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

Abstract

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.

Early Terminal Complement Blockade and C6 Deficiency Are Protective in Enterohemorrhagic Escherichia coli–Infected Mice

“…Bacteremia is not a feature of EHEC infection, and Stx, which gains access to the bloodstream via the damaged intestine, circulates bound to blood cells (10,11) and within blood cell-derived extracellular microvesicles (12), thereby reaching target organs. In the kidney the toxin damages both glomerular endothelial cells as well as tubular epithelial cells (4,13).…”

“…In the kidney the toxin damages both glomerular endothelial cells as well as tubular epithelial cells (4,13). The renal lesion in HUS patients consists of thrombi in glomerular capillaries, swelling and detachment of endothelial cells from the basement membrane (14), and extensive tubular injury (4,12). Mouse models of EHEC infection or Stx injection reproduce certain aspects of this lesion with platelet deposition in glomeruli (15,16), damaged glomerular capillaries (17), and pronounced tubular damage (7,18).…”

The Antibiotic Polymyxin B Impairs the Interactions between Shiga Toxins and Human Neutrophils

“…Thus, the same antibiotic could impair the interaction of two potent bacterial virulence factors with target cells involved in the pathogenesis of HUS. This could be particularly important in preventing the generation of tissue factor-bearing leukocyte/platelet-derived microvesicles, also containing Stx2, which are involved in HUS pathogenesis (24,36,37). Microvesicles have been shown to constitute a novel mechanism of transfer of Stx2 to glomerular endothelial cells in vitro and in patients with HUS (37).…”

“…This could be particularly important in preventing the generation of tissue factor-bearing leukocyte/platelet-derived microvesicles, also containing Stx2, which are involved in HUS pathogenesis (24,36,37). Microvesicles have been shown to constitute a novel mechanism of transfer of Stx2 to glomerular endothelial cells in vitro and in patients with HUS (37). They are produced in vitro upon incubation with LPS and Stx2 both interacting with TLR4 present on the membrane of the different target cells, that is, neutrophils, monocytes, and platelets (24).…”

“…Various arguments have been put forward as a causative role: 1) free Stx have never been found in sera of patients with HUS (27)(28)(29), and, conversely, the toxins have been detected on the surface of their neutrophils (19,27,30); 2) patients' neutrophils are activated, degranulated, and hyporesponsive to other stimuli (19,31); 3) neutrophilia is a common finding in patients with HUS and is considered a poor prognostic factor (32-34); 4) the greater the amount of toxins in patients' feces, the greater the saturation of neutrophil receptors by Stx (35); 5) the transfer of the toxins from neutrophils to target endothelial cells has been demonstrated in vitro (18) during leukocyte transmigration (22); 6) HUS patients with lower amounts of toxins in their neutrophils had severe renal involvement (19,27,30); 7) HUS with only mild renal involvement developed in patients with a high detectable amount of toxins on neutrophil membrane, although they were at risk for neurologic complications (19,27,30); 8) Stx2 has been found on leukocytes (monocyte or neutrophil)/platelet aggregates circulating in patients with HUS (24); and 9) leukocyte-or platelet-derived microvesicles containing Stx2 have been found in the blood patients with HUS (24,36) and in their renal cortex (37).…”