Inhibition of retrograde transport protects mice from lethal ricin challenge.

Bacterial Shiga-like toxins are virulence factors that constitute a significant public health threat worldwide, and the plant toxin ricin is a potential bioterror weapon. To gain access to their cytosolic target, ribosomal RNA, these toxins follow the retrograde transport route from the plasma membrane to the endoplasmic reticulum, via endosomes and the Golgi apparatus. Here, we used high-throughput screening to identify small molecule inhibitors that protect cells from ricin and Shiga-like toxins. We identified two compounds that selectively block retrograde toxin trafficking at the early endosome-TGN interface, without affecting compartment morphology, endogenous retrograde cargos, or other trafficking steps, demonstrating an unexpected degree of selectivity and lack of toxicity. In mice, one compound clearly protects from lethal nasal exposure to ricin. Our work discovers the first small molecule that shows efficacy against ricin in animal experiments and identifies the retrograde route as a potential therapeutic target.

Shiga toxins: from structure and mechanism to applications.

Shiga toxins are a group of type 2 ribosome-inactivating proteins (RIPs) produced in several types of bacteria. The toxins possess an AB5 structure, which comprises a catalytic A chain with N-glycosidase activity, and five identical B chains and recognize and bind to the target cells with specific carbohydrate moieties. In humans, the major molecular target which recognizes the Shiga toxins is the Gb3 receptor, which is mainly expressed on the cell surface of endothelial cells of the intestine, kidney, and the brain. This causes these organs to be susceptible to the toxicity of Shiga toxins. When a person is infected by Shiga toxin-producing bacteria, the toxin is produced in the gut, translocated to the circulatory system, and carried to the target cells. Toxicity of the toxin causes inflammatory responses and severe cell damages in the intestine, kidneys, and brain, bringing about the hemolytic uremic syndrome (HUS), which can be fatal. The Shiga toxin requires a couple of steps to exert its toxicity to the target cells. After binding with the target cell surface receptor, the toxin requires a complicated process to be transported into the cytosol of the cell before it can approach the ribosomes. The mechanisms for the interactions of the toxin with the cells are described in this review. The consequences of the toxin on the cells are also discussed. It gives an overview of the steps for the toxin to be produced and transported, expression of catalytic activity, and the effects of the toxin on the target cells, as well as effects on the human body.

Novel actions of 2-deoxy-D-glucose: protection against Shiga toxins and changes in cellular lipids.

2-Deoxy-D-glucose (2DG) is a structural analogue of glucose with well-established applications as an inhibitor of glycolysis and N-glycosylation. Importantly, 2DG has been shown to improve the efficacy of several cancer chemotherapeutic agents in vivo and thus it is in clinical studies in combination with chemotherapy and radiotherapy. However, although 2DG has been demonstrated to modulate many cellular functions, including autophagy, apoptosis and cell cycle control, little is known about the effects of 2DG on intracellular transport, which is of great importance when predicting the effects of 2DG on therapeutic agents. In addition to proteins, lipids play important roles in cellular signalling and in controlling cellular trafficking. We have, in the present study, investigated the effects of 2DG on cellular lipid composition and by use of protein toxins we have studied 2DG-mediated changes in intracellular trafficking. By quantifying more than 200 individual lipid species from 17 different lipid classes, we have found that 2DG treatment changes the levels and/or species composition of several lipids, such as phosphatidylinositol (PI), diacylglycerol (DAG), cholesteryl ester (CE), ceramide (Cer) and lysophospho-lipids. Moreover, 2DG becomes incorporated into the carbohydrate moiety of glycosphingolipids (GSLs). In addition, we have discovered that 2DG protects cells against Shiga toxins (Stxs) and inhibits release of the cytotoxic StxA1 moiety in the endoplasmic reticulum (ER). The data indicate that the 2DG-induced protection against Stx is independent of inhibition of glycolysis or N-glycosylation, but rather mediated via the depletion of Ca(2+) from cellular reservoirs by 2DG. In conclusion, our results reveal novel actions of 2DG on cellular lipids and Stx toxicity.

Shiga toxins and stx phages: highly diverse entities.

