Shiga Toxins Stimulate Secretion of Interleukin-8 from Intestinal Epithelial Cells

In the 1980s, Shiga toxin (Stx)-producing Escherichia coli O157:H7 (STEC) was identified as a cause of hemorrhagic colitis in the United States and was found to be associated with hemolytic uremic syndrome (HUS), a microangiopathic hemolytic anemia characterized by thrombocytopenia and renal failure. The precise way that Stxs cause hemorrhagic colitis and HUS is unclear. Stxs have been thought to cause disease by killing or irreversibly harming sensitive cells through a nonspecific blockade of mRNA translation, eventually resulting in cytotoxicity by preventing synthesis of critical molecules needed to maintain cell integrity. Because STEC is noninvasive, we have been exploring the host-toxin response at the level of the gastrointestinal mucosa, where STEC infection begins. We have found that Stx is capable of interleukin-8 (IL-8) superinduction in a human colonic epithelial cell line. Despite a general blockade of mRNA translation, Stx treatment results in increased IL-8 mRNA as well as increased synthesis and secretion of IL-8 protein. Our data suggest that an active Stx A subunit is required for this activity. Ricin, which has the same enzymatic activity and trafficking pathway as Stx, has similar effects. Exploration of the effects of other protein synthesis inhibitors (cycloheximide, anisomycin) suggests a mechanism of gene regulation that is distinct from a general translational blockade. Use of the specific p38/RK inhibitor SB202190 showed that blocking of this pathway results in decreased Stx-mediated IL-8 secretion. Furthermore, Stxs induced mRNA of the primary response gene c-jun, which was subsequently partially blocked by SB202190. These data suggest a novel model of how Stxs contribute to disease, namely that Stxs may alter regulation of host cell processes in sensitive cells via activation of at least one member of the mitogen-activated protein kinase family in the p38/RK cascade and induction of c-jun mRNA. Stx-induced increases in chemokine synthesis from intestinal epithelial cells could be important in augmenting the host mucosal inflammatory response to STEC infection.     

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-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 Toxin Translocation across Intestinal Epithelial Cells Is Enhanced by Neutrophil Transmigration

Shiga toxin-producing E. coli (STEC) is a food-borne pathogen that causes serious illness, including hemolytic-uremic syndrome (HUS). STEC colonizes the lower intestine and produces Shiga toxins (Stxs). Stxs appear to translocate across intestinal epithelia and affect sensitive endothelial cell beds at various sites. We have previously shown that Stxs cross polarized intestinal epithelial cells (IECs) via a transcellular route and remain biologically active. Since acute inflammatory infiltration of the gut and fecal leukocytes is seen in many STEC-infected patients and since polymorphonuclear leukocyte (PMN) transmigration across polarized IECs diminishes the IEC barrier function in vitro, we hypothesized that PMN transmigration may enhance Stx movement across IECs. We found that basolateral-to-apical transmigration of neutrophils significantly increased the movement of Stx1 and Stx2 across polarized T84 IECs in the opposite direction. The amount of Stx crossing the T84 barrier was proportional to the degree of neutrophil transmigration, and the increase in Stx translocation appears to be due to increases in paracellular permeability caused by migrating PMNs. STEC clinical isolates applied apically induced PMN transmigration across and interleukin-8 (IL-8) secretion from T84 cells. Of the 10 STEC strains tested, three STEC strains lacking eae and espB (eae- and espB-negative STEC strains) induced significantly more neutrophil transmigration and significantly greater IL-8 secretion than eae- and espB-positive STEC or enteropathogenic E. coli. This study suggests that STEC interaction with intestinal epithelia induces neutrophil recruitment to the intestinal lumen, resulting in neutrophil extravasation across IECs, and that during this process Stxs may pass in greater amounts into underlying tissues, thereby increasing the risk of HUS.     

