Cellular signaling pathway of Shiga toxin-induced ATP release.

BACKGROUND: Shiga toxin (Stx) is the main virulence factor of enterohemorrhagic Escherichia coli, a food-borne pathogen that colonizes the intestine causing gastroenteritis and, in severe cases, hemolytic uremic syndrome. Stx was shown to induce ATP release in vivo and in vitro and blockade of purinergic P2X receptors inhibited its cytotoxicity. Here we investigated the intracellular signaling events preceding ATP release. METHODS: Inhibitors included pertussis toxin, wortmannin, manoalide, 2-aminoethoxydiphenylborate (2-APB), BAPTA-AM and Ca(2+)-free medium. The inositol 1,4,5-triphosphate receptor (IP(3)R) was silenced. Stx-induced apoptosis was detected by caspase 3/7 activation. BALB/c mice were injected with Stx2 i.p. Certain mice were pretreated with alpelisib (1 h before and 24 h after Stx2). Kidneys collected after 4 days were stained for phosphatidylinositol 4,5-bisphosphate (PIP2). RESULTS: Stx1-mediated ATP release in HeLa cells was blocked by pertussis toxin affecting the Gi/o family of G-protein coupled receptors. ATP release was also abrogated by wortmannin, an inhibitor of phosphoinositide 3-kinase (PI3K), by manoalide, inhibiting phospholipase C, by 2-APB inhibiting IP(3)R, and by reduction of intracellular calcium (BAPTA-AM) and extracellular calcium (Ca(2+)-free medium). Blocking or silencing the IP(3)R protected HeLa cells from Stx1-induced apoptosis. Likewise, blocking the IP(3)R reduced Stx2-induced apoptosis. Stx2-challenged mice had distinct PIP2 glomerular staining that decreased in the presence of the PI3K inhibitor alpelisib. CONCLUSION: Stx interaction with HeLa cells initiates a signaling pathway involving membrane G protein, PI3K, phospholipase C and IP(3)R, ultimately leading to ATP release and promoting cytotoxic effects. The PI3K inhibitor alpelisib altered PIP2 expression in Stx2-challenged mice.