Shiga toxins are the main virulence factors of a group of Escherichia coli strains [Shiga toxin-producing E. coli (STEC)] that cause severe human diseases, such as haemorrhagic colitis and haemolytic-uraemic syndrome. The Shiga toxin family comprises several toxin subtypes, which have been differentially related to clinical manifestations. In addition, the phages that carry the Shiga toxin genes (stx phages) are also diverse. These phages play an important role not only in the dissemination of Shiga toxin genes and the emergence of new STEC strains, but also in the regulation of Shiga toxin production. Consequently, differences in stx phages may affect the dissemination of stx genes as well as the virulence of STEC strains. In addition to presenting an overview of Shiga toxins and stx phages, in this review we highlight current knowledge about the diversity of stx phages, with emphasis on its impact on STEC virulence. We consider that this diversity should be taken into account when developing STEC infection treatments and diagnostic approaches, and when conducting STEC control in reservoirs.

Safe and effective means of detecting and quantitating Shiga-like toxins in attomole amounts.

Shiga-like toxins (verotoxins) are a class of AB5 holotoxins that are primarily responsible for the virulence associated with Shiga-like toxin producing Escherichia coli (STEC) infections. The holotoxins are composed of a pentamer of identical subunits (B subunit) responsible for delivering the catalytic subunit (A subunit) to a host cell and facilitating endocytosis of the toxin into the cell. The B subunits are not associated with toxicity. We developed a multiple reaction monitoring method based on analyzing conserved peptides, derived from the tryptic digestion of the B subunits. Stable-isotope-labeled analogues were prepared and used as internal standards to identify and quantify these characteristic peptides. We were able to detect and quantify Shiga toxins (Stx), Shiga-like toxin type 1 (Stx1) and type 2 (Stx2) subtypes, and to distinguish among most of the known subtypes. The limit of detection for digested pure standards was in the low attomole range/injection (~10 attomoles), which corresponded to a concentration of 1.7 femtomol/mL. A matrix effect was observed when dilute samples were digested in the buffer, Luria broth, or mouse plasma (LOD ~ 30 attomol/injection = 5 femtomol/mL). In addition, we determined that the procedures necessary to perform our mass spectrometry-based analysis completely inactivate the toxins present in the sample. This is a safe and effective method of detecting and quantitating Stx, Stx1, and Stx2, since it does not require the use of intact toxins.

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.

Relevance of biofilms in the pathogenesis of Shiga-toxin-producing Escherichia coli infection.

The present study was designed to determine the relationships among biofilm formation, cellular stress and release of Shiga toxin (Stx) by three different clinical Shiga toxin-producing Escherichia coli (STEC) strains. The biofilm formation was determined using crystal violet stain in tryptic soy broth or thioglycollate medium with the addition of sugars (glucose or mannose) or hydrogen peroxide. The reactive oxygen species (ROSs) were detected by the reduction of nitro blue tetrazolium and reactive nitrogen intermediates (RNI) determined by the Griess assay. In addition, the activities of two antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT), were studied. For the cytotoxicity studies, Vero cells were cultured with Stx released of STEC biofilms. The addition of sugars in both culture mediums resulted in an increase in biofilm biomass, with a decrease in ROS and RNI production, low levels of SOD and CAT activity, and minimal cytotoxic effects. However, under stressful conditions, an important increase in the antioxidant enzyme activity and high level of Stx production were observed. The disturbance in the prooxidant-antioxidant balance and its effect on the production and release of Stx evaluated under different conditions of biofilm formation may contribute to a better understanding of the relevance of biofilms in the pathogenesis of STEC infection.

The Fatal Role of Enterohaemorrhagic Escherichia coli Shiga Toxin-associated Extracellular Vesicles in Host Cells.