Shiga Toxins Induce, Superinduce, and Stabilize a Variety of C-X-C Chemokine mRNAs in Intestinal Epithelial Cells, Resulting in Increased Chemokine Expression

Exposure of humans to Shiga toxins (Stxs) is a risk factor for hemolytic-uremic syndrome (HUS). Because Stx-producing Escherichia coli (STEC) is a noninvasive enteric pathogen, the extent to which Stxs can cross the host intestinal epithelium may affect the risk of developing HUS. We have previously shown that Stxs can induce and superinduce IL-8 mRNA and protein in intestinal epithelial cells (IECs) in vitro via a ribotoxic stress response. We used cytokine expression arrays to determine the effect of Stx1 on various C-X-C chemokine genes in IECs. We observed that Stx1 induces multiple C-X-C chemokines at the mRNA level, including interleukin-8 (IL-8), GRO-alpha, GRO-beta, GRO-gamma, and ENA-78. Like that of IL-8, GRO-alpha and ENA-78 mRNAs are both induced and superinduced by Stx1. Furthermore, Stx1 induces both IL-8 and GRO-alpha protein in a dose-response fashion, despite an overall inhibition in host cell protein synthesis. Stx1 treatment stabilizes both IL-8 and GRO-alpha mRNA. We conclude that Stxs are able to increase mRNA and protein levels of multiple C-X-C chemokines in IECs, with increased mRNA stability at least one mechanism involved. We hypothesize that ribotoxic stress is a pathway by which Stxs can alter host signal transduction in IECs, resulting in the production of multiple chemokine mRNAs, leading to increased expression of specific proteins. Taken together, these data suggest that exposing IECs to Stxs may stimulate a proinflammatory response, resulting in influx of acute inflammatory cells and thus contributing to the intestinal tissue damage seen in STEC infection.     

Activation of the Classical Mitogen-Activated Protein Kinases Is Part of the Shiga Toxin-Induced Ribotoxic Stress Response and May Contribute to Shiga Toxin-Induced Inflammation

Infection with enterohemorrhagic Escherichia coli (EHEC) can result in severe disease, including hemorrhagic colitis and the hemolytic uremic syndrome. Shiga toxins (Stx) are the key EHEC virulence determinant contributing to severe disease. Despite inhibiting protein synthesis, Shiga toxins paradoxically induce the expression of proinflammatory cytokines from various cell types in vitro, including intestinal epithelial cells (IECs). This effect is mediated in large part by the ribotoxic stress response (RSR). The Shiga toxin-induced RSR is known to involve the activation of the stress-activated protein kinases (SAPKs) p38 and JNK. In some cell types, Stx also can induce the classical mitogen-activated protein kinases (MAPKs) or ERK1/2, but the mechanism(s) by which this activation occurs is unknown. In this study, we investigated the mechanism by which Stx activates ERK1/2s in IECs and the contribution of ERK1/2 activation to interleukin-8 (IL-8) expression. We demonstrate that Stx1 activates ERK1/2 in a biphasic manner: the first phase occurs in response to StxB1 subunit, while the second phase requires StxA1 subunit activity. We show that the A subunit-dependent ERK1/2 activation is mediated through ZAK-dependent signaling, and inhibition of ERK1/2 activation via the MEK1/2 inhibitors U0126 and PD98059 results in decreased Stx1-mediated IL-8 mRNA. Finally, we demonstrate that ERK1/2 are activated in vivo in the colon of Stx2-intoxicated infant rabbits, a model in which Stx2 induces a primarily neutrophilic inflammatory response. Together, our data support a role for ERK1/2 activation in the development of Stx-mediated intestinal inflammation.     

Simultaneous induction of apoptotic and survival signaling pathways in macrophage-like THP-1 cells by Shiga toxin 1

