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 toxin 1 triggers a ribotoxic stress response leading to p38 and JNK activation and induction of apoptosis in intestinal epithelial cells

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 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.     

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.     

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.     

Intestinal Shiga toxin-producing Escherichia coli bacteria mitigate bovine leukemia virus infection in experimentally infected sheep

Ruminants often carry gastrointestinal Shiga toxin (Stx)-producing Escherichia coli (STEC). Stxs belong to a large family of ribosome-inactivating proteins (RIPs), found in many plants and some bacteria. Plant RIPs, secreted into extracellular spaces, limit the spread of viruses through plant tissues by penetrating and killing virally infected cells. Previously, we showed Stx activity against bovine leukemia virus (BLV)-infected cells in vitro and hypothesized that STEC bacteria have antiviral activity in ruminant hosts. Here, we investigated the impact of STEC on the initial phases of BLV infection in sheep. Sheep were treated with biweekly oral doses of E. coli O157:H7 (an STEC) or an isogenic stx mutant strain. A different group of sheep were similarly treated with five naturally occurring ovine STEC isolates or stx-negative E. coli. Intestinal STEC bacteria were enumerated and identified by standard fecal culture and DNA hybridization. Oral STEC treatment did not always result in carriage of STEC, although many animals consistently presented with >10(4) CFU/g feces. BLV viremia was assessed by spontaneous lymphocyte proliferation (SLP) in cultures of blood mononuclear cells and by syncytium formation in cocultures of the same with F-81 indicator cells. SLP was lower (P < 0.05) and syncytia were fewer (P < 0.05) in STEC-treated sheep than in untreated sheep. Both lower SLP and fewer syncytia positively correlated with fecal STEC numbers. Average weight gain post-BLV challenge was higher in STEC-treated sheep than in untreated sheep (P < 0.05). These results support the hypothesis that in ruminants, intestinal STEC bacteria have antiviral activity and mitigate BLV-induced disease.     

Comparison of a shiga toxin enzyme-linked immunosorbent assay and two types of PCR for detection of shiga toxin-producing Escherichia coli in human stool specimens

Shiga toxin (Stx)-producing Escherichia coli (STEC) is a major cause of sporadic cases of disease as well as serious outbreaks worldwide. The spectrum of illnesses includes mild nonbloody diarrhea, hemorrhagic colitis, and hemolytic-uremic syndrome. STEC produces one or more Stxs, which are subdivided into two major classes, Stx1 and Stx2. The ingestion of contaminated food or water, person-to-person spread, and contact with animals are the major transmission modes. The infective dose of STEC may be less than 100 organisms. Effective prevention of infection is dependent on rapid detection of the causative bacterial pathogen. In the present study, we examined 295 stool specimens for the presence of Stx-producing E. coli by three different methods: an Stx enzyme-linked immunosorbent assay, a conventional PCR assay, and a LightCycler PCR (LC-PCR) assay protocol recently developed by our laboratory at the Institute of Medical Microbiology at Hannover Medical School. Our intent was to compare these three methods and to examine the utility of the STEC LC-PCR protocol in a clinical laboratory. The addition of a control DNA to each sample to clearly discriminate inhibited specimens from negative ones enhanced the accuracy of the LC-PCR protocol. From our results, it can be concluded that LC-PCR is a very useful tool for the rapid and safe detection of STEC in clinical samples.     

Probiotic bifidobacteria protect mice from lethal infection with Shiga toxin-producing Escherichia coli O157:H7

The anti-infectious activity of probiotic Bifidobacteria against Shiga toxin-producing Escherichia coli (STEC) O157:H7 was examined in a fatal mouse STEC infection model. Stable colonization of the murine intestines was achieved by the oral administration of Bifidobacterium breve strain Yakult (naturally resistant to streptomycin sulfate) as long as the mice were treated with streptomycin in their drinking water (5 mg/ml). The pathogenicity of STEC infection, characterized by marked body weight loss and subsequent death, observed in the infected controls was dramatically inhibited in the B. breve-colonized group. Moreover, Stx production by STEC cells in the intestine was almost completely inhibited in the B. breve-colonized group. A comparison of anti-STEC activity among several Bifidobacterium strains with natural resistance to streptomycin revealed that strains such as Bifidobacterium bifidum ATCC 15696 and Bifidobacterium catenulatum ATCC 27539(T) did not confer an anti-infectious activity, despite achieving high population levels similar to those of effective strains, such as B. breve strain Yakult and Bifidobacterium pseudocatenulatum DSM 20439. The effective strains produced a high concentration of acetic acid (56 mM) and lowered the pH of the intestine (to pH 6.75) compared to the infected control group (acetic acid concentration, 28 mM; pH, 7.15); these effects were thought to be related to the anti-infectious activity of these strains because the combination of a high concentration of acetic acid and a low pH was found to inhibit Stx production during STEC growth in vitro.     

