate the expression pattern and bioactivity in human glomerular endothelial cells. Methodology/Principal Findings: Immunohistochemistry was performed on kidney sections from ADAMTS13 wild-type and ADAMTS13-deficient mice. Phenotypic differences were examined by ultramorphology. ADAMTS13 expression in human glomerular endothelial cells and dermal microvascular endothelial cells was investigated by real-time PCR, flow cytometry, immunofluorescence and immunoblotting. VWF cleavage was demonstrated by multimer structure analysis and immunoblotting. ADAMTS13 was demonstrated in glomerular endothelial cells in Adamts13+/+ mice but no staining was visible in tissue from Adamts13-/- mice. Thickening of glomerular capillaries with platelet deposition on the vessel wall was detected in Adamts13-/- mice. ADAMTS13 mRNA and protein were detected in both human endothelial cells and the protease was secreted. ADAMTS13 activity was demonstrated in glomerular endothelial cells as cleavage of VWF. Conclusions/Significance: Glomerular endothelial cells express and secrete ADAMTS13. The proteolytic activity could have a protective effect preventing deposition of platelets along capillary lumina under the conditions of high shear stress present in glomerular capillaries. © 2011 Tati et al.published_or_final_versio

Platelet activation and tissue factor release in hemolytic uremic syndrome

Hemolytic uremic syndrome (HUS) is a clinical syndrome characterized by microangiopathic hemolytic anemia, thrombocytopenia and acute renal failure. There are two subtypes: typical HUS associated with enterohemorrhagic E. coli (EHEC) and atypical HUS associated with uninhibited activation of the alternative pathway of complement. EHEC produce virulence factors such as lipopolysaccharide (LPS) and Shiga toxin. Atypical HUS is associated with mutations in complement regulators, mainly in factor H. During HUS platelets are activated and consumed leading to thrombocytopenia. The overall aim of this thesis was to investigate how platelet activation occurs and if tissue factor is released during typical and atypical HUS, as pathogenetic mechanisms explaining the formation of thrombotic microangiopathy. LPS was shown to bind to platelets through a novel receptor complex composed of TLR4/CD62. Binding led to platelet activation and aggregation. Shiga toxin bound to activated platelets and monocytes. In whole blood LPS and Shiga toxin induced platelet-leukocyte complex formation, generation of tissue factor-expressing platelet microparticles and release of functional tissue factor into plasma. Patients with typical HUS were shown to have LPS and Shiga toxin bound to their platelets and increased levels of platelet-leukocyte complexes, tissue factor-expressing platelet microparticles and circulating tissue factor were demonstrated. Mutated factor H was incapable of protecting platelets from complement activation. This was demonstrated using serum from patients with atypical HUS and defined mutations in factor H. Purified mutated factor H exhibited decreased binding to the platelet surface and allowed complement activation to occur. Complement activation on platelets led to their activation and release of tissue factor-expressing microparticles. Clusterin is one of the regulators of the terminal complement pathway. A novel mutation in clusterin (Q433P) was found in a child with atypical HUS. This mutated protein could not bind C5b-7 and serum from the patient induced complement activation on normal platelets. The results presented in this thesis show that LPS and complement deposition on platelets can activate platelets, release tissue factor and ultimately result in a prothrombotic state

Enterohemorrhagic Escherichia coli Pathogenesis and the Host Response

Enterohemorrhagic Escherichia coli (EHEC) is a highly pathogenic bacterial strain capable of causing watery or bloody diarrhea, the latter termed hemorrhagic colitis, and hemolytic-uremic syndrome (HUS). HUS is defined as the simultaneous development of non-immune hemolytic anemia, thrombocytopenia, and acute renal failure. The mechanism by which EHEC bacteria colonize and cause severe colitis, followed by renal failure with activated blood cells, as well as neurological symptoms, involves the interaction of bacterial virulence factors and specific pathogen-associated molecular patterns with host cells as well as the host response. The innate immune host response comprises the release of antimicrobial peptides as well as cytokines and chemokines in addition to activation and/or injury to leukocytes, platelets, and erythrocytes and activation of the complement system. Some of the bacterial interactions with the host may be protective in nature, but, when excessive, contribute to extensive tissue injury, inflammation, and thrombosis, effects that may worsen the clinical outcome of EHEC infection. This article describes aspects of the host response occurring during EHEC infection and their effects on specific organs

Isolation and Characterization of Shiga Toxin-Associated Microvesicles

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

Exosomes and microvesicles in normal physiology, pathophysiology, and renal diseases

Extracellular vesicles are cell-derived membrane particles ranging from 30 to 5,000 nm in size, including exosomes, microvesicles, and apoptotic bodies. They are released under physiological conditions, but also upon cellular activation, senescence, and apoptosis. They play an important role in intercellular communication. Their release may also maintain cellular integrity by ridding the cell of damaging substances. This review describes the biogenesis, uptake, and detection of extracellular vesicles in addition to the impact that they have on recipient cells, focusing on mechanisms important in the pathophysiology of kidney diseases, such as thrombosis, angiogenesis, tissue regeneration, immune modulation, and inflammation. In kidney diseases, extracellular vesicles may be utilized as biomarkers, as they are detected in both blood and urine. Furthermore, they may contribute to the pathophysiology of renal disease while also having beneficial effects associated with tissue repair. Because of their role in the promotion of thrombosis, inflammation, and immune-mediated disease, they could be the target of drug therapy, whereas their favorable effects could be utilized therapeutically in acute and chronic kidney injury