Cheleste M. Thorpe

The Emerging Clinical Importance of Non-O157 Shiga Toxin—Producing Escherichia coli

In 1982, hemorrhagic colitis and hemolytic-uremic syndrome were linked to infection with Escherichia coli O157:H7, a serotype now classified as Shiga toxin-producing E. coli (STEC). Thereafter, hemorrhagic colitis and hemolytic-uremic syndrome associated with non-O157 STEC serogroups were reported, with the frequency of non-O157 STEC illness rivaling that of O157:H7 in certain geographic regions. In the United States, non-O157 E. coli may account for up to 20%-50% of all STEC infections. A high index of suspicion, paired with options to test for non-O157 STEC infection, are necessary for early recognition and appropriate treatment of these infections. Supportive care without the use of antibiotics is currently considered to be optimal treatment for all STEC infections. This commentary provides a perspective on the non-O157 STEC as human pathogens, how and when the clinician should approach the diagnosis of these organisms, and the challenges ahead.

Ab5 Subtilase Cytotoxin Inactivates the Endoplasmic Reticulum Chaperone Bip

AB5 toxins are produced by pathogenic bacteria and consist of enzymatic A subunits that corrupt essential eukaryotic cell functions, and pentameric B subunits that mediate uptake into the target cell. AB5 toxins include the Shiga, cholera and pertussis toxins and a recently discovered fourth family, subtilase cytotoxin, which is produced by certain Shiga toxigenic strains of Escherichia coli. Here we show that the extreme cytotoxicity of this toxin for eukaryotic cells is due to a specific single-site cleavage of the essential endoplasmic reticulum chaperone BiP/GRP78. The A subunit is a subtilase-like serine protease; structural studies revealed an unusually deep active-site cleft, which accounts for its exquisite substrate specificity. A single amino-acid substitution in the BiP target site prevented cleavage, and co-expression of this resistant protein protected transfected cells against the toxin. BiP is a master regulator of endoplasmic reticulum function, and its cleavage by subtilase cytotoxin represents a previously unknown trigger for cell death.

Shiga Toxin 1 Triggers a Ribotoxic Stress Response Leading to p38 and JNK Activation and Induction of Apoptosis in Intestinal Epithelial Cells

ABSTRACT Shiga toxins made by Shiga toxin-producing Escherichia coli (STEC) are associated with hemolytic uremic syndrome. Shiga toxins (Stxs) may access the host systemic circulation by absorption across the intestinal epithelium. The effects of Stxs on this cell layer are not completely understood, although animal models of STEC infection suggest that, in the gut, Stxs may participate in both immune activation and apoptosis. Stxs have one enzymatically active A subunit associated with five identical B subunits. The A subunit inactivates ribosomes by cleaving a specific adenine from the 28S rRNA. We have previously shown that Stxs can induce multiple C-X-C chemokines in intestinal epithelial cells in vitro, including interleukin-8 (IL-8), and that Stx-induced IL-8 expression is linked to induction of c-Jun mRNA and p38 mitogen-activated protein (MAP) kinase pathway activity. We now report Stx1 induction of both primary response genes c-jun and c-fos and activation of the stress-activated protein kinases, JNK/SAPK and p38, in the intestinal epithelial cell line HCT-8. By 1 h of exposure to Stx1, mRNAs for c-jun and c-fos are induced, and both JNK and p38 are activated; activation of both kinases persisted up to 24 h. Stx1 enzymatic activity was required for kinase activation; a catalytically defective mutant toxin did not activate either. Stx1 treatment of HCT-8 cells resulted in cell death that was associated with caspase 3 cleavage and internucleosomal DNA fragmentation; this cytotoxicity also required Stx1 enzymatic activity. Blocking Stx1-induced p38 and JNK activation with the inhibitor SB202190 prevented cell death and diminished Stx1-associated caspase 3 cleavage. In summary, these data link the Stx1-induced ribotoxic stress response with both chemokine expression and apoptosis in the intestinal epithelial cell line HCT-8 and suggest that blocking host cell MAP kinases may prevent these Stx-associated events.

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

ABSTRACT 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 lackingeae and espB (eae- andespB-negative STEC strains) induced significantly more neutrophil transmigration and significantly greater IL-8 secretion thaneae- 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 toxin-producing Escherichia coli infection.

Large-scale outbreaks of Shiga toxin-producing Escherichia coli (STEC) infection have revealed the great disease-causing potential of this organism, especially among children and elderly persons. Approximately 5%-10% of people with STEC infection will develop hemolytic-uremic syndrome (HUS), approximately 10% of those who develop HUS will die or have permanent renal failure, and up to 50% of those who develop HUS will develop some degree of renal impairment. Important concepts in understanding the pathogenesis and prevention of STEC-associated HUS are emerging, although no specific therapy yet exists. Optimal management of STEC infection includes intravenous hydration, avoidance of antimotility agents and antimicrobials, and monitoring for sequelae. Antimicrobials may have a potentially harmful role, possibly by inducing intestinal production of Shiga toxin during the diarrheal phase of illness. A recent clinical trial evaluating an intraluminal Shiga toxin-binding agent to ameliorate HUS showed no improvement in outcome. Interventions to prevent HUS from developing in STEC-infected children are under investigation. Prevention of exposure to STEC remains important, and animal vaccines to prevent stool shedding of STEC among food animals are in development.

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

ABSTRACT Exposure of humans to Shiga toxins (Stxs) is a risk factor for hemolytic-uremic syndrome (HUS). Because Stx-producingEscherichia 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-α, GRO-β, GRO-γ, and ENA-78. Like that of IL-8, GRO-α and ENA-78 mRNAs are both induced and superinduced by Stx1. Furthermore, Stx1 induces both IL-8 and GRO-α protein in a dose-response fashion, despite an overall inhibition in host cell protein synthesis. Stx1 treatment stabilizes both IL-8 and GRO-α 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.

