Abstract
Subtilase cytotoxin (SubAB) is an AB(5) cytotoxin produced by some strains of Shiga-toxigenic Escherichia coli. The A subunit is a subtilase-like serine protease and cleaves an endoplasmic reticulum (ER) chaperone, BiP, leading to transient inhibition of protein synthesis and cell cycle arrest at G(1) phase, and inducing caspase-dependent apoptosis via mitochondrial membrane damage in Vero cells. Here we investigated the mechanism of mitochondrial permeabilization in HeLa cells. SubAB-induced cytochrome c release into cytosol did not depend on mitochondrial permeability transition pore (PTP), since cyclosporine A did not suppress cytochrome c release. SubAB did not change the expression of anti-apoptotic Bcl-2 or Bcl-XL and pro-apoptotic Bax or Bak, but triggered Bax and Bak conformational changes and association of Bax with Bak. Silencing using siRNA of both bax and bak genes, but not bax, bak, or bim alone, resulted in reduction of cytochrome c release, caspase-3 activation, DNA ladder formation and cytotoxicity, indicating that Bax and Bak were involved in apoptosis. SubAB activated ER transmembrane transducers, Ire1alpha, and cJun N-terminal kinase (JNK), and induced C/EBF-homologue protein (CHOP). To investigate whether these signals were involved in cytochrome c release by Bax activation, we silenced ire1alpha, jnk or chop; however, silencing did not decrease SubAB-induced cytochrome c release, suggesting that these signals were not necessary for SubAB-induced mitochondrial permeabilization by Bax activation.