Enterohemorrhagic Escherichia coli (EHEC) is a specific subset of Shiga toxin-producing Escherichia coli (STEC) strains that are characterized by their ability to cause bloody diarrhea (hemorrhagic colitis) and potentially life-threatening, extraintestinal complications such as hemolytic uremic syndrome (HUS), which is associated with acute renal failure., contributing to severe clinical outcomes. The Shiga toxins (Stxs), produced by EHEC, are primary virulence factors. These potent cytotoxins are composed of one enzymatically active A subunit (StxA) and five receptor-binding B subunits (StxB). Although the toxins are primarily associated with cytotoxic effects, they also elicit other pathogenic consequences due to their induction of a number of biological processes, including apoptosis through ER-stress, pro-inflammatory responses, autophagy, and post-translational modification (PTM). Moreover, several studies have reported the association between Stxs and extracellular vesicles (EVs), including microvesicles and exosomes, demonstrating that Stx-containing EVs secreted by intoxicated macrophages are taken up by recipient cells, such as toxin-sensitive renal proximal tubular epithelial cells. This mechanism likely contributes to the spreading of Stxs within the host, and may exacerbate gastrointestinal illnesses and kidney dysfunction. In this review, we summarize recent findings relating to the host responses, in different types of cells in vitro and in animal models, mediated by Stxs-containing exosomes. Due to their unique properties, EVs have been explored as therapeutic agents, drug delivery systems, and diagnostic tools. Thus, potential therapeutic applications of EVs in EHEC Stxs-mediated pathogenesis are also briefly reviewed.

Recent Advancements in Technologies to Detect Enterohaemorrhagic Escherichia coli Shiga Toxins.

Shiga toxin (Stxs)-producing enterohaemorrhagic Escherichia coli (EHEC) and Shigella dysenteriae serotype 1 are major causative agents of severe bloody diarrhea (known as hemorrhagic colitis) and hemolytic uremic syndrome (HUS) associated with extraintestinal complications such as acute renal failure and neurologic impairment in infected patients under 9 years of age. Extreme nephrotoxicity of Stxs in HUS patients is associated with severe outcomes, highlighting the need to develop technologies to detect low levels of the toxin in environmental or food samples. Currently, the conventional polymerase chain reaction (PCR) or immunoassay is the most broadly used assay to detect the toxin. However, these assays are laborious, time-consuming, and costly. More recently, numerous studies have described novel, highly sensitive, and portable methods for detecting Stxs from EHEC. To contextualize newly emerging Stxs detection methods, we briefly explain the basic principles of these methods, including lateral flow assays, optical detection, and electrical detection. We subsequently describe existing and newly emerging rapid detection technologies to identify and measure Stxs.

Enterohemorrhagic Escherichia coli O157:H7 Shiga toxins inhibit gamma interferon-mediated cellular activation.

Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 is a food-borne pathogen that causes significant morbidity and mortality in developing and industrialized nations. EHEC infection of host epithelial cells is capable of inhibiting the gamma interferon (IFN-γ) proinflammatory pathway through the inhibition of Stat-1 phosphorylation, which is important for host defense against microbial pathogens. The aim of this study was to determine the bacterial factors involved in the inhibition of Stat-1 tyrosine phosphorylation. Human HEp-2 and Caco-2 epithelial cells were challenged directly with either EHEC or bacterial culture supernatants and stimulated with IFN-γ, and then the protein extracts were analyzed by immunoblotting. The data showed that IFN-γ-mediated Stat-1 tyrosine phosphorylation was inhibited by EHEC secreted proteins. Using two-dimensional difference gel electrophoresis, EHEC Shiga toxins were identified as candidate inhibitory factors. EHEC Shiga toxin mutants were then generated and complemented in trans, and mutant culture supernatant was supplemented with purified Stx to confirm their ability to subvert IFN-γ-mediated cell activation. We conclude that while other factors are likely involved in the suppression of IFN-γ-mediated Stat-1 tyrosine phosphorylation, E. coli-derived Shiga toxins represent a novel mechanism by which EHEC evades the host immune system.

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.

AB5 Enterotoxin-Mediated Pathogenesis: Perspectives Gleaned from Shiga Toxins.

