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Shiga Toxin Uptake and Sequestration in Extracellular Vesicles Is Mediated by Its B-Subunit

Willysson, Annie LU ; Ståhl, Anne-Lie LU ; Gillet, Daniel ; Barbier, Julien ; Cintrat, Jean-Christophe ; Chambon, Valérie ; Billet, Anne ; Johannes, Ludger and Karpman, Diana LU orcid (2020) In Toxins 12(7).
Abstract

Shiga toxin (Stx)-stimulated blood cells shed extracellular vesicles (EVs) which can transfer the toxin to the kidneys and lead to hemolytic uremic syndrome. The toxin can be taken up by renal cells within EVs wherein the toxin is released, ultimately leading to cell death. The mechanism by which Stx is taken up, translocated, and sequestered in EVs was addressed in this study utilizing the B-subunit that binds to the globotriaosylceramide (Gb3) receptor. We found that Stx1B was released in EVs within minutes after stimulation of HeLa cells or red blood cells, detected by live cell imaging and flow cytometry. In the presence of Retro-2.1, an inhibitor of intracellular retrograde trafficking, a continuous release of Stx-positive EVs... (More)

Shiga toxin (Stx)-stimulated blood cells shed extracellular vesicles (EVs) which can transfer the toxin to the kidneys and lead to hemolytic uremic syndrome. The toxin can be taken up by renal cells within EVs wherein the toxin is released, ultimately leading to cell death. The mechanism by which Stx is taken up, translocated, and sequestered in EVs was addressed in this study utilizing the B-subunit that binds to the globotriaosylceramide (Gb3) receptor. We found that Stx1B was released in EVs within minutes after stimulation of HeLa cells or red blood cells, detected by live cell imaging and flow cytometry. In the presence of Retro-2.1, an inhibitor of intracellular retrograde trafficking, a continuous release of Stx-positive EVs occurred. EVs from HeLa cells possess the Gb3 receptor on their membrane, and EVs from cells that were treated with a glycosylceramide synthase inhibitor, to reduce Gb3, bound significantly less Stx1B. Stx1B was detected both on the membrane and within the shed EVs. Stx1B was incubated with EVs derived from blood cells, in the absence of cells, and was shown to bind to, and be taken up by, these EVs, as demonstrated by electron microscopy. Using a membrane translocation assay we demonstrated that Stx1B was taken up by blood cell- and HeLa-derived EVs, an effect enhanced by chloropromazine or methyl-ß-cyclodextrin, suggesting toxin transfer within the membrane. This is a novel mechanism by which EVs derived from blood cells can sequester their toxic content, possibly to evade the host response.

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author
; ; ; ; ; ; ; and
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Erythrocytes/metabolism, Extracellular Vesicles/metabolism, Female, HeLa Cells, Humans, Protein Subunits, Protein Transport, Receptors, Cell Surface/metabolism, Shiga Toxin 1/chemistry, Time Factors, Trihexosylceramides/metabolism, Uterine Cervical Neoplasms/metabolism
in
Toxins
volume
12
issue
7
article number
449
publisher
MDPI AG
external identifiers
  • scopus:85088030907
  • pmid:32664382
ISSN
2072-6651
DOI
10.3390/toxins12070449
language
English
LU publication?
yes
id
f390a468-b6cd-4c2f-b477-f5f968ae9d9a
date added to LUP
2020-07-29 10:39:21
date last changed
2024-05-29 17:11:51
@article{f390a468-b6cd-4c2f-b477-f5f968ae9d9a,
  abstract     = {{<p>Shiga toxin (Stx)-stimulated blood cells shed extracellular vesicles (EVs) which can transfer the toxin to the kidneys and lead to hemolytic uremic syndrome. The toxin can be taken up by renal cells within EVs wherein the toxin is released, ultimately leading to cell death. The mechanism by which Stx is taken up, translocated, and sequestered in EVs was addressed in this study utilizing the B-subunit that binds to the globotriaosylceramide (Gb3) receptor. We found that Stx1B was released in EVs within minutes after stimulation of HeLa cells or red blood cells, detected by live cell imaging and flow cytometry. In the presence of Retro-2.1, an inhibitor of intracellular retrograde trafficking, a continuous release of Stx-positive EVs occurred. EVs from HeLa cells possess the Gb3 receptor on their membrane, and EVs from cells that were treated with a glycosylceramide synthase inhibitor, to reduce Gb3, bound significantly less Stx1B. Stx1B was detected both on the membrane and within the shed EVs. Stx1B was incubated with EVs derived from blood cells, in the absence of cells, and was shown to bind to, and be taken up by, these EVs, as demonstrated by electron microscopy. Using a membrane translocation assay we demonstrated that Stx1B was taken up by blood cell- and HeLa-derived EVs, an effect enhanced by chloropromazine or methyl-ß-cyclodextrin, suggesting toxin transfer within the membrane. This is a novel mechanism by which EVs derived from blood cells can sequester their toxic content, possibly to evade the host response.</p>}},
  author       = {{Willysson, Annie and Ståhl, Anne-Lie and Gillet, Daniel and Barbier, Julien and Cintrat, Jean-Christophe and Chambon, Valérie and Billet, Anne and Johannes, Ludger and Karpman, Diana}},
  issn         = {{2072-6651}},
  keywords     = {{Erythrocytes/metabolism; Extracellular Vesicles/metabolism; Female; HeLa Cells; Humans; Protein Subunits; Protein Transport; Receptors, Cell Surface/metabolism; Shiga Toxin 1/chemistry; Time Factors; Trihexosylceramides/metabolism; Uterine Cervical Neoplasms/metabolism}},
  language     = {{eng}},
  month        = {{07}},
  number       = {{7}},
  publisher    = {{MDPI AG}},
  series       = {{Toxins}},
  title        = {{Shiga Toxin Uptake and Sequestration in Extracellular Vesicles Is Mediated by Its B-Subunit}},
  url          = {{http://dx.doi.org/10.3390/toxins12070449}},
  doi          = {{10.3390/toxins12070449}},
  volume       = {{12}},
  year         = {{2020}},
}