Novel Subtilase Cytotoxin Produced by Shiga-Toxigenic Escherichia coli Induces Apoptosis in Vero Cells via Mitochondrial Membrane Damage

Subtilase cytotoxin (SubAB) is an AB₅ cytotoxin produced by some strains of Shiga-toxigenic Escherichia coli. The A subunit is a subtilase-like serine protease and cleaves an endoplasmic reticulum chaperone, BiP, leading to transient inhibition of protein synthesis and cell cycle arrest at G₁ phase. Here we show that SubAB, but not the catalytically inactive mutant SubAB(S272A), induced apoptosis in Vero cells, as detected by DNA fragmentation and annexin V binding. SubAB induced activation of caspase-3, -7, and -8. Caspase-3 appeared earlier than caspase-8, and by use of specific caspase inhibitors, it was determined that caspase-3 may be upstream of caspase-8. A general caspase inhibitor blocked SubAB-induced apoptosis, detected by annexin V binding. SubAB also stimulated cytochrome c release from mitochondria, which was not suppressed by caspase inhibitors. In HeLa cells, Apaf-1 small interfering RNA inhibited caspase-3 activation, suggesting that cytochrome c might form an apoptosome, leading to activation of caspase-3. These data suggested that SubAB induced caspase-dependent apoptosis in Vero cells through mitochondrial membrane damage.Shiga-toxigenic Escherichia coli (STEC) is an etiologic agent of hemorrhagic colitis. Gastrointestinal disease caused by STEC may progress to systemic complications, including hemolytic uremic syndrome (HUS), which is characterized by thrombocytopenia, microangiopathic hemolytic anemia, and renal failure (13, 23). Shiga toxin 1 (Stx1) and Stx2 are both produced by STEC. However, whether Shiga toxins are the only factors responsible for these devastating diseases is still not clear.A new member of the AB₅ toxin family, named subtilase cytotoxin (SubAB), was identified (22, 23) in E. coli O113:H21 strain 98NK2, which produced Stx2 and was responsible for an outbreak of HUS. SubAB consists of one A subunit and five B subunits, which form a pentamer, similar to the case for Stx. The SubAB A subunit, with a molecular size of 35 kDa, shares sequence homology with a subtilase-like serine protease of Bacillus anthracis, and the toxin was named “subtilase cytotoxin.” The A subunit cleaves at a specific single site of endoplasmic reticulum (ER) chaperone BiP (21). The B subunits bind to some N-glycosylated membrane proteins, and α2β1 integrin has been shown to one of the receptors for vacuolating activity of B subunits (18, 30). Recently, it was reported that B subunits specifically bound to glycans terminating in the sialic acid N-glycolylneuraminic acid (3). SubAB is lethal for mice, causing extensive microvascular thrombosis as well as necrosis in the brain, kidney, and liver and apoptosis in the spleen, kidney, and liver. These findings are similar to the histopathologic, biochemical, and hematologic changes seen in human HUS (22, 26).SubAB is cytotoxic to Vero cells. BiP cleavage by the A subunit is necessary for Vero cell death (17, 18, 21, 22). BiP is known as a master regulator of ER function and homeostasis (11). SubAB induces ER stress (17, 27), as shown by activation of double-stranded RNA-activated protein kinase-like ER kinase (PERK) and eukaryotic initiation factor 2α (eIF2α), leading to transient protein synthesis inhibition and stress-inducible C/EBP-homologous protein (CHOP) induction, with cell cycle arrest in G₁ phase as a result of downregulation of cyclin D1 (17).Apoptosis, or programmed cell death, is a physiological event important in a diverse array of biological processes ranging from embryo development to bacterial infection (7, 31, 33). Morphologically, cells undergoing apoptosis demonstrate nuclear/cytoplasmic condensation and membrane protrusions. Biochemically, apoptotic cells are characterized by reduction in the mitochondrial transmembrane potential, intracellular acidification, production of reactive oxygen species, externalization of phosphatidylserine residues in membrane bilayers, selective proteolysis of a subset of cellular proteins, and internucleosomal degradation of DNA, resulting in a typical fragmentation pattern (28). There are multiple potential participants described for ER stress-induced apoptosis; however, the precise mechanisms of ER stress-induced apoptosis have not been fully elucidated (29). Recently, SubAB-induced apoptosis was partially described (27). We report here that SubAB triggers apoptosis in Vero cells initiated via mitochondrial membrane damage, followed by activation of a caspase-dependent cell death pathway.