Foodborne diseases affect an estimated 600 million people worldwide annually, with the majority of these illnesses caused by Norovirus, Vibrio, Listeria, Campylobacter, Salmonella, and Escherichia coli. To elicit infections in humans, bacterial pathogens express a combination of virulence factors and toxins. AB5 toxins are an example of such toxins that can cause various clinical manifestations, including dehydration, diarrhea, kidney damage, hemorrhagic colitis, and hemolytic uremic syndrome (HUS). Treatment of most bacterial foodborne illnesses consists of fluid replacement and antibiotics. However, antibiotics are not recommended for infections caused by Shiga toxin-producing E. coli (STEC) because of the increased risk of HUS development, although there are conflicting views and results in this regard. Lack of effective treatment strategies for STEC infections pose a public health threat during outbreaks; therefore, the debate on antibiotic use for STEC infections could be further explored, along with investigations into antibiotic alternatives. The overall goal of this review is to provide a succinct summary on the mechanisms of action and the pathogenesis of AB5 and related toxins, as expressed by bacterial foodborne pathogens, with a primary focus on Shiga toxins (Stx). The role of Stx in human STEC disease, detection methodologies, and available treatment options are also briefly discussed.

Shiga toxin subtypes display dramatic differences in potency.

Purified Shiga toxin (Stx) alone is capable of producing systemic complications, including hemolytic-uremic syndrome (HUS), in animal models of disease. Stx includes two major antigenic forms (Stx1 and Stx2), with minor variants of Stx2 (Stx2a to -h). Stx2a is more potent than Stx1. Epidemiologic studies suggest that Stx2 subtypes also differ in potency, but these differences have not been well documented for purified toxin. The relative potencies of five purified Stx2 subtypes, Stx2a, Stx2b, Stx2c, Stx2d, and activated (elastase-cleaved) Stx2d, were studied in vitro by examining protein synthesis inhibition using Vero monkey kidney cells and inhibition of metabolic activity (reduction of resazurin to fluorescent resorufin) using primary human renal proximal tubule epithelial cells (RPTECs). In both RPTECs and Vero cells, Stx2a, Stx2d, and elastase-cleaved Stx2d were at least 25 times more potent than Stx2b and Stx2c. In vivo potency in mice was also assessed. Stx2b and Stx2c had potencies similar to that of Stx1, while Stx2a, Stx2d, and elastase-cleaved Stx2d were 40 to 400 times more potent than Stx1.

Escherichia coli Shiga Toxins and Gut Microbiota Interactions.

Escherichia coli (EHEC) and Shigella dysenteriae serotype 1 are enterohemorrhagic bacteria that induce hemorrhagic colitis. This, in turn, may result in potentially lethal complications, such as hemolytic uremic syndrome (HUS), which is characterized by thrombocytopenia, acute renal failure, and neurological abnormalities. Both species of bacteria produce Shiga toxins (Stxs), a phage-encoded exotoxin inhibiting protein synthesis in host cells that are primarily responsible for bacterial virulence. Although most studies have focused on the pathogenic roles of Stxs as harmful substances capable of inducing cell death and as proinflammatory factors that sensitize the host target organs to damage, less is known about the interface between the commensalism of bacterial communities and the pathogenicity of the toxins. The gut contains more species of bacteria than any other organ, providing pathogenic bacteria that colonize the gut with a greater number of opportunities to encounter other bacterial species. Notably, the presence in the intestines of pathogenic EHEC producing Stxs associated with severe illness may have compounding effects on the diversity of the indigenous bacteria and bacterial communities in the gut. The present review focuses on studies describing the roles of Stxs in the complex interactions between pathogenic Shiga toxin-producing E. coli, the resident microbiome, and host tissues. The determination of these interactions may provide insights into the unresolved issues regarding these pathogens.

Primary Human Renal Proximal Tubular Epithelial Cells (pHRPTEpiCs): Shiga Toxin (Stx) Glycosphingolipid Receptors, Stx Susceptibility, and Interaction with Membrane Microdomains.