Critical Roles for stx2, eae, and tir in Enterohemorrhagic Escherichia coli-Induced Diarrhea and Intestinal Inflammation in Infant Rabbits

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) is a group of food-borne pathogens that can cause diarrhea, colitis, and the hemolytic uremic syndrome (HUS). The importance of several of the proposed EHEC virulence factors lacks experimental verification in animal models. The limitations of current animal models led us to reexamine the infant rabbit model for the study of EHEC pathogenicity. Here, we report that intragastric inoculation of a Shiga toxin 2 (Stx2)-producing E. coli O157:H7 clinical isolate into infant rabbits led to severe diarrhea and intestinal inflammation but no signs of HUS. We constructed a set of isogenic derivatives of this isolate with deletions in several putative virulence genes, including stx2, eae, tir, and ehxA, to investigate the contribution of individual virulence factors to EHEC pathogenicity. stx2 increased the severity and duration of EHEC-induced diarrhea. Furthermore, although stx2 had no role in EHEC intestinal colonization nor was it required for EHEC-induced inflammation, stx2 altered how the host responded to EHEC infection by promoting heterophilic infiltration of the colonic epithelium and lamina propria. Intragastric inoculation of purified Stx2 also induced inflammation and diarrhea in this model. Diarrhea and intestinal inflammation were also dependent on EHEC colonization, as EHEC derivatives with deletions in eae and tir did not colonize, form attaching and effacing lesions, or develop clinical signs of disease. Our studies indicate that infant rabbits are a useful model for investigation of the intestinal stage of EHEC pathogenesis and suggest that Shiga toxin may play a critical role in causing diarrhea and inflammation in patients infected with EHEC.

Subtilase cytotoxin activates PERK, IRE1 and ATF6 endoplasmic reticulum stress-signalling pathways

Subtilase cytotoxin (SubAB) is the prototype of a new family of AB5 cytotoxins produced by Shiga toxigenic Escherichia coli. Its cytotoxic activity is due to its capacity to enter cells and specifically cleave the essential endoplasmic reticulum (ER) chaperone BiP (GRP78). In the present study, we have examined its capacity to trigger the three ER stress‐signalling pathways in Vero cells. Activation of PKR‐like ER kinase was demonstrated by phosphorylation of eIF2α, which occurred within 30 min of toxin treatment, and correlated with inhibition of global protein synthesis. Activation of inositol‐requiring enzyme 1 was demonstrated by splicing of X‐box‐binding protein 1 mRNA, while activating transcription factor 6 activation was demonstrated by depletion of the 90 kDa uncleaved form, and appearance of the 50 kDa cleaved form. The rapidity with which ER stress‐signalling responses are triggered by exposure of cells to SubAB is consistent with the hypothesis that cleavage by the toxin causes BiP to dissociate from the signalling molecules.

ZAK: a MAP3Kinase that transduces Shiga toxin- and ricin-induced proinflammatory cytokine expression.

Shiga toxins (Stxs) and ricin initiate damage to host cells by cleaving a single adenine residue on the α‐sarcin loop of the 28S ribosomal RNA. This molecular insult results in a cascade of intracellular events termed the ribotoxic stress response (RSR). Although Stxs and ricin have been shown to cause the RSR, the mitogen‐activated protein kinase kinase kinase (MAP3K) that transduces the signal from intoxicated ribosomes to activate SAPKinases has remained elusive. We show in vitro that DHP‐2 (7‐[3‐fluoro‐4‐aminophenyl‐(4‐(2‐pyridin‐2‐yl‐5,6‐dihydro‐4H‐pyrrolo[1,2‐b]pyrazol‐3‐yl))]‐quinoline), a zipper sterile‐α‐motif kinase (ZAK)‐specific inhibitor, blocks Stx2/ricin‐induced SAPKinase activation. Treatment of cells with DHP‐2 also blocks Stx2/ricin‐mediated upregulation of the proinflammatory cytokine interleukin‐8 and results in a modest but statistically significant improvement in cell viability following Stx2/ricin treatment. Finally we show that siRNA directed against the N‐terminus of ZAK diminishes Stx2/Ricin‐induced SAPKinase activation. Together, these data demonstrate that a ZAK isoform(s) is the MAP3Kinase that transduces the RSR. Therefore, ZAKα and/or β isoforms may act as potential therapeutic target(s) for treating Stx/ricin‐associated illnesses. Furthermore, a small molecule inhibitor like DHP‐2 may prove valuable in preventing the Stx/ricin‐induced proinflammatory and/or apoptotic effects that are thought to contribute to pathogenesis by Stx‐producing Escherichia coli and ricin.

Clathrin-dependent trafficking of subtilase cytotoxin, a novel AB5 toxin that targets the endoplasmic reticulum chaperone BiP.

Subtilase cytotoxin (SubAB) is the prototype of a new family of AB5 cytotoxins produced by Shiga toxigenic Escherichia coli. Its cytotoxic activity is due to its capacity to enter cells and specifically cleave the endoplasmic reticulum (ER) chaperone BiP. However, its trafficking within target cells has not been investigated previously. In Vero cells, fluorescence colocalization with subcellular markers established that SubAB is trafficked from the cell surface to the ER via a retrograde pathway similar, but not identical, to those of Shiga toxin (Stx) and cholera toxin (Ctx), with their pathways converging at the Golgi. The clathrin inhibitor phenylarsine oxide prevented SubAB entry and BiP cleavage in SubAB‐treated Vero, HeLa and N2A cells, while cholesterol depletion did not, demonstrating that, unlike either Stx or Ctx, SubAB internalization is exclusively clathrin‐dependent.