Shiga toxins have been shown to induce apoptosis in many cell types. However, Shiga toxin 1 (Stx1) induced only limited apoptosis of macrophage-like THP-1 cells in vitro. The mechanisms regulating macrophage death or survival following toxin challenge are unknown. Differentiated THP-1 cells expressed tumor necrosis factor receptors and membrane-associated tumor necrosis factor alpha (TNF-alpha) and produced soluble TNF-alpha after exposure to Stx1. However, the cells were refractory to apoptosis induced by TNF-alpha, although the cytokine modestly increased apoptosis in the presence of Stx1. Despite the partial resistance of macrophage-like THP-1 cells to Stx1-mediated killing, treatment of these cells with Stx1 activated a broad array of caspases, disrupted the mitochondrial membrane potential (DeltaPsi(m)), and released cytochrome c into the cytoplasm. The DeltaPsi(m) values were greatest in cells that had detached from plastic surfaces. Specific caspase inhibitors revealed that caspase-3, caspase-6, caspase-8, and caspase-9 were primarily involved in apoptosis induction. The antiapoptotic factors involved in macrophage survival following toxin challenge include inhibitors of apoptosis proteins and X-linked inhibitor of apoptosis protein. NF-kappaB and JNK mitogen-activated protein kinases (MAPKs) appeared to activate survival pathways, while p38 MAPK was involved in proapoptotic signaling. The JNK and p38 MAPKs were shown to be upstream signaling pathways which may regulate caspase activation. Finally, the protein synthesis inhibitors Stx1 and anisomycin triggered limited apoptosis and prolonged JNK and p38 MAPK activation, while macrophage-like cells treated with cycloheximide remained viable and showed transient activation of MAPKs. Collectively, these data suggest that Stx1 activates both apoptotic and cell survival signaling pathways in macrophage-like THP-1 cells.     

Protective effect of Lactobacillus casei strain Shirota on Shiga toxin-producing Escherichia coli O157:H7 infection in infant rabbits

We examined colonization patterns of Shiga toxin-producing Escherichia coli (STEC), concentrations of Shiga toxins (Stxs) and specific immunoglobulin A (lgA) against Stxs and STEC bacterial cell surface antigen in various portions of the gastrointestinal tract in an infant rabbit infection model. After inoculation of 3-day-old infant rabbits with STEC strain 89020087 at low doses (approximately 10(3) CFU/body), numbers of colonizing STEC bacteria and concentrations of Stxs in the intestine increased dramatically and the animals developed diarrhea within a couple of days after infection. Daily administration of Lactobacillus casei from the day of birth dramatically decreased the severity of diarrhea and lowered STEC colonization levels in the gastrointestinal tract 100-fold day 7 after infection. Both Stx1 and Stx2 concentrations in the intestines and histological damage to the intestinal mucus induced by STEC infection were decreased by the administration of L. casei. Examination of the concentrations of volatile fatty acids and pH of the intestinal contents revealed that the protective effect of L. casei administration against STEC infection was not due to fermented products such as lactic acid in the gastrointestinal tract. Administration of L. casei increased levels of lgAs against Stx1, Stx2, and formalin-killed STEC cells in the colon approximately two-, four-, and threefold, respectively, compared with those of the untreated controls by day 7 after infection. These results suggest that administration of L. casei strain Shirota enhances the local immune responses to STEC cells and Stxs and leads to elimination of STEC and thus decreases Stx concentrations in the intestines.     

Shiga toxins 1 and 2 translocate differently across polarized intestinal epithelial cells

Shiga toxin-producing Escherichia coli (STEC) is an important food-borne pathogen that causes hemolytic-uremic syndrome. Following ingestion, STEC cells colonize the intestine and produce Shiga toxins (Stx), which appear to translocate across the intestinal epithelium and subsequently reach sensitive endothelial cell beds. STEC cells produce one or both of two major toxins, Stx1 and Stx2. Stx2-producing STEC is more often associated with disease for reasons as yet undetermined. In this study, we used polarized intestinal epithelial cells grown on permeable filters as a model to compare Stx1 and Stx2 movement across the intestinal epithelium. We have previously shown that biologically active Stx1 is able to translocate across cell monolayers in an energy-dependent, saturable manner. This study demonstrates that biologically active Stx2 is also capable of movement across the epithelium without affecting barrier function, but significantly less Stx2 crossed monolayers than Stx1. Chilling the monolayers to 4 degrees C reduced the amount of Stx1 and Stx2 movement by 200-fold and 20-fold respectively. Stx1 movement was clearly directional, favoring an apical-to-basolateral translocation, whereas Stx2 movement was not. Colchicine reduced Stx1, but not Stx2, translocation. Monensin reduced the translocation of both toxins, but the effect was more pronounced with Stx1. Brefeldin A had no effect on either toxin. Excess unlabeled Stx1 blocks the movement of (125)I-Stx1. Excess Stx2 failed to have any effect on Stx1 movement. Our data suggests that, despite the many common physical and biochemical properties of the two toxins, they appear to be crossing the epithelial cell barrier by different pathways.     