Tubular epithelial cells of the human kidney are considered as targets of Shiga toxins (Stxs) in the Stx-mediated pathogenesis of hemolytic-uremic syndrome (HUS) caused by Stx-releasing enterohemorrhagic Escherichia coli (EHEC). Analysis of Stx-binding glycosphingolipids (GSLs) of primary human renal proximal tubular epithelial cells (pHRPTEpiCs) yielded globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer) with Cer (d18:1, C16:0), Cer (d18:1, C22:0), and Cer (d18:1, C24:1/C24:0) as the dominant lipoforms. Investigation of detergent-resistant membranes (DRMs) and nonDRMs, serving as equivalents for the liquid-ordered and liquid-disordered membrane phase, respectively, revealed the prevalence of Gb3Cer and Gb4Cer together with cholesterol and sphingomyelin in DRMs, suggesting lipid raft association. Stx1a and Stx2a exerted strong cellular damage with half-maximal cytotoxic doses (CD50) of 1.31 × 102 pg/mL and 1.66 × 103 pg/mL, respectively, indicating one order of magnitude higher cellular cytotoxicity of Stx1a. Surface acoustic wave (SAW) real-time interaction analysis using biosensor surfaces coated with DRM or nonDRM fractions gave stronger binding capability of Stx1a versus Stx2a that correlated with the lower cytotoxicity of Stx2a. Our study underlines the substantial role of proximal tubular epithelial cells of the human kidney being associated with the development of Stx-mediated HUS at least for Stx1a, while the impact of Stx2a remains somewhat ambiguous.

Role of Shiga Toxins in Cytotoxicity and Immunomodulatory Effects of Escherichia coli O157:H7 during Host-Bacterial Interactions in vitro.

Enterohemorrhagic Escherichia coli (EHEC) strains are food-borne pathogens that can cause different clinical conditions. Shiga toxin 2a and/or 2c (Stx2)-producing E. coli O157:H7 is the serotype most frequently associated with severe human disease. In this work we analyzed the hypothesis that host cells participate in Stx2 production, cell damage, and inflammation during EHEC infection. With this aim, macrophage-differentiated THP-1 cells and the intestinal epithelial cell line HCT-8 were incubated with E. coli O157:H7. A time course analysis of cellular and bacterial survival, Stx2 production, stx2 transcription, and cytokine secretion were analyzed in both human cell lines. We demonstrated that macrophages are able to internalize and kill EHEC. Simultaneously, Stx2 produced by internalized bacteria played a major role in macrophage death. In contrast, HCT-8 cells were completely resistant to EHEC infection. Besides, macrophages and HCT-8 infected cells produce IL-1ß and IL-8 inflammatory cytokines, respectively. At the same time, bacterial stx2-specific transcripts were detected only in macrophages after EHEC infection. The interplay between bacteria and host cells led to Stx production, triggering of inflammatory response and cell damage, all of which could contribute to a severe outcome after EHEC infections.

Tissue Responses to Shiga Toxin in Human Intestinal Organoids.

BACKGROUND & AIMS: Shiga toxin (Stx)-producing Escherichia coli (eg, O157:H7) infection produces bloody diarrhea, while Stx inhibits protein synthesis and causes the life-threatening systemic complication of hemolytic uremic syndrome. The murine intestinal tract is resistant to O157:H7 and Stx, and human cells in culture fail to model the complex tissue responses to intestinal injury. We used genetically identical, human stem cell-derived intestinal tissues of varying complexity to study Stx toxicity in vitro and in vivo. METHODS: In vitro susceptibility to apical or basolateral exposure to Stx was assessed using human intestinal organoids (HIOs) derived from embryonic stem cells, or enteroids derived from multipotent intestinal stem cells. HIOs contain a lumen, with a single layer of differentiated epithelium surrounded by mesenchymal cells. Enteroids only contain epithelium. In vivo susceptibility was assessed using HIOs, with or without an enteric nervous system, transplanted into mice. RESULTS: Stx induced necrosis and apoptotic death in both epithelial and mesenchymal cells. Responses that require protein synthesis (cellular proliferation and wound repair) also were observed. Epithelial barrier function was maintained even after epithelial cell death was seen, and apical to basolateral translocation of Stx was seen. Tissue cross-talk, in which mesenchymal cell damage caused epithelial cell damage, was observed. Stx induced mesenchymal expression of the epithelial marker E-cadherin, the initial step in mesenchymal-epithelial transition. In vivo responses of HIO transplants injected with Stx mirrored those seen in vitro. CONCLUSIONS: Intestinal tissue responses to protein synthesis inhibition by Stx are complex. Organoid models allow for an unprecedented examination of human tissue responses to a deadly toxin.

