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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-1β (IL-1β) and chemokines IL-8, growth-regulated protein-β, macrophage inflammatory protein-1α (MIP-1α), and MIP-1β. 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.

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-1β. 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.

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.

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.

Shiga toxins (Stxs) are bacterial cytotoxins produced by the enteric pathogens Shigella dysenteriae serotype 1 and some serotypes of Escherichia coli that cause bacillary dysentery and hemorrhagic colitis, respectively. To date, approaches to studying the capacity of Stxs to alter gene expression in intoxicated cells have been limited to individual genes. However, it is known that many of the signaling pathways activated by Stxs regulate the expression of multiple genes in mammalian cells. To expand the scope of analysis of gene expression and to better understand the underlying mechanisms for the various effects of Stxs on host cell functions, we carried out comparative microarray analyses to characterize the global transcriptional response of human macrophage-like THP-1 cells to Shiga toxin type 1 (Stx1) and lipopolysaccharides. The data were analyzed by using a rigorous combinatorial approach with three separate statistical algorithms. A total of 36 genes met the criteria of upregulated expression in response to Stx1 treatment, with 14 genes uniquely upregulated by Stx1. Microarray data were validated by real-time reverse transcriptase PCR for genes encoding early growth response 1 (Egr-1) (transcriptional regulator), cyclooxygenase 2 (COX-2; inflammation), and dual specificity phosphatase 1 (DUSP1), DUSP5, and DUSP10 (regulation of mitogen-activated protein kinase signaling). Stx1-mediated signaling through extracellular signal-regulated kinase 1/2 and Egr-1 appears to be involved in the increased expression and production of the proinflammatory mediator tumor necrosis factor alpha. Activation of COX-2 is associated with the increased production of proinflammatory and vasoactive eicosanoids. However, the capacity of Stx1 to increase the expression of genes encoding phosphatases suggests that mechanisms to dampen the macrophage proinflammatory response may be built into host response to the toxins.

Shiga toxins (Stxs), which are proteins expressed by the enteric pathogens Shigella dysenteriae serotype 1 and some serotypes of Escherichia coli , are potent protein synthesis inhibitors. Stx-producing organisms cause bloody diarrhea with the potential to progress to acute renal failure and central nervous system complications. Studies using animal models of these diseases have shown that Stxs are major virulence factors, and purified toxins have been shown to be capable of killing many types of cells in vitro. We showed that Stx type 1 (Stx1) rapidly induced apoptosis in undifferentiated, monocytic THP-1 cells through a mechanism involving the endoplasmic reticulum (ER) stress response. Rapid apoptosis correlated with increased expression of C/EBP homologous protein (CHOP), TRAIL, and DR5, while expression of the antiapoptotic factor Bcl-2 was downregulated. Stx1 treatment of differentiated, macrophage-like THP-1 cells was associated with cytokine production and delayed apoptosis. The mechanisms contributing to cell maturation-dependent differences in responses to Stx1 are unknown. We show here that in macrophage-like cells, Stx1 activated the proximal ER stress sensors RNA-dependent protein kinase-like ER kinase and inositol-requiring ER signal kinase 1α but did not activate activating transcription factor 6. Proapoptotic signaling pathways mediated by CHOP and by Bax and Bak were activated by Stx1. However, the toxin also activated prosurvival signaling through increased expression, mitochondrial translocation, and alternative phosphorylation of Bcl-2.