Inhibition of O-GlcNAcylation protects from Shiga toxin-mediated cell injury and lethality in host.

Shiga toxins (Stxs) produced by enterohemorrhagic Escherichia coli (EHEC) are the major virulence factors responsible for hemorrhagic colitis, which can lead to life-threatening systemic complications including acute renal failure (hemolytic uremic syndrome) and neuropathy. Here, we report that O-GlcNAcylation, a type of post-translational modification, was acutely increased upon induction of endoplasmic reticulum (ER) stress in host cells by Stxs. Suppression of the abnormal Stx-mediated increase in O-GlcNAcylation effectively inhibited apoptotic and inflammatory responses in Stx-susceptible cells. The protective effect of O-GlcNAc inhibition for Stx-mediated pathogenic responses was also verified using three-dimensional (3D)-cultured spheroids or organoids mimicking the human kidney. Treatment with an O-GlcNAcylation inhibitor remarkably improved the major disease symptoms and survival rate for mice intraperitoneally injected with a lethal dose of Stx. In conclusion, this study elucidates O-GlcNAcylation-dependent pathogenic mechanisms of Stxs and demonstrates that inhibition of aberrant O-GlcNAcylation is a potential approach to treat Stx-mediated diseases.

Exosomes released from Shiga toxin 2a-treated human macrophages modulate inflammatory responses and induce cell death in toxin receptor expressing human cells.

Shiga toxins (Stxs) produced by Stx-producing Escherichia coli are the primarily virulence factors of hemolytic uremic syndrome and central nervous system (CNS) impairment. Although the precise mechanisms of toxin dissemination remain unclear, Stxs bind to extracellular vesicles (EVs). Exosomes, a subset of EVs, may play a key role in Stx-mediated renal injury. To test this hypothesis, we isolated exosomes from monocyte-derived macrophages in the presence of Stx2a or Stx2 toxoids. Macrophage-like differentiated THP-1 cells treated with Stxs secreted Stx-associated exosomes (Stx-Exo) of 90-130 nm in diameter, which induced cytotoxicity in recipient cells in a toxin receptor globotriaosylceramide (Gb3 )-dependent manner. Stx2-Exo engulfed by Gb3 -positive cells were translocated to the endoplasmic reticulum in the human proximal tubule epithelial cell line HK-2. Stx2-Exo contained pro-inflammatory cytokine mRNAs and proteins and induced more severe inflammation than purified Stx2a accompanied by greater death of target cells such as human renal or retinal pigment epithelial cells. Blockade of exosome biogenesis using the pharmacological inhibitor GW4869 reduced Stx2-Exo-mediated human renal cell death. Stx2-Exo isolated from human primary monocyte-derived macrophages activated caspase 3/7 and resulted in significant cell death in primary human renal cortical epithelial cells. Based on these results, we speculate that Stx-containing exosomes derived from macrophages may exacerbate cytotoxicity and inflammation and trigger cell death in toxin-sensitive cells. Therapeutic interventions targeting Stx-containing exosomes may prevent or ameliorate Stx-mediated acute vascular dysfunction.

Roles of Shiga Toxins in Immunopathology.

Shigella species and Shiga toxin-producing Escherichia coli (STEC) are agents of bloody diarrhea that may progress to potentially lethal complications such as diarrhea-associated hemolytic uremic syndrome (D+HUS) and neurological disorders. The bacteria share the ability to produce virulence factors called Shiga toxins (Stxs). Research over the past two decades has identified Stxs as multifunctional toxins capable of inducing cell stress responses in addition to their canonical ribotoxic function inhibiting protein synthesis. Notably, Stxs are not only potent inducers of cell death, but also activate innate immune responses that may lead to inflammation, and these effects may increase the severity of organ injury in patients infected with Stx-producing bacteria. In the intestines, kidneys, and central nervous system, excessive or uncontrolled host innate and cellular immune responses triggered by Stxs may result in sensitization of cells to toxin mediated damage, leading to immunopathology and increased morbidity and mortality in animal models (including primates) and human patients. Here, we review studies describing Stx-induced innate immune responses that may be associated with tissue damage, inflammation, and complement activation. We speculate on how these processes may contribute to immunopathological responses to the toxins.

Shiga Toxins Induce Apoptosis and ER Stress in Human Retinal Pigment Epithelial Cells.