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-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.     

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.Shiga toxins (Stxs) are major virulence factors expressed by the enteric pathogens Shigella dysenteriae serotype 1 and certain Escherichia coli serotypes referred to as Shiga toxin-producing E. coli (STEC). Infections with Stx-producing bacteria are associated with watery diarrhea that may progress to bloody diarrhea, acute renal failure, and central nervous system complications such as lethargy, seizures, and paralysis (60). STEC is a particular public health concern in developed nations, with approximately 73,000 cases annually of hemorrhagic colitis caused by E. coli O157:H7 and 37,000 annual cases caused by STEC non-O157 serotypes in the United States (42). The histopathological hallmark of disease caused by Stxs is damage to endothelial cells lining colonic capillaries, renal glomeruli and arterioles, and central nervous system (CNS) blood vessels (46). The essential role of Stxs in pathogenesis has been confirmed using animal models in which the infusion of the toxins causes extensive microvascular thromboses in the kidney and CNS and, in some cases, ataxia and limb paralysis (43, 61). S. dysenteriae serotype 1 produces Shiga toxin, while STEC may express one or more toxin variants categorized as Shiga toxin type 1 (Stx1) or Shiga toxin type 2 (Stx2) based on their antigenic similarity to Shiga toxin (56). All Stxs possess an AB₅ structure composed of a monomeric A subunit in noncovalent association with a pentamer of B subunits (17). The B subunits mediate toxin binding by interaction with the membrane neutral glycolipid globotriaosylceramide (Gb₃) (38). The toxins are then internalized and undergo a complex series of intracellular routing events, collectively termed retrograde transport, which ultimately deliver the toxins to the endoplasmic reticulum (ER) lumen (50). In the ER, the A subunit is proteolytically processed, and a fragment of the A subunit retrotranslocates into the cytosol. The N-glycosidase activity associated with the processed A subunit catalyzes the inactivation of eukaryotic ribosomes and inhibits protein synthesis (12, 51).In addition to the capacity to inhibit protein synthesis, Stxs have been shown to induce apoptosis, or programmed cell death, in many cell types (5). The toxins appear to activate apoptotic signaling through an extrinsic (death receptor-mediated signaling) or an intrinsic (mitochondrion-mediated signaling) pathway. For example, the toxins have been shown to be capable of directly activating initiator and executioner caspase cascades but also to generate truncated BID (tBID) which translocates to mitochondrial membranes, leading to increased mitochondrial membrane permeability, release of cytochrome c, and formation of the apoptosome (6, 18, 34). As a result of signaling through the intrinsic or extrinsic pathway, intoxicated cells display characteristics of apoptosis such as DNA fragmentation, cell shrinkage, membrane blebbing, and chromatin condensation.We previously showed that Stx1 induced apoptosis in the human myelogenous leukemia cell line THP-1 in a cell maturation-dependent manner. Undifferentiated, nonadherent monocytic THP-1 cells underwent rapid apoptosis when treated with Stx1, while differentiation to the adherent, macrophage-like state was associated with increased resistance to the cytotoxic action of the toxins, with only approximately 30% of cells undergoing delayed apoptosis (22). The induction of apoptosis by Stx1 involved the activation of the ER stress response in both monocytic and macrophage-like THP-1 cells (33, 36). Stx1 induced the expression of the ER stress effectors C/EBP homologous protein (CHOP), tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), and death receptor 5 (DR5) in monocytic THP-1 cells. Delivery of functional Stx1 into the cytosol of monocytic THP-1 cells led to downregulated expression of the prosurvival factor Bcl-2, while the delayed-apoptosis phenotype in macrophage-like cells was associated with increased Bcl-2 expression, phosphorylation, and mitochondrial translocation.Increased expression of the apoptosis-inducing factor TRAIL and its death-inducing receptor, DR5, enhances cell death signals triggered during a prolonged ER stress response (23, 68). TRAIL may be membrane associated or may be cleaved from the cell surface by proteases to generate a soluble ligand (26, 40). Engagement of TRAIL with its cognate receptor DR5 activates the extrinsic pathway of apoptosis through DR5 aggregation, the recruitment of the Fas-associated death domain (FADD), and the formation of the death-inducing signaling complex (DISC) (31, 53). The observation that expression of TRAIL and DR5 was upregulated by Stx1 treatment of monocytic THP-1 cells suggested that this receptor-ligand pair may contribute to rapid apoptosis induced by the toxin in these cells. However, we also showed that calpains were rapidly activated by Stx1 in monocytic THP-1 cells, and calpains may directly cleave caspase-3 (36). The studies reported here were designed to characterize the roles of TRAIL/DR5 and calpains in the rapid apoptosis response of monocytic cells and in delayed apoptosis in macrophage-like cells. We show that Stx1-induced apoptotic signaling is amplified by the addition of soluble TRAIL (sTRAIL) and inhibited by exposure of cells to neutralizing anti-TRAIL antibodies prior to intoxication. A reduction in CHOP or DR5 expression using RNA interference (RNAi) techniques markedly protected cells from apoptosis induced by Stx1, linking activation of the ER stress response with apoptosis in this system. Signaling through CHOP and DR5 led to activation of the initiator caspase, caspase-8, and the executioner caspase, caspase-3, in macrophage-like THP-1 cells, but the effect of CHOP and DR5 knockdown on caspase activation and apoptosis of monocytic cells was minimal. In contrast, the rate of calpain activation (cleavage) was directly correlated with the rapid onset of apoptosis in monocytic THP-1 cells.     

