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.     

Efficacy and safety of ramucirumab and docetaxel in previously treated patients with squamous cell lung cancer: a multicenter retrospective cohort study

Investigational New Drugs - Objective. Ramucirumab plus docetaxel (RAM/DOC) therapy is currently the standard for previously treated advanced non-small cell lung cancer (NSCLC), irrespective of...     

Therapeutic efficacy of double filtration plasmapheresis in patients with anti-aquaporin-4 antibody-positive multiple sclerosis

Multiple sclerosis (MS) in Asian countries, including Japan, is classified into two types: conventional MS (C-MS), characterized mainly by cerebral lesions, and opticospinal MS (OS-MS) or neuromyelitis optica (NMO), characterized by selective involvement of the optic nerve and spinal cord. Recently, a serum immunoglobulin-G-antibody was discovered in patients with NMO that targets aquaporin-4 (AQP4). The existence of the anti-AQP4 antibody shows the pathogenetic role of humoral immune factors in OS-MS/NMO. We treated eight patients with anti-AQP4 antibody-positive MS with double filtration plasmapheresis (DFPP) to remove the antibody. Improvement of vision was observed in two patients. Motion improvement was seen in seven patients. Sensory improvement was observed in four patients. In total, six out of eight patients (75%) showed therapeutic improvement after DFPP treatment. We propose that DFPP might be an effective therapeutic option for patients with anti-AQP4 antibody-positive MS.     

Subtilase cytotoxin induces apoptosis in HeLa cells by mitochondrial permeabilization via activation of Bax/Bak,independent of C/EBF-homologue protein (CHOP), Ire1α or JNK signaling

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 (ER) chaperone, BiP, leading to transient inhibition of protein synthesis and cell cycle arrest at G₁ 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, Ire1α, 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 ire1α, 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.     

Milk whey protein enhances the bone breaking force in ovariectomized rats

We studied the effects of whey protein (WP) and fractionated WP (HWP; heat-stable WP, LWP; low M.W. WP, EWP; ethanol-precipitated WP) on calcium and bone metabolism in ovariectomized (OVX) rats. Six-week-old female Sprague-Dawley rats were ovariectomized and fed a low-calcium diet (0.03% Ca, 0.3% P) for 4 weeks. The rats were divided into five groups, Cont, WP, HWP, LWP and EWP group, and were fed a Cont diet (20% casein, 0.3% Ca) or a diet (19% casein, 0.3% Ca) containing 1% WP, HWP, LWP or EWP for 4 weeks. There were no significant differences in the calcium balance, serum calcium and calcitonin levels among the experimental groups. However, serum ALP activity of the HWP and EWP groups at 14 wks. were lower than that of the Cont group. The bone breaking strength and energy of femur of the HWP, LWP and EWP groups were higher than those of the Cont group. As for the amount of calcium, phosphorus, and magnesium in the femur, there were no significant differences among the experimental groups; however, the amounts of total amino acids in the femur of the HWP, LWP and EWP groups were higher than that of the Cont group. The amounts of proline and hydroxyproline (typical amino acids of collagen) in the femur of the HWP, LWP and EWP groups were also higher than those of the Cont group. These data indicate that milk WP contains active components that influence bone metabolism in OVX rats by increasing in bone protein such as collagen and enhance the bone breaking force (strength and energy). These results suggest that the active components are existed in heatstable, low M.W. and 30–70% ethanol-precipitated fraction, respectively.     

Atrial septal defect in apical hypertrophic cardiomyopathy associated with coronary spasm

Apical hypertrophic cardiomyopathy (HCM) is a well-known myocardial disease, but the additional coexistence of an atrial septal defect (ASD) and coronary spasm is quite rare. We report here on a 62-year-old man suffering from congestive heart failure due to apical HCM complicated by coronary spasm and secundum-type ASD. The transthoracic, transesophageal echocardiography and cardiac catheterization were useful for diagnosing and evaluating of the patient's status. A calcium channel blocker was given to prevent coronary spasm, and a surgical patch closure operation was successfully performed. Afterwards, his symptoms were alleviated.     

Analysis of Helicobacter pylori vacA Gene and Serum Antibodies to VacA in Japan

Vacuolating cytotoxin, VacA, is one of the most important pathogenetic factors produced by Helicobacter pylori. However, it is not clear whether the diversity in disease outcome may be ascribed to variations in strain and/or to the host responses to virulence factors. In this study, we analyzed the vacA middle region sequence among 65 Japanese isolates to clarify the variation in strain and assayed antibody titer to VacA by ELISA using purified VacA to evaluate the host response to cytotoxin. The nucleotide sequence identities compared among Japanese isolates were 92.8 ± 3.56%, and compared to 88.3 ± 2.89% in tox⁺ strains reported in GenBank. Positive correlation was found between the antibody titers and the severity of atrophic change of the stomach. In Japan the nucleotide sequences of the vacA middle region were highly homologous and genetically closer to tox⁺ strains. Antibody titers and host response to cytotoxin may be associated with atrophy of the stomach.