Shiga toxins (Stxs) produced by Shiga toxin-producing bacteria Shigella dysenteriae serotype 1 and select serotypes of Escherichia coli are the most potent known virulence factors in the pathogenesis of hemorrhagic colitis progressing to potentially fatal systemic complications such as acute renal failure, blindness and neurological abnormalities. Although numerous studies have defined apoptotic responses to Shiga toxin type 1 (Stx1) or Shiga toxin type 2 (Stx2) in a variety of cell types, the potential significance of Stx-induced apoptosis of photoreceptor and pigmented cells of the eye following intoxication is unknown. We explored the use of immortalized human retinal pigment epithelial (RPE) cells as an in vitro model of Stx-induced retinal damage. To the best of our knowledge, this study is the first report that intoxication of RPE cells with Stxs activates both apoptotic cell death signaling and the endoplasmic reticulum (ER) stress response. Using live-cell imaging analysis, fluorescently labeled Stx1 or Stx2 were internalized and routed to the RPE cell endoplasmic reticulum. RPE cells were significantly sensitive to wild type Stxs by 72 h, while the cells survived challenge with enzymatically deficient mutant toxins (Stx1A- or Stx2A-). Upon exposure to purified Stxs, RPE cells showed activation of a caspase-dependent apoptotic program involving a reduction of mitochondrial transmembrane potential (Δψm), increased activation of ER stress sensors IRE1, PERK and ATF6, and overexpression CHOP and DR5. Finally, we demonstrated that treatment of RPE cells with Stxs resulted in the activation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38MAPK), suggesting that the ribotoxic stress response may be triggered. Collectively, these data support the involvement of Stx-induced apoptosis in ocular complications of intoxication. The evaluation of apoptotic responses to Stxs by cells isolated from multiple organs may reveal unique functional patterns of the cytotoxic actions of these toxins in the systemic complications that follow ingestion of toxin-producing bacteria.

Toxins: State of the Journal Report, 2017.

On behalf of the Toxins editorial team, we are happy to report that the impact factor for Toxins for 2015 is 3.571, 5-year impact factor: 3.942, which places the journal at a ranking of 16th out of 89 journals covering the field of toxicology. [...].

Announcing the 2017 Toxins Travel Awards for Post-Doctoral Fellows and Ph.D. Students.

n/a.

Shiga Toxins as Multi-Functional Proteins: Induction of Host Cellular Stress Responses, Role in Pathogenesis and Therapeutic Applications.

Shiga toxins (Stxs) produced by Shiga toxin-producing bacteria Shigella dysenteriae serotype 1 and select serotypes of Escherichia coli are primary virulence factors in the pathogenesis of hemorrhagic colitis progressing to potentially fatal systemic complications, such as hemolytic uremic syndrome and central nervous system abnormalities. Current therapeutic options to treat patients infected with toxin-producing bacteria are limited. The structures of Stxs, toxin-receptor binding, intracellular transport and the mode of action of the toxins have been well defined. However, in the last decade, numerous studies have demonstrated that in addition to being potent protein synthesis inhibitors, Stxs are also multifunctional proteins capable of activating multiple cell stress signaling pathways, which may result in apoptosis, autophagy or activation of the innate immune response. Here, we briefly present the current understanding of Stx-activated signaling pathways and provide a concise review of therapeutic applications to target tumors by engineering the toxins.

Shiga Toxins Activate the NLRP3 Inflammasome Pathway To Promote Both Production of the Proinflammatory Cytokine Interleukin-1ß and Apoptotic Cell Death.

Shiga toxin (Stx)-mediated immune responses, including the production of the proinflammatory cytokines tumor necrosis-α (TNF-α) and interleukin-1ß (IL-1ß), may exacerbate vascular damage and accelerate lethality. However, the immune signaling pathway activated in response to Stx is not well understood. Here, we demonstrate that enzymatically active Stx, which leads to ribotoxic stress, triggers NLRP3 inflammasome-dependent caspase-1 activation and IL-1ß secretion in differentiated macrophage-like THP-1 (D-THP-1) cells. The treatment of cells with a chemical inhibitor of glycosphingolipid biosynthesis, which suppresses the expression of the Stx receptor globotriaosylceramide and subsequent endocytosis of the toxin, substantially blocked activation of the NLRP3 inflammasome and processing of caspase-1 and IL-1ß. Processing and release of both caspase-1 and IL-1ß were significantly reduced or abolished in Stx-intoxicated D-THP-1 cells in which the expression of NLRP3 or ASC was stably knocked down. Furthermore, Stx mediated the activation of caspases involved in apoptosis in an NLRP3- or ASC-dependent manner. In Stx-intoxicated cells, the NLRP3 inflammasome triggered the activation of caspase-8/3, leading to the initiation of apoptosis, in addition to caspase-1-dependent pyroptotic cell death. Taken together, these results suggest that Stxs trigger the NLRP3 inflammasome pathway to release proinflammatory IL-1ß as well as to promote apoptotic cell death.

