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

Department of Molecular Infectiology, Graduate School of Medicine, Chiba University, Chiba, Japan.
Infection and immunity (Impact Factor: 3.73). 05/2009; 77(7):2919-24. DOI: 10.1128/IAI.01510-08
Source: PubMed


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.

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    • "In previous studies, SubAB toxin was found to be lethal for mice, resulting in extensive microvascular damage, thrombosis and necrosis in several organs, including the kidneys [6], [28], features observed in Stx-induced HUS in humans. The holotoxin was also proved to be highly toxic for several cell lines, including Vero cells [6], [23]–[27]. However, the role that SubAB plays in the pathogenesis of HUS remains to be elucidated. "
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    ABSTRACT: Shiga toxin (Stx)-producing Escherichia coli (STEC) cause post-diarrhea Hemolytic Uremic Syndrome (HUS), which is the most common cause of acute renal failure in children in many parts of the world. Several non-O157 STEC strains also produce Subtilase cytotoxin (SubAB) that may contribute to HUS pathogenesis. The aim of the present work was to examine the cytotoxic effects of SubAB on primary cultures of human cortical renal tubular epithelial cells (HRTEC) and compare its effects with those produced by Shiga toxin type 2 (Stx2), in order to evaluate their contribution to renal injury in HUS. For this purpose, cell viability, proliferation rate, and apoptosis were assayed on HRTEC incubated with SubAB and/or Stx2 toxins. SubAB significantly reduced cell viability and cell proliferation rate, as well as stimulating cell apoptosis in HRTEC cultures in a time dependent manner. However, HRTEC cultures were significantly more sensitive to the cytotoxic effects of Stx2 than those produced by SubAB. No synergism was observed when HRTEC were co-incubated with both SubAB and Stx2. When HRTEC were incubated with the inactive SubAA272B toxin, results were similar to those in untreated control cells. Similar stimulation of apoptosis was observed in Vero cells incubated with SubAB or/and Stx2, compared to HRTEC. In conclusion, primary cultures of HRTEC are significantly sensitive to the cytotoxic effects of SubAB, although, in a lesser extent compared to Stx2.
    Full-text · Article · Jan 2014 · PLoS ONE
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    • "SubAB action on eukaryotic cells involves highly specific A-subunit-mediated proteolytic cleavage of the endoplasmic reticulum (ER) chaperone BiP (GRP78) [29]. This triggers a massive ER stress response, ultimately leading to apoptosis [30–32]. Recently, it was shown that this toxin increases the tissue factor-dependent factor Xa generation in cultured human umbilical vein endothelial cells and human macrophages, suggesting a direct procoagulant effect [33]. "
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    ABSTRACT: The hemolytic uremic syndrome (HUS) associated with diarrhea is a complication of Shiga toxin (Stx)-producing Escherichia coli (STEC) infection. In Argentina, HUS is endemic and responsible for acute and chronic renal failure in children younger than 5 years old. The human kidney is the most affected organ due to the presence of very Stx-sensitive cells, such as microvascular endothelial cells. Recently, Subtilase cytotoxin (SubAB) was proposed as a new toxin that may contribute to HUS pathogenesis, although its action on human glomerular endothelial cells (HGEC) has not been described yet. In this study, we compared the effects of SubAB with those caused by Stx2 on primary cultures of HGEC isolated from fragments of human pediatric renal cortex. HGEC were characterized as endothelial since they expressed von Willebrand factor (VWF) and platelet/endothelial cell adhesion molecule 1 (PECAM-1). HGEC also expressed the globotriaosylceramide (Gb3) receptor for Stx2. Both, Stx2 and SubAB induced swelling and detachment of HGEC and the consequent decrease in cell viability in a time-dependent manner. Preincubation of HGEC with C-9 -a competitive inhibitor of Gb3 synthesis-protected HGEC from Stx2 but not from SubAB cytotoxic effects. Stx2 increased apoptosis in a time-dependent manner while SubAB increased apoptosis at 4 and 6 h but decreased at 24 h. The apoptosis induced by SubAB relative to Stx2 was higher at 4 and 6 h, but lower at 24 h. Furthermore, necrosis caused by Stx2 was significantly higher than that induced by SubAB at all the time points evaluated. Our data provide evidence for the first time how SubAB could cooperate with the development of endothelial damage characteristic of HUS pathogenesis.
    Full-text · Article · Jul 2013 · PLoS ONE
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    • "Morinaga et al. [22] also reported transient inhibition of protein synthesis in SubAB-teated Vero cells due to PERK-mediated eIF2α phosphorylation; the toxin also induced cell cycle arrest in G1 phase, possibly through down-regulation of cyclin D1 due to a combination of translational inhibition and proteasomal degradation. More recently, Matsuura et al. [23] demonstrated SubAB-induced apoptosis in Vero cells by DNA fragmentation and annexin V labeling, as well as activation of caspase-3, caspase-7 and caspase-8; release of cytochrome c from mitochondria was also demonstrated. "
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    ABSTRACT: Subtilase cytotoxin (SubAB) is the prototype of a new AB(5) toxin family produced by a subset of Shiga toxigenic Escherichia coli (STEC) strains. Its A subunit is a subtilase-like serine protease and cytotoxicity for eukaryotic cells is due to a highly specific, single-site cleavage of BiP/GRP78, an essential Hsp70 family chaperone located in the endoplasmic reticulum (ER). This cleavage triggers a severe and unresolved ER stress response, ultimately triggering apoptosis. The B subunit has specificity for glycans terminating in the sialic acid N-glycolylneuraminic acid. Although its actual role in human disease pathogenesis is yet to be established, SubAB is lethal for mice and induces pathological features overlapping those seen in the haemolytic uraemic syndrome, a life-threatening complication of STEC infection. The toxin is also proving to be a useful tool for probing the role of BiP and ER stress in a variety of cellular functions.
    Full-text · Article · Feb 2010 · Toxins
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