Responses of human intestinal microvascular endothelial cells to Shiga toxins 1 and 2 and pathogenesis of hemorrhagic colitis

Endothelial damage is characteristic of infection with Shiga toxin (Stx)-producing Escherichia coli (STEC). Because Stx-mediated endothelial cell damage at the site of infection may lead to the characteristic hemorrhagic colitis of STEC infection, we compared the effects of Stx1 and Stx2 on primary and transformed human intestinal microvascular endothelial cells (HIMEC) to those on macrovascular endothelial cells from human saphenous vein (HSVEC). Adhesion molecule, interleukin-8 (IL-8), and Stx receptor expression, the effects of cytokine activation and Stx toxins on these responses, and Stx1 and Stx2 binding kinetics and bioactivity were measured. Adhesion molecule and IL-8 expression increased in activated HIMEC, but these responses were blunted in the presence of toxin, especially in the presence of Stx1. In contrast to HSVEC, unstimulated HIMEC constitutively expressed Stx receptor at high levels, bound large amounts of toxin, were highly sensitive to toxin, and were not further sensitized by cytokines. Although the binding capacities of HIMEC for Stx1 and Stx2 were comparable, the binding affinity of Stx1 to HIMEC was 50-fold greater than that of Stx2. Nonetheless, Stx2 was more toxic to HIMEC than an equivalent amount of Stx1. The decreased binding affinity and increased toxicity for HIMEC of Stx2 compared to those of Stx1 may be relevant to the preponderance of Stx2-producing STEC involved in the pathogenesis of hemorrhagic colitis and its systemic complications. The differences between primary and transformed HIMEC in these responses were negligible. We conclude that transformed HIMEC lines could represent a simple physiologically relevant model to study the role of Stx in the pathogenesis of hemorrhagic colitis.     