Distinct renal pathology and a chemotactic phenotype after enterohemorrhagic Escherichia coli shiga toxins in non-human primate models of hemolytic uremic syndrome.

Enterohemorrhagic Escherichia coli cause approximately 1.5 million infections globally with 176,000 cases occurring in the United States annually from ingesting contaminated food, most frequently E. coli O157:H7 in ground beef or fresh produce. In severe cases, the painful prodromal hemorrhagic colitis is complicated by potentially lethal hemolytic uremic syndrome (HUS), particularly in children. Bacterial Shiga-like toxins (Stx1, Stx2) are primarily responsible for HUS and the kidney and neurologic damage that ensue. Small animal models are hampered by the inability to reproduce HUS with thrombotic microangiopathy, hemolytic anemia, and acute kidney injury. Earlier, we showed that nonhuman primates (Papio) recapitulated clinical HUS after Stx challenge and that novel therapeutic intervention rescued the animals. Here, we present detailed light and electron microscopic pathology examination of the kidneys from these Stx studies. Stx1 challenge resulted in more severe glomerular endothelial injury, whereas the glomerular injury after Stx2 also included prominent mesangiolysis and an eosinophilic inflammatory infiltration. Both toxins induced glomerular platelet-rich thrombi, interstitial hemorrhage, and tubular injury. Analysis of kidney and other organs for inflammation biomarkers showed a striking chemotactic profile, with extremely high mRNA levels for IL-8, monocyte chemoattractant protein 1, and macrophage inflammatory protein 1α and elevated urine chemokines at 48 hours after challenge. These observations give unique insight into the pathologic consequences of each toxin in a near human setting and present potential pathways for therapeutic intervention.

Shiga toxins expressed by human pathogenic bacteria induce immune responses in host cells.

Shiga toxins are a family of genetically and structurally related toxins that are the primary virulence factors produced by the bacterial pathogens Shigella dysenteriae serotype 1 and certain Escherichia coli strains. The toxins are multifunctional proteins inducing protein biosynthesis inhibition, ribotoxic and ER stress responses, apoptosis, autophagy, and inflammatory cytokine and chemokine production. The regulated induction of inflammatory responses is key to minimizing damage upon injury or pathogen-mediated infections, requiring the concerted activation of multiple signaling pathways to control cytokine/chemokine expression. Activation of host cell signaling cascades is essential for Shiga toxin-mediated proinflammatory responses and the contribution of the toxins to virulence. Many studies have been reported defining the inflammatory response to Shiga toxins in vivo and in vitro, including production and secretion of tumor necrosis factor alpha (TNF-α), interleukin-1ß (IL-1ß), macrophage inflammatory protein-1α/ß (MIP-1α/ß), macrophage chemoattractant monocyte chemoattractant protein 1 (MCP-1), interleukin 8 (IL-8), interleukin 6 (IL-6), and Groß. These cytokines and chemokines may contribute to damage in the colon and development of life threatening conditions such as acute renal failure (hemolytic uremic syndrome) and neurological abnormalities. In this review, we summarize recent findings in Shiga toxin-mediated inflammatory responses by different types of cells in vitro and in animal models. Signaling pathways involved in the inflammatory responses are briefly reviewed.

Regulation of cytokine and chemokine expression by the ribotoxic stress response elicited by Shiga toxin type 1 in human macrophage-like THP-1 cells.

