Shiga toxin of enterohaemorrhagicEscherichia colidirectly injures developing human erythrocytes

Abstract

Haemolytic anaemia is one of the characteristics of life-threatening extraintestinal complications in humans during infection with enterohaemorrhagic Escherichia coli (EHEC). Shiga toxins (Stxs) of EHEC preferentially damage microvascular endothelial cells of the kidney and the brain, whereby occluded small blood vessels may elicit anaemia through mechanical erythrocyte disruption. Here we show for the first time that Stx2a, the major virulence factor of EHEC, is also capable of direct targeting developing human erythrocytes. We employed an ex vivo erythropoiesis model using mobilized CD34(+) haematopoietic stem/progenitor cells from human blood and monitored expression of Stx receptors and Stx2a-mediated cellular injury of developing erythrocytes. CD34(+) haematopoietic stem/progenitor cells were negative for Stx2a receptors and resistant towards the toxin. Expression of Stx2a-binding glycosphingolipids and toxin sensitivity was apparent immediately after initiation of erythropoietic differentiation, peaked for basophilic and polychromatic erythroblast stages and declined during maturation into orthochromatic erythroblasts and reticulocytes, which became highly refractory to Stx2a. The observed Stx-mediated toxicity towards erythroblasts during the course of erythropoiesis might contribute, although speculative at this stage of research, to the anaemia caused by Stx-producing pathogens.

A Topographical Atlas of Shiga Toxin 2e Receptor Distribution in the Tissues of Weaned Piglets

“…The advantage of using antibodies lies in the fact that they can detect even trace quantities of GSLs in complex GSL mixtures as outlined in previous publications [38,42,43,44]. Although being considerably less sensitive in receptor detection, we employed Stx2e in combination with anti-Stx2 antibody in addition to the antibody-mediated detection of Stx2e receptors.…”

“…The Stx2e-containing STEC supernatant was used undiluted. The anti-Stx2 antibody was applied in 1:1000 dilution and the secondary AP-conjugated antibodies were used as 1:2000 dilutions (both in 1% BSA in PBS) as previously described [25,43,44,78,79,80]. Bound secondary antibodies were detected with 0.05% ( w / v ) 5-bromo-4-chloro-3-indolyl phosphate p -toluidine salt (Roth, Karlsruhe, Germany) in glycine buffer (pH 10.4) (Serva), which generates a blue precipitate at sites of antibody binding on the TLC plate.…”

“…Electrospray ionization mass spectrometry (ESI-MS) was carried out with a quadrupole time-of-flight mass spectrometer endowed with a nanospray manipulator (Micromass, Manchester, UK) in the positive ion mode as described previously [25,38,44,79]. Dried GSL extracts from silica gel were dissolved in methanol containing 1% ( v / v ) formic acid (Sigma-Aldrich, Steinheim, Germany) and analyzed in the positive ion mode by ESI-MS 1 .…”

Emergence of CD43-Expressing Hematopoietic Progenitors from Human Induced Pluripotent Stem Cells

“…CB CD34+ cells were collected after normal deliveries. CD34+ cells were further purified by immunomagnetic sorting via MACS (Milteny, Bergisch-Gladbach, Germany), and cells from three donations (adult PB and CB each) were subsequently applied to ex vivo erythropoiesis as biological replicates as previously described [21]. Written informed consent was obtained from all donors; the study was approved by the local ethics committee (Medical University Graz, Austria EK: 27-165 ex 14/15).…”

Membrane assembly of Shiga toxin glycosphingolipid receptors and toxin refractiveness of MDCK II epithelial cells

“…TLC overlay assays using polyclonal chicken anti-Lc2Cer, anti-Gb3Cer and anti-Gb4Cer antibodies, the monoclonal rat IgM anti-Forssman GSL antibody and bacterial supernatants containing Stx1a, Stx2a, and Stx2e subtypes were done as previously described (45,75,82,87,88,96). In short, after GSL separation the silica gel layer requires fixation with polyisobutylmethacrylate (Plexigum P28, …”