Shiga toxin 1 induces apoptosis in the human myelogenous leukemia cell line THP-1 by a caspase-8-dependent, tumor necrosis factor receptor-independent mechanism

Shiga toxins (Stxs) induce apoptosis in a variety of cell types. Here, we show that Stx1 induces apoptosis in the undifferentiated myelogenous leukemia cell line THP-1 in the absence of tumor necrosis factor alpha (TNF-alpha) or death receptor (TNF receptor or Fas) expression. Caspase-8 and -3 inhibitors blocked, and caspase-6 and -9 inhibitors partially blocked, Stx1-induced apoptosis. Stx1 induced the mitochondrial pathway of apoptosis, as activation of caspase-8 triggered the (i) cleavage of Bid, (ii) disruption of mitochondrial membrane potential, and (iii) release of cytochrome c into the cytoplasm. Caspase-8, -9, and -3 cleavage and functional activities began 4 h after toxin exposure and peaked after 8 h of treatment. Caspase-6 may also contribute to Stx1-induced apoptosis by directly acting on caspase-8. It appears that functional Stx1 holotoxins must be transported to the endoplasmic reticulum to initiate apoptotic signaling through the ribotoxic stress response. These data suggest that Stxs may activate monocyte apoptosis via a novel caspase-8-dependent, death receptor-independent mechanism.     

Subtype-specific suppression of Shiga toxin 2 released from Escherichia coli upon exposure to protein synthesis inhibitors

Shiga toxins (Stx) are important virulence factors in the pathogenesis of severe disease including hemolytic-uremic syndrome, caused by Stx-producing Escherichia coli (STEC). STEC strains increase the release of Stx in vitro following the addition of fluoroquinolones, whereas protein synthesis inhibitors previously have been reported to suppress the release of Stx. The amount of Stx released from wild-type STEC strains incubated with protein synthesis inhibitors was examined by a Vero cell cytotoxicity assay. The amounts released were compared to the Stx type (Stx1 or Stx2) and additionally to the individual subtypes and toxin variants of Stx2. In general, Stx2 release was suppressed significantly upon exposure to protein synthesis inhibitors at MICs, which was not observed in the case of Stx1. Also, the average amount of different Stx2 toxin variants released was suppressed to various levels ranging from 14.0% (Stx2-O157-EDL933) to 94.7% (Stx2d-O8-C466-01B). Clinical studies exploring protein synthesis inhibitors as future candidates for treatment of intestinal infections caused by Stx2-producing STEC should therefore include knowledge of the toxin variant in addition to the subtype.     

Bovine lymphocytes express functional receptors for Escherichia coli Shiga toxin 1

Interactions of Shiga toxins (Stxs) and immune cells contribute to the pathogenesis of diseases due to Stx-producing Escherichia coli (STEC) infections in humans and facilitate the persistence of infection in asymptomatically infected cattle. Our recent findings that bovine B and T lymphocytes express Gb(3)/CD77, the human Stx-receptor, prompted us to determine whether the bovine homologue also mediates binding and internalization of Stx1. In fact, Stx1 holotoxin and recombinant B subunit (rStxB1) bound to stimulated bovine peripheral blood mononuclear cells, especially to those subpopulations (B cells, BoCD8(+) T cells) that are highly sensitive to Stx1. Competition and HPTLC-binding studies confirmed that Stx1 binds to bovine Gb(3), but different receptor isoforms with varying affinities for rStxB1 were expressed during the course of lymphocyte activation. At least one of these isoforms mediated toxin uptake. An anti-StxB1 mouse monoclonal antibody, used as a model for bovine serum antibodies specific for Stx1, modulated rather than generally prevented rStxB1 binding to and internalization by the receptors. The presence of functional Stx1-receptors on bovine lymphocytes explains the immunomodulatory effect of Stx1 observed in cattle at a molecular level. Furthermore, expression of such receptors by bovine but not human T cells enlightens the background for the differential outcome of STEC infections in cattle and man, i.e., persistent infection and development of disease, respectively.     