Shiga toxins (Stxs) are cytotoxins produced by the enteric pathogens Shigella dysenteriae serotype 1 and Shiga toxin-producing Escherichia coli (STEC). Stxs bind to a membrane glycolipid receptor, enter cells, and undergo retrograde transport to ultimately reach the cytosol, where the toxins exert their protein synthesis-inhibitory activity by depurination of a single adenine residue from the 28S rRNA component of eukaryotic ribosomes. The depurination reaction activates the ribotoxic stress response, leading to signaling via the mitogen-activated protein kinase (MAPK) pathways (Jun N-terminal protein kinase [JNK], p38, and extracellular signal-regulated kinase [ERK]) in human epithelial, endothelial, and myeloid cells. We previously showed that treatment of human macrophage-like THP-1 cells with Stxs resulted in increased cytokine and chemokine expression. In the present study, we show that individual inactivation of ERK, JNK, and p38 MAPKs using pharmacological inhibitors in the presence of Stx1 resulted in differential regulation of the cytokines tumor necrosis factor alpha and interleukin-1ß (IL-1ß) and chemokines IL-8, growth-regulated protein-ß, macrophage inflammatory protein-1α (MIP-1α), and MIP-1ß. THP-1 cells exposed to Stx1 upregulate the expression of select dual-specificity phosphatases (DUSPs), enzymes that dephosphorylate and inactivate MAPKs in mammalian cells. In this study, we confirmed DUSP1 protein production by THP-1 cells treated with Stx1. DUSP1 inhibition by triptolide showed that ERK and p38 phosphorylation is regulated by DUSP1, while JNK phosphorylation is not. Inhibition of p38 MAPK signaling blocked the ability of Stx1 to induce DUSP1 mRNA expression, suggesting that an autoregulatory signaling loop may be activated by Stxs. Thus, Stxs appear to be capable of eliciting signals which both activate and deactivate signaling for increased cytokine/chemokine production in human macrophage-like cells.

Coxiella burnetii induces apoptosis during early stage infection via a caspase-independent pathway in human monocytic THP-1 cells.

The ability of Coxiella burnetii to modulate host cell death may be a critical factor in disease development. In this study, human monocytic THP-1 cells were used to examine the ability of C. burnetii Nine Mile phase II (NMII) to modulate apoptotic signaling. Typical apoptotic cell morphological changes and DNA fragmentation were detected in NMII infected cells at an early stage of infection. FACS analysis using Annexin-V-PI double staining showed the induction of a significant number of apoptotic cells at an early stage of NMII infection. Double staining of apoptotic cell DNA and intracellular C. burnetii indicates that NMII infected cells undergoing apoptosis. Interestingly, caspase-3 was not cleaved in NMII infected cells and the caspase-inhibitor Z-VAD-fmk did not prevent NMII induced apoptosis. Surprisingly, the caspase-3 downstream substrate PARP was cleaved in NMII infected cells. These results suggest that NMII induces apoptosis during an early stage of infection through a caspase-independent pathway in THP-1 cells. In addition, NMII-infected monocytes were unable to prevent exogenous staurosporine-induced apoptotic death. Western blot analysis indicated that NMII infection induced the translocation of AIF from mitochondria into the nucleus. Cytochrome c release and cytosol-to-mitochondrial translocation of the pore-forming protein Bax in NMII infected cells occurred at 24 h post infection. These data suggest that NMII infection induced caspase-independent apoptosis through a mechanism involving cytochrome c release, cytosol-to-mitochondrial translocation of Bax and nuclear translocation of AIF in THP-1 monocytes. Furthermore, NMII infection increased TNF-α production and neutralization of TNF-α in NMII infected cells partially blocked PARP cleavage, suggesting TNF-α may play a role in the upstream signaling involved in NMII induced apoptosis. Antibiotic inhibition of C. burnetii RNA synthesis blocked NMII infection-induced PARP activation. These results suggest that both intracellular C. burnetii replication and secreted TNF-α contribute to NMII infection-triggered apoptosis during an early stage of infection.

Activation of cell stress response pathways by Shiga toxins.

