Shiga toxin signals via ATP and its effect is blocked by purinergic receptor antagonism

Abstract

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 2 Triggers C3a-Dependent Glomerular and Tubular Injury through Mitochondrial Dysfunction in Hemolytic Uremic Syndrome

“…The inhibition of C3a, by normalizing VDAC and ATP5I levels, also limited mitochondrial functional alterations, which are instrumental to cell apoptosis. On the basis of our data, however, we cannot rule out the possibility that cytosolic ATP production and extracellular ATP release may be modulated in Stx2/LPS mice, possibly through C3a, as other groups have described [ 62 , 63 ]. These findings in experimental HUS are consistent with our previous data, which showed the detrimental role of C3a on podocytes, through mitochondrial dysfunction, in mice with diabetic nephropathy [ 32 ].…”

Shiga Toxin-Bearing Microvesicles Exert a Cytotoxic Effect on Recipient Cells Only When the Cells Express the Toxin Receptor

“…Cells were incubated with microvesicles, free Stx2 or PBS for a total of 3 h and 15 min, washed with PBS three times and incubated with Lhomopropargylglycine (HPG, 1:1,000) diluted in methioninefree RPMI medium (Gibco) for 45 min before protein synthesis was measured using the Click-iT HPG Protein Synthesis Assay Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. In total, cells were exposed to Stx2 for 4 h. This time point was selected as a previous study showed that Stx localized to the ER after 2 h (Johansson et al, 2019). Protein synthesis measurements were carried out using Alexa Fluor 647 azide for CHO-Gb3 and CHO-control cells or Alexa Fluor 488 azide for HeLa cells and DLD-1 cells.…”

Molecular mechanisms of Shigella effector proteins: a common pathogen among diarrheic pediatric population

“…ATP release mediates ATP signaling through purinergic receptor P2X, which in turn leads to Ca+ influx and cellular damage. Finally, it produces Shiga toxin containing microvesicle [ 202 ]. Shiga toxin mediates unfolded protein response mediated by ER and yields apoptosis for epithelial, lymphoid, and endothelial cells [ 203 ].…”