Neuronal apoptosis and inflammatory responses in the central nervous system of a rabbit treated with Shiga toxin-2

Background  

Shiga toxins (Stxs) are the major agents responsible for hemorrhagic colitis and hemolytic-uremic syndrome (HUS) during infections caused by Stx-producing Escherichia coli (STEC) such as serotype O157:H7. Central nervous system (CNS) involvement is an important determinant of mortality in diarrhea associated-HUS. It has been suggested that vascular endothelial injuries caused by Stxs play a crucial role in the development of the disease. The current study investigates the relationship between the cytotoxic effects of Stxs and inflammatory responses in a rabbit brain treated with Stx2.     

Shiga toxin 2 and lipopolysaccharide induce human microvascular endothelial cells to release chemokines and factors that stimulate platelet function

Shiga toxins (Stxs) produced by Shigella dysenteriae type 1 and enterohemorrhagic Escherichia coli are the most common cause of hemolytic-uremic syndrome (HUS). It is well established that vascular endothelial cells, mainly those located in the renal microvasculature, are targets for Stxs. The aim of the present research was to evaluate whether E. coli-derived Shiga toxin 2 (Stx2) incubated with human microvascular endothelial cells (HMEC-1) induces release of chemokines and other factors that might stimulate platelet function. HMEC-1 were exposed for 24 h in vitro to Stx2, lipopolysaccharide (LPS), or the Stx2-LPS combination, and chemokine production was assessed by immunoassay. More interleukin-8 was released than stromal cell-derived factor 1alpha (SDF-1alpha) or SDF-1beta and RANTES. The Stx2-LPS combination potentiated chemokine release, but Stx2 alone caused more release of SDF-1alpha at 24 h than LPS or Stx2-LPS did. In the presence of low ADP levels, HMEC-1 supernatants activated platelet function assessed by classical aggregometry, single-particle counting, granule secretion, P-selectin exposure, and the formation of platelet-monocyte aggregates. Supernatants from HMEC-1 exposed only to Stx2 exhibited enhanced exposure of platelet P-selectin and platelet-THP-1 cell interactions. Blockade of platelet cyclooxygenase by indomethacin prevented functional activation. The chemokine RANTES enhanced platelet aggregation induced by SDF-1alpha, macrophage-derived chemokine, or thymus and activation-regulated chemokine in the presence of very low ADP levels. These data support the hypothesis that microvascular endothelial cells exposed to E. coli O157:H7-derived Stx2 and LPS release chemokines and other factors, which when combined with low levels of primary agonists, such as ADP, cause platelet activation and promote the renal thrombosis associated with HUS.     

Shiga toxin 1 from Escherichia coli blocks activation and proliferation of bovine lymphocyte subpopulations in vitro

Shiga toxin-producing Escherichia coli (STEC) is widespread in the cattle population, but the clinical significance of Shiga toxins (Stx's) for the bovine species remains obscure. Since Stx's exert immunomodulating effects in other species, we examined the effect of purified Stx1 on a bovine B lymphoma cell line (BL-3) and peripheral blood mononuclear cells (PBMC) isolated from adult bovine blood by viability assays and flow cytometry analysis. Stx1 markedly induced apoptosis in stimulated BL-3 cells. The susceptibility of this B-cell-derived cell line was induced only by either lipopolysaccharide (LPS) or pokeweed mitogen, while cultures stimulated with T-cell mitogens were unaffected by the toxin. In contrast, Stx1 did not induce cellular death-neither apoptosis nor necrosis-in primary cultures of PBMC but hindered the mitogen-induced increase in metabolic activity. The influence of Stx1 on single PBMC subpopulations varied with the type of mitogenic stimulus applied. Stimulation with phytohemagglutinin P particularly induced the proliferation of bovine CD8-expressing (BoCD8(+)) cells, and this proliferative response was blocked by Stx1. On the other hand, Stx1 reduced the portion of viable B cells in the presence of LPS. Modulation of activation marker expression (BoCD25 and BoCD71) by Stx1 indicated that the toxin hindered the proliferation of cells by blocking their activation. In conclusion, we assume that Stx1 contributes to the pathogenesis of STEC-associated diarrhea in calves by suppressing the mucosa-associated immune response. The usefulness of cattle as a model in which to study Stx-induced immunomodulation is discussed.