Shiga toxin-producing bacteria cause widespread outbreaks of bloody diarrhoea that may progress to life-threatening systemic complications. Shiga toxins (Stxs), the main virulence factors expressed by the pathogens, are ribosome-inactivating proteins which inhibit protein synthesis by removing an adenine residue from 28S rRNA. Recently, Stxs were shown to activate multiple stress-associated signalling pathways in mammalian cells. The ribotoxic stress response is activated following the depurination reaction localized to the α-sarcin/ricin loop of eukaryotic ribosomes. The unfolded protein response (UPR) may be initiated by toxin unfolding within the endoplasmic reticulum, and maintained by production of truncated, misfolded proteins following intoxication. Activation of the ribotoxic stress response leads to signalling through MAPK cascades, which appears to be critical for activation of innate immunity and regulation of apoptosis. Precise mechanisms linking ribosomal damage with MAPK activation require clarification but may involve recognition of ribosomal conformational changes and binding of protein kinases to ribosomes, which activate MAP3Ks and MAP2Ks. Stxs appear capable of activating all ER membrane localized UPR sensors. Prolonged signalling through the UPR induces apoptosis in some cell types. The characterization of stress responses activated by Stxs may identify targets for the development of interventional therapies to block cell damage and disease progression.

The induction of apoptosis by Shiga toxins and ricin.

Shiga toxins and ricin are ribosome-inactivating proteins which share the property of inhibiting protein synthesis by catalytic inactivation of eukaryotic ribosomes. There is now abundant evidence that Shiga toxins and ricin induce apoptosis in epithelial, endothelial, lymphoid and myeloid cells in vitro, and in multiple organs in animals when administered these toxins. Many studies suggest that protein synthesis inhibition and apoptosis induction mediated by Shiga toxins and ricin may be dissociated. In some cells, non-enzymatic toxin components (Shiga toxin B-subunits, ricin B-chain) appear capable of inducing apoptosis. The toxins appear capable of activating components of both the extrinsic or death receptor-mediated and intrinsic or mitochondrial-mediated pathways of apoptosis induction. Although the toxins have been shown to be capable of activating several cell stress response pathways, the precise signaling mechanisms by which Shiga toxins and ricin induce apoptosis remain to be fully characterized. This chapter provides an overview of studies describing Shiga toxin- and ricin-induced apoptosis and reviews evidence that signaling through the ribotoxic stress response and the unfolded protein response may be involved in apoptosis induction in some cell types.

Shiga toxins induce autophagy leading to differential signalling pathways in toxin-sensitive and toxin-resistant human cells.

The bacterial virulence factors Shiga toxins (Stxs) are expressed by Shigella dysenteriae serotype 1 and certain Escherichia coli strains. Stxs are protein synthesis inhibitors and induce apoptosis in many cell types. Stxs induce apoptosis via prolonged endoplasmic reticulum stress signalling to activate both extrinsic and intrinsic pathways in human myeloid cells. Studies have shown that autophagy, a lysosome-dependent catabolic process, may be associated with activation of pro-survival or death processes. It is currently unknown if autophagy contributes to apoptosis or protects cells from Stxs. To study cellular responses to Stxs, we intoxicated toxin-sensitive cells (THP-1 and HK-2 cells), and toxin-resistant cells (primary human monocyte-derived macrophages) and examined toxin intracellular trafficking and autophagosome formation. Stxs translocated to different cell compartments in toxin-resistant versus toxin-sensitive cells. Confocal microscopy revealed autophagosome formation in both toxin-resistant and toxin-sensitive cells. Proteolytic cleavage of Atg5 and Beclin-1 plays pivotal roles in switching non-cytotoxic autophagy to cell death signalling. We detected cleaved forms of Atg5 and Beclin-1 in Stx-treated toxin-sensitive cells, while cleaved caspases, calpains, Atg5 and Beclin-1 were not detected in toxin-resistant primary human monocytes and macrophages. These findings suggest that toxin sensitivity correlates with caspase and calpain activation, leading to Atg5 and Beclin-1 cleavage.

Differential response of the human renal proximal tubular epithelial cell line HK-2 to Shiga toxin types 1 and 2.

