Asymptomatic healthy slaughterhouse workers in South Korea carrying Shiga toxin-producing Escherichia coli

A total of 1602 stool samples from healthy employees in a slaughter company were screened by PCR for Shiga toxin (Stx)-producing Escherichia coli (STEC). The PCR product of Stx-encoding genes was detected in 90 (5.6%) of 1602 stool samples. Among the 90 stx -positive workers, the Residual Products Handlers and Slaughterers had rates of 8.0% and 6.0%– higher than Inspectors, Grading Testers and Livestock Hygiene Controllers at 3.3%, 2.0% and 3.5%, respectively. Forty-nine (54.4%) were shown to have stx 2; 25 (27.7%) carried stx 1 and 16 (17.7%) had both stx 1 and 2. Distribution of the stx PCR-positive workers by age revealed an increase in STEC infection with age ( P <0.05). Phenotypic and genotypic traits of nine STEC strains isolated from eight slaughter plant workers were characterized. A variety of serotypes, five O serogroups (O8, O54, O59, O103 and O153) and two H serogroups (H7 and H32) were found, but none of the strains belonged to the serogroup O157. Eight Vero cell cytotoxicity assay-positive strains were isolated from the workers and these workers were asymptomatic and healthy. The results of the study show that slaughter plant workers are at high risk of STEC infection.     

Production of Shiga toxin by Shiga toxin-expressing Escherichia coli (STEC) in broth media: from divergence to definition

AIMS: To determine the suitability of eight different commercial broth media for Shiga toxin (Stx) production. METHODS AND RESULTS: Shiga toxin-producing Escherichia coli (STEC) strains producing Stx1 or Stx2 were grown at 37 degrees C (250 rev min(-1)) for 24 h in brain heart infusion broth, E. coli broth, Evans medium, Luria-Bertani broth, Penassay broth, buffered-peptone water, syncase broth and trypticase soy broth. Toxin production was measured by enzyme-linked immunosorbent assay (ELISA) in polymyxin-treated cell pellets and/or supernatants of cultures, ELISA optical densities reached 1 when isolates were grown for 2-4 h in E. coli broth in the presence of antibiotic. Besides, a collection of STEC-expressing Stx strains was evaluated and the Stx production was assayed in the supernatants and in polymyxin-treated pellets of bacterial growth after 4 h of cultivation in E. coli broth in the presence of antibiotic. CONCLUSIONS: The most suitable medium for Stx production was E. coli broth when the bacterial isolates were grown for 4 h in the presence of ciprofloxacin and the Stx production is detected in the supernatant. SIGNIFICANCE AND IMPACT OF THE STUDY: This study presents the first comprehensive comparison of different broth media with regard to Stx production to establish optimal culture conditions for STEC detection in routine diagnostic laboratories.     

Urinary concentrating defect in rats given Shiga toxin: elevation in urinary AQP2 level associated with polyuria

Shiga toxin (Stx) plays a central role in the etiology of hemolytic uremic syndrome (HUS) associated with Stx-producing Escherichia coli infection. The deposition of Stx2 in the renal collecting duct epithelial cells of rats administered Stx2 intravenously has been demonstrated by immunohistochemistry, and these rats were shown to develop substantial morphological changes in the kidney tubules, associated with polyuria. Severe polyuria was observed as an early event with no other obvious sequelae after Stx administration, in parallel with elevated urinary level of aquaporin 2 (AQP2) water channel protein that was determined by a sandwich EIA assay. Immunoblotting revealed that Stx treatment markedly induced an elevation in urinary AQP2 level and reduction in AQP2 protein in the renal plasma membranes. Elevated urinary AQP2 level was a more sensitive marker to assess Stx-induced renal tubular damage than urinary beta2-microglobulin or N-acetyl-beta-D-glucosaminidase in rats. Stx2 caused more severe renal tubular impairment than Stx1. Change in urinary AQP2 level by Stx1 and Stx2 at non-lethal doses of 40 ng/kg and 10 ng/kg, respectively, was reversed at 7 days in association with recovery of urinary concentrating ability, suggesting that there is a causative link.     

Occurrence of phages infecting Escherichia coli O157:H7 carrying the Stx 2 gene in sewage from different countries

Shiga toxin-converting bacteriophages are involved in the pathogenicity of some enteric bacteria, such as Escherichia coli O157:H7. Recent studies have demonstrated a relatively high presence of Shiga toxin 2 phages in sewage from Spain, but no data on sewage from other areas were available. In order to evaluate the presence of such phages in sewage from diverse geographical origins, 33 sewage samples, including samples from eight different European countries as well as from New Zealand and South Africa were analysed. Using an experimental approach based on the detection of Stx 2 gene by a phage enrichment culture followed by PCR, bacteriophages infecting E. coli O157:H7 carrying the Shiga toxin 2 gene were detected in 15 of the samples studied. Results presented here show that the presence of phages carrying the Stx 2 gene is common in sewage from developed countries.     

Monoclonal antibodies against Stx1B subunit of Escherichia coli O157:H7 distinguish the bacterium from other bacteria

Aims: The Shiga‐like toxins (Stx) are critical virulence factors of enterohaemorrhagic Escherichia coli (EHEC). Stx1B subunit plays important roles in EHEC infection. This work aims to generate and characterize monoclonal antibodies (mAbs) against the Stx1B and to investigate their utility in discrimination ELISA. Methods and Results: Two newly identified mAbs (designated 2H8 and 1B10, respectively) against the Stx1B protein were prepared via hybridoma techniques. The immunoreactivity of both mAbs to the Stx1B protein was confirmed in ELISA and Western blot. Moreover, they differentiate EHEC from Salmonella enteritis, non‐Stx1‐producing E. coli, Mycobacterium tuberculosis, Listeria monocytogenes, Streptococcus agalactiae and Staphylococcus aureus. Conclusions: The anti‐STx1B mAbs are valuable diagnostic reagents for distinguishing EHEC from other bacteria. Significance and Impact of the Study: This is the first report regarding the usage of anti‐STx1B mAbs in discrimination ELISA. The established ELISA may have potential in clinical surveillance of EHEC infection.     

Inducible stx2 phages are lysogenized in the enteroaggregative and other phenotypic Escherichia coli O86:HNM isolated from patients

We characterized two Shiga toxin-producing Escherichia coli (STEC) O86:HNM isolates from a patient with hemolytic uremic syndrome (HUS) or bloody diarrhea. Both of them did not possess the eaeA gene. However, the isolate from a HUS patient carried genetic markers of enteroaggregative E. coli (EAEC) and showed aggregative adherence pattern to HEp-2 cells. The other isolate from bloody diarrhea, which was negative with EAEC markers, was diffusely adhered to HEp-2 cells. The stx2 gene in both E. coli O86:HNM strains was encoded in each infectious phage, which was partially homologous to that of strain EDL933, a STEC O157:H7. These results will help to explain the genotypic divergences of STEC.     

The effect of probiotic treatment with Clostridium butyricum on enterohemorrhagic Escherichia coli O157:H7 infection in mice

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 has been considered as an agent responsible for outbreak of hemorrhagic colitis and the hemolytic uremic syndrome. We examined the effect of the probiotic agent Clostridium butyricum MIYAIRI strain 588 on EHEC O157:H7 infections in vitro and in vivo using gnotobiotic mice. The growth of EHEC O157:H7 and the production of Shiga-like toxins in broth cultures were inhibited by co-incubation with C. butyricum. The antibacterial effects of butyric and lactic acid were demonstrated in a dose-dependent manner. In addition, the inhibitory effect of butyric acid on the viability of EHEC was demonstrated not only at low pH, but also at neutral pH adjusted to 7.0. Flowcytometric analysis showed that pre-incubation of Caco-2 cells with C. butyricum and E. coli K12 inhibited the adhesion of EHEC O157:H7. However, the effect of C. butyricum on adhesion of EHEC to Caco-2 cells was more inhibitory than that of E. coli K12. Gnotobiotic mice mono-associated with EHEC O157:H7 died within 4-7 days after the infection. On the other hand, all gnotobiotic mice prophylactically pre-treated with C. butyricum survived exposure to EHEC O157:H7 and of the gnotobiotic mice therapeutically post-treated with C. butyricum, 50% survived. Both counts of EHEC O157:H7 and the amounts of shiga-like toxins (Stx1 and Stx2) in fecal contents of gnotobiotic mice di-associated with EHEC O157:H7 and C. butyricum were less than those of gnotobiotic mice mono-associated with EHEC O157:H7. These results indicated that the probiotic bacterium C. butyricum MIYAIRI strain 588 has preventive and therapeutic effects on EHEC O157:H7 infection in gnotobiotic mice.     

Bacteriophages: A possible solution to combat enteropathogenic Escherichia coli infections in neonatal goats

Due to awareness and benefits of goat rearing in developing economies, goats' significance is increasing. Unfortunately, these ruminants are threatened via multiple bacterial pathogens such as enteropathogenic Escherichia coli (EPEC). In goat kids and lambs, EPEC causes gastrointestinal disease leading to substantial economic losses for farmers and may also pose a threat to public health via the spread of zoonotic diseases. Management of infection is primarily based on antibiotics, but the need for new therapeutic measures as an alternative to antibiotics is becoming vital because of the advent of antimicrobial resistance (AMR). The prevalence of EPEC was established using bfpA gene, uspA gene and Stx1 gene, followed by phylogenetic analysis using Stx1 gene. The lytic activity of the isolated putative coliphages was tested on multi-drug resistant strains of EPEC. It was observed that a PCR based approach is more effective and rapid as compared to phenotypic tests of Escherichia coli virulence. It was also established that the isolated bacteriophages exhibited potent antibacterial efficacy in vitro, with some of the isolates (16%) detected as T4 and T4-like phages based on gp23 gene. Hence, bacteriophages as therapeutic agents may be explored as an alternative to antibiotics in managing public, livestock and environmental health in this era of AMR.     

Invasion by activated macrophages requires delivery of nascent membrane‐type‐1 matrix metalloproteinase through late endosomes/lysosomes to the cell surface

Macrophage migration into injured or infected tissue is a key aspect in the pathophysiology of many diseases where inflammation is a driving factor. Membrane‐type‐1 matrix metalloproteinase (MT1‐MMP) cleaves extracellular matrix components to facilitate invasion. Here we show that, unlike the constitutive MT1‐MMP surface recycling seen in cancer cells, unactivated macrophages express low levels of MT1‐MMP. Upon lipopolysaccharide (LPS) activation, MT1‐MMP synthesis dramatically increases 10‐fold at the surface by 15 hours. MT1‐MMP is trafficked from the Golgi complex to the surface via late endosomes/lysosomes in a pathway regulated by the late endosome/lysosome R‐SNAREs VAMP7 and VAMP8. These form two separate complexes with the surface Q‐SNARE complex Stx4/SNAP23 to regulate MT1‐MMP delivery to the plasma membrane. Loss of either one of these SNAREs leads to a reduction in surface MT1‐MMP, gelatinase activity and reduced invasion. Thus, inhibiting MT1‐MMP transport through this pathway could reduce macrophage migration and the resulting inflammation.     

单克隆抗体S2C4对2型志贺毒素及其亚型毒性的中和作用

纯化的2型志贺毒素(Shiga toxin 2,Stx2)经福尔马林脱毒后免疫BALB/c小鼠制备Stx2单克隆抗体,用体外中和试验对具有中和活性的阳性抗体克隆进行初筛,对所获得的中和抗体的重、轻链同种型及结合特异性进行鉴定,其中和保护作用通过体内、体外中和试验加以验证,最后,中和抗体对Stx2亚型Stx2c和Stx2vha的中和谱用体内中和试验验证．结果显示,12株抗Stx2的阳性抗体克隆中,只有1株具有中和活性,命名为S2C4,其重、轻链同种型为G₁/κ,其靶分子为Stx2的A亚单位,与Stx2的B亚单位或Stx1不结合．在体外中和试验中S2C4可有效中和Stx2对Vero细胞的杀伤作用,同样,S2C4可中和致死量的Stx2及其亚型Stx2c和Stx2vha对小鼠的毒性作用．该抗体有望成为治疗产志贺毒素大肠杆菌感染的候选分子．     

Shiga toxin 1 interaction with enterocytes causes apical protein mistargeting through the depletion of intracellular galectin-3

Shiga toxins (Stx) 1 and 2 are responsible for intestinal and systemic sequelae of infection by enterohemorrhagic Escherichia coli (EHEC). However, the mechanisms through which enterocytes are damaged remain unclear. While secondary damage from ischemia and inflammation are postulated mechanisms for all intestinal effects, little evidence excludes roles for more primary toxin effects on intestinal epithelial cells. We now document direct pathologic effects of Stx on intestinal epithelial cells. We study a well-characterized rabbit model of EHEC infection, intestinal tissue and stool samples from EHEC-infected patients, and T84 intestinal epithelial cells treated with Stx1. Toxin uptake by intestinal epithelial cells in vitro and in vivo causes galectin-3 depletion from enterocytes by increasing the apical galectin-3 secretion. This Shiga toxin-mediated galectin-3 depletion impairs trafficking of several brush border structural proteins and transporters, including villin, dipeptidyl peptidase IV, and the sodium-proton exchanger 2, a major colonic sodium absorptive protein. The mistargeting of proteins responsible for the absorptive function might be a key event in Stx1-induced diarrhea. These observations provide new evidence that human enterocytes are directly damaged by Stx1. Conceivably, depletion of galectin-3 from enterocytes and subsequent apical protein mistargeting might even provide a means whereby other pathogens might alter intestinal epithelial absorption and produce diarrhea.     

Abnormal Rab11‐Rab8‐vesicles cluster in enterocytes of patients with microvillus inclusion disease

Microvillus inclusion disease (MVID) is a congenital enteropathy characterized by accumulation of vesiculo‐tubular endomembranes in the subapical cytoplasm of enterocytes, historically termed “secretory granules.” However, neither their identity nor pathophysiological significance is well defined. Using immunoelectron microscopy and tomography, we studied biopsies from MVID patients (3× Myosin 5b mutations and 1× Syntaxin3 mutation) and compared them to controls and genome‐edited CaCo2 cell models, harboring relevant mutations. Duodenal biopsies from 2 patients with novel Myosin 5b mutations and typical clinical symptoms showed unusual ultrastructural phenotypes: aberrant subapical vesicles and tubules were prominent in the enterocytes, though other histological hallmarks of MVID were almost absent (ectopic intra‐/intercellular microvilli, brush border atrophy). We identified these enigmatic vesiculo‐tubular organelles as Rab11‐Rab8‐positive recycling compartments of altered size, shape and location harboring the apical SNARE Syntaxin3, apical transporters sodium‐hydrogen exchanger 3 (NHE3) and cystic fibrosis transmembrane conductance regulator. Our data strongly indicate that in MVID disrupted trafficking between cargo vesicles and the apical plasma membrane is the primary cause of a defect of epithelial polarity and subsequent facultative loss of brush border integrity, leading to malabsorption. Furthermore, they support the notion that mislocalization of transporters, such as NHE3 substantially contributes to the reported sodium loss diarrhea.      

The critical role of lipopolysaccharide in the upregulation of aquaporin 4 in glial cells treated with Shiga toxin

Background

In 2011, there was an outbreak of Shiga toxin-producing Escherichia coli (STEC) infections in Japan. Approximately 62 % of patients with hemolytic-uremic syndrome also showed symptoms of encephalopathy. To determine the mechanisms of onset for encephalopathy during STEC infections, we conducted an in vitro study with glial cell lines and primary glial cells.

Results

Shiga toxin 2 (Stx-2) in combination with lipopolysaccharide (LPS), or LPS alone activates nuclear factor-κB (NF-κB) signaling in glial cells. Similarly, Stx-2 in combination with LPS, or LPS alone increases expression levels of aquaporin 4 (AQP4) in glial cells. It is possible that overexpression of AQP4 results in a rapid and increased influx of osmotic water across the plasma membrane into cells, thereby inducing cell swelling and cerebral