Reduction of influenza virus titer and protection against influenza virus infection in infant mice fed Lactobacillus casei Shirota

We investigated whether oral administration of Lactobacillus casei strain Shirota to neonatal and infant mice ameliorates influenza virus (IFV) infection in the upper respiratory tract and protects against influenza infection. In a model of upper respiratory IFV infection, the titer of virus in the nasal washings of infant mice administered L. casei Shirota (L. casei Shirota group) was significantly (P < 0.05) lower than that in infant mice administered saline (control group) (10(2.48) +/- 10(0.31) and 10(2.78) +/- 10(0.4), respectively). Further, the survival rate of the L. casei Shirota group was significantly (P < 0.05) higher than that of the control group (14.3 versus 40.0%). One day after infection, pulmonary NK cell activity and interleukin-12 production by mediastinal lymph node cells of mice in the L. casei Shirota group were significantly greater than those of mice in the control group. These findings suggest that oral administration of L. casei Shirota activates the immature immune system of neonatal and infant mice and protects against IFV infection. Therefore, oral administration of L. casei Shirota may accelerate the innate immune response of the respiratory tract and protect against various respiratory infections in neonates, infants, and children, a high risk group for viral and bacterial infections.     

Virulence inhibition by zinc in shiga-toxigenic Escherichia coli

Previously, our laboratories reported that zinc inhibited expression of several important virulence factors in enteropathogenic Escherichia coli (EPEC) and reduced EPEC-induced intestinal damage in vivo. Since EPEC is genetically related to Shiga-toxigenic E. coli (STEC), we wondered whether the beneficial effects of zinc extended to STEC as well. Treatment options for STEC infection are very limited, since antibiotics tend to exacerbate disease via enhanced toxin production, so a safe intervention for this infection would be welcome. In this study, we report that in STEC strains zinc inhibits adherence to cultured cells as well as expression of EHEC secreted protein A (EspA). In addition, zinc inhibits the expression of Shiga toxin (Stx) at both the protein and the RNA level. Zinc inhibits basal and antibiotic-induced Stx production and inhibits both Stx1 and Stx2 by ≥90% at a concentration of 0.4 mM zinc. Rabbit EPEC strains were selected for acquisition of Stx-encoding bacteriophages, and these rabbit STEC strains (designated RDEC-H19A and E22-stx2) were used to test the effects of zinc in vivo in ligated rabbit intestinal loops. In vivo, zinc reduced fluid secretion into loops, inhibited mucosal adherence, reduced the amount of toxin in the loops, and reduced STEC-induced histological damage (villus blunting). Zinc has beneficial inhibitory effects against STEC strains that parallel those observed in EPEC. In addition, zinc strongly inhibits Stx expression; since Stx is responsible for the extraintestinal effects of STEC infection, such as hemolytic-uremic syndrome (HUS), zinc might be capable of preventing severe sequelae of STEC infection.     

Recent Progress of Shiga Toxin Neutralizer for Treatment of Infections by Shiga Toxin-Producing Escherichia coli

Infection with Shiga toxin (Stx)-producing Escherichia coli (STEC), including O157:H7, causes bloody diarrhea and hemorrhagic colitis in humans, occasionally resulting in fatal systemic complications, such as neurological damage and hemolytic-uremic syndrome. Because Stx is a major virulence factor of the infectious disease, a series of Shiga toxin neutralizers with various structural characteristics has been developed as promising therapeutic agents. Most of these agents function to bind to the toxin directly and inhibit the binding to its receptor present on the target cells. Other neutralizers do not inhibit receptor binding but induce aberrant intracellular transport of the toxin, resulting in effective detoxification. Such a novel type of Stx neutralizer provides a new therapeutic strategy against STEC infections. Here, recent progress of the development of Stx neutralizers is reviewed.     

Stx2-specific human monoclonal antibodies protect mice against lethal infection with Escherichia coli expressing Stx2 variants

Shiga toxin-producing Escherichia coli (STEC) strains are responsible for causing hemolytic-uremic syndrome (HUS), and systemic administration of Shiga toxin (Stx)-specific human monoclonal antibodies (HuMAbs) is considered a promising approach for prevention or treatment of the disease in children. The goal of the present study was to investigate the ability of Stx2-specific HuMAbs to protect against infections with STEC strains that produce Stx2 variants. Dose-response studies on five HuMAbs, using the mouse toxicity model, revealed that only the three directed against the A subunit were protective against Stx2 variants, and 5C12 was the most effective among the three tested. Two HuMAbs directed against the B subunit, while highly effective against Stx2, were ineffective against Stx2 variants. In a streptomycin-treated mouse model, parenteral administration of 5C12 significantly protected mice up to 48 h after oral bacterial challenge. We conclude that 5C12, reactive against the Stx2 A subunit, is an excellent candidate for immunotherapy against HUS and that antibodies directed against the A subunit of Stx2 have broad-spectrum activity that includes Stx2 variants, compared with those directed against the B subunit.     

Critical roles for stx2, eae, and tir in enterohemorrhagic Escherichia coli-induced diarrhea and intestinal inflammation in infant rabbits

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.     

Antibody response to Shiga toxins Stx2 and Stx1 in children with enteropathic hemolytic-uremic syndrome

A Western blot (immunoblot) assay (WBA) for the detection of immunoglobulin G antibodies to Shiga toxins Stx2 and Stx1 in sera from 110 patients with enteropathic hemolytic-uremic syndrome (53 culture confirmed to have Shiga toxin-producing Escherichia coli [STEC] infection) and 110 age-matched controls was established by using a chemiluminescence detection system. Thirty-nine (74%) of the 53 culture-confirmed cases were infections with STEC serotype O157, and 14 (26%) were associated with infection by other STEC serotypes. The frequency of an anti-Stx2 response following infection by a Stx2-producing strain (34 of 48 cases; 71%) was higher than that of an anti-Stx1 response following Stx1-producing STEC infection (4 of 10). Furthermore, the frequency of an anti-Stx2 response in 110 control sera (10%) was significantly higher than the frequency of an anti-Stx1 response (1.8%) (P = 0.0325). For STEC O157 culture-confirmed cases WBA for toxin detection had a diagnostic sensitivity of 71% and a specificity of 90%. Because of its high specificity the assay might be a helpful tool for diagnosing suspected STEC infection when tests of stool samples or serological tests against various lipopolysaccharide antigens are negative. Furthermore, the prevalence of anti-Stx antibodies in healthy controls probably reflects the population immunity to systemic Stx-associated disease. It can thus serve as a basis for comparing immunity levels in different populations and for considering future Stx toxoid immunization strategies.     

Analysis of urinary Escherichia coli isolates for ability to produce Shiga toxin

The frequency of Shiga toxin (Stx)-producing Escherichia coli (STEC) in the urinary tract, which can precipitate the hemolytic-uremic syndrome, is unknown. We tested 597 urinary E. coli isolates by Stx immunoassay and found no STEC. The routine screening of urinary E. coli for the ability to produce Stx is not warranted.     

Comparative pathogenicity of Escherichia coli O157 and intimin-negative non-O157 Shiga toxin-producing E coli strains in neonatal pigs

We compared the pathogenicity of intimin-negative non-O157:H7 Shiga toxin (Stx)-producing Escherichia coli (STEC) O91:H21 and O104:H21 strains with the pathogenicity of intimin-positive O157:H7 and O157:H(-) strains in neonatal pigs. We also examined the role of Stx2d-activatable genes and the large hemolysin-encoding plasmid of O91:H21 strain B2F1 in the pathogenesis of STEC disease in pigs. We found that all E. coli strains that made wild-type levels of Stx caused systemic illness and histological lesions in the brain and intestinal crypts, whereas none of the control Stx-negative E. coli strains evoked comparable central nervous system signs or intestinal lesions. By contrast, the absence of intimin, hemolysin, or motility had little impact on the overall pathogenesis of systemic disease during STEC infection. The most striking differences between pigs inoculated with non-O157 STEC strains and pigs inoculated with O157 STEC strains were the absence of attaching and effacing intestinal lesions in pigs inoculated with non-O157:H7 strains and the apparent association between the level of Stx2d-activatable toxin produced by an STEC strain and the severity of lesions.     

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.     

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.     

Cohort study of Guinean children: incidence,pathogenicity, conferred protection,and attributable risk for enteropathogens during the first 2 years of life

We recruited 200 children shortly after birth and collected stool specimens weekly, irrespective of whether the children had diarrhea, until up to 2 years of age. All children were recruited during the first year of the study and were monitored for a median of 18.4 months. To measure pathogenicity, the odds ratio for diarrhea, adjusted for age, sex, and coinfections with other enteropathogens, was determined by logistic regression. Standard estimation of the population attributable risk indicated that rotavirus, enterotoxigenic Escherichia coli that produced only the heat-stable toxin ST, Isospora spp., Cryptosporidium parvum, Shiga toxin (Stx)-producing E. coli (STEC), and Shigella spp. or enteroinvasive E. coli were the most important contributors to diarrhea in this population. Stx2- but not Stx1-producing STEC strains were pathogenic. Enteroaggregative E. coli, diffusely adherent E. coli, and attaching-and-effacing E. coli strains, which were the most commonly isolated microorganisms, were not associated with diarrhea. For most of the microorganisms, primary infections did not confer protection against reinfection with the same organism, but some conferred protection against diarrhea from reinfection.     

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.