Shiga toxins (Stxs) are expressed by the enteric pathogens Shigella dysenteriae serotype 1 and certain serotypes of Escherichia coli. Stx-producing bacteria cause bloody diarrhea with the potential to progress to acute renal failure. Stxs are potent protein synthesis inhibitors and are the primary virulence factors responsible for renal damage that may follow diarrheal disease. We explored the use of the immortalized human proximal tubule epithelial cell line HK-2 as an in vitro model of Stx-induced renal damage. We showed that these cells express abundant membrane Gb(3) and are differentially susceptible to the cytotoxic action of Stxs, being more sensitive to Shiga toxin type 1 (Stx1) than to Stx2. At early time points (24 h), HK-2 cells were significantly more sensitive to Stxs than Vero cells; however, by 72 h, Vero cell monolayers were completely destroyed while some HK-2 cells survived toxin challenge, suggesting that a subpopulation of HK-2 cells are relatively toxin resistant. Fluorescently labeled Stx1 B subunits localized to both lysosomal and endoplasmic reticulum (ER) compartments in HK-2 cells, suggesting that differences in intracellular trafficking may play a role in susceptibility to Stx-mediated cytotoxicity. Although proinflammatory cytokines were not upregulated by toxin challenge, Stx2 selectively induced the expression of two chemokines, macrophage inflammatory protein-1α (MIP-1α) and MIP-1ß. Stx1 and Stx2 differentially activated components of the ER stress response in HK-2 cells. Finally, we demonstrated significant poly(ADP-ribose) polymerase (PARP) cleavage after exposure to Stx1 or Stx2. However, procaspase 3 cleavage was undetectable, suggesting that HK-2 cells may undergo apoptosis in response to Stxs in a caspase 3-independent manner.

Role of tumor necrosis factor alpha in disease using a mouse model of Shiga toxin-mediated renal damage.

Mice have been extensively employed as an animal model of renal damage caused by Shiga toxins. In this study, we examined the role of the proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha) in the development of toxin-mediated renal disease in mice. Mice pretreated with TNF-alpha and challenged with Shiga toxin type 1 (Stx1) showed increased survival compared to that of mice treated with Stx1 alone. Conversely, mice treated with Stx1 before TNF-alpha administration succumbed more quickly than mice given Stx1 alone. Increased lethality in mice treated with Stx1 followed by TNF-alpha was associated with evidence of glomerular damage and the loss of renal function. No differences in renal histopathology were noted between animals treated with Stx1 alone and the TNF-alpha pretreatment group, although we noted a sparing of renal function when TNF-alpha was administered before toxin. Compared to that of treatment with Stx1 alone, treatment with TNF-alpha after toxin altered the renal cytokine profile so that the expression of proinflammatory cytokines TNF-alpha and interleukin-1beta (IL-1beta) increased, and the expression of the anti-inflammatory cytokine IL-10 decreased. Increased lethality in mice treated with Stx1 followed by TNF-alpha was associated with higher numbers of dUTP-biotin nick end labeling-positive renal tubule cells, suggesting that increased lethality involved enhanced apoptosis. These data suggest that the early administration of TNF-alpha is a candidate interventional strategy blocking disease progression, while TNF-alpha production after intoxication exacerbates disease.

Signaling through C/EBP homologous protein and death receptor 5 and calpain activation differentially regulate THP-1 cell maturation-dependent apoptosis induced by Shiga toxin type 1.

Shiga toxins (Stxs) induce apoptosis via activation of the intrinsic and extrinsic pathways in many cell types. Toxin-mediated activation of the endoplasmic reticulum (ER) stress response was shown to be instrumental in initiating apoptosis in THP-1 myeloid leukemia cells. THP-1 cells responded to Shiga toxin type 1 (Stx1) in a cell maturation-dependent manner, undergoing rapid apoptosis in the undifferentiated state but reduced and delayed apoptosis in differentiated cells. The onset of apoptosis was associated with calpain activation and changes in expression of C/EBP homologous protein (CHOP), Bcl-2 family members, and death receptor 5 (DR5). Ligation of DR5 by tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) activates the extrinsic pathway of apoptosis. We show here that expression of TRAIL and DR5 is increased by Stx1 treatment. Addition of exogenous TRAIL enhances, and anti-TRAIL antibodies inhibit, Stx1-induced apoptosis of THP-1 cells. Silencing of CHOP or DR5 expression selectively prevented caspase activation, loss of mitochondrial membrane potential, and Stx1-induced apoptosis of macrophage-like THP-1 cells. In contrast, the rapid kinetics of apoptosis induction in monocytic THP-1 cells correlated with rates of calpain cleavage. The results suggest that CHOP-DR5 signaling and calpain activation differentially contribute to cell maturation-dependent Stx1-induced apoptosis. Inhibition of these signaling pathways may protect cells from Stx cytotoxicity.