The type 4 pili of enterohemorrhagic Escherichia coli O157:H7 are multipurpose structures with pathogenic attributes.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 produces long bundles of polar type 4 pili (T4P) called HCP (for hemorrhagic coli pili) that form physical bridges between bacteria associating with human and animal epithelial cells. Here, we sought to further investigate whether HCP possessed other pathogenicity attributes associated with T4P production. Comparative studies performed with wild-type EHEC EDL933 and an isogenic hcpA mutant revealed that HCP play different roles in the biology of this organism. We found that in addition to promoting bacterial attachment to host cells, HCP mediate (i) invasion of epithelial cells, (ii) hemagglutination of rabbit erythrocytes, (iii) interbacterial connections conducive to biofilm formation, (iv) specific binding to host extracellular matrix proteins laminin and fibronectin but not collagen, and (v) twitching motility. Nonadherent laboratory E. coli strain HB101 complemented with hcpABC genes on plasmid pJX22, which specifies for HCP overproduction in EDL933, became hyperadherent and invasive and produced a thick biofilm, suggesting that the presence of HCP confers HB101(pJX22) new attributes otherwise not exhibited by HB101. Analogous to other bacteria in which T4P are involved in the pathogenesis of several infectious diseases, our data strongly suggest that HCP display multiple functions that may contribute to EHEC colonization of different hosts and to virulence, survival, and transmission of this food-borne pathogen.

Interactions between Shiga toxins and human polymorphonuclear leukocytes.

Human intestinal infections by Shiga toxin (Stx)-producing Escherichia coli cause hemorrhagic colitis and hemolytic uremic syndrome (HUS), which represents the main cause of acute renal failure in early childhood. In HUS, Stx released in the gut enter the bloodstream and are targeted to renal endothelium. The mechanism of toxin delivery is still a matter of debate, although the role of polymorphonuclear leukocytes (PMN) as a Stx carrier has been indicated. The aim of this paper was to better define the interactions between Stx and human PMN. Direct and indirect flow cytometric analysis and binding experiments with radiolabeled toxins demonstrated that Stx bind to the surface of human mature PMN but not to immature PMN from G-CSF-treated donors. The use of the human myeloid leukemia cell (HL-60) model for inducible cell differentiation confirmed that the toxin binding occurs only after granulocytic differentiation. Stx binding caused a delay of the spontaneous apoptosis of PMN, as shown by the delayed appearance of apoptotic nuclei and activation of caspase 3 and by the higher number of cells negative to the annexin V-binding assay after 48 h. Moreover, flow cytometric analysis of mixed Stx-positive and Stx-negative PMN populations showed that the toxins were transferred from positive to negative PMN. The delayed, spontaneous apoptosis and the passage of the toxic ligand from older PMN to new, mature cells entering the circulation from the bone marrow may explain the previously reported persistence of Stx in the blood of children with HUS.

[The apoptosis of vero cells induced by Shiga toxin 2e].

OBJECTIVE: Shiga-like toxin-producing Escherichia coli (STEC) causes edema disease in piglets and hemolytic uremic syndrome in human. Shiga-like toxins (Stxs) produced by STEC induce mammalian cells death via either necrosis or apoptosis. However, the ability of stx2e, separated from edema disease (Stx2e), to trigger apoptosis and the sequence of intracellular signaling events have not yet been completely defined. In this study we investigated the apoptotic effects of Stx2e on Vero cells. METHODS: Vero cells were treated with different concentrations of Stx2e for different time and the apoptotic cells were characterized by acridine orange and ethidium bromide fluorescent dye staining. The fragmentation of chromatin from Vero cells treated with Stx2e were detected by agarose gel electrophoresis. The expression patterns of apoptosis-associated factors were assayed by Western blotting. RESULTS: Stx2e-treated cells showed characteristic features of apoptosis, including membrane blebbing, DNA fragmentation, chromatin condensation, and the formation of apoptotic bodies, whereas ricin did not induce apoptosis of Vero cells even at a high dose. Fluorescent dye staining showed that Stx2e induced apoptosis of Vero cells in dose- and time-dependent manners. Caspase-3 was activated whereas expression levels of bcl2 associated X protein (Bax) and caspase-9 had no change compared with the negative control. CONCLUSION: Stx2e induced intensively apoptosis of Vero cells, which was mediated through the mitochondrion-independent pathway and might be throught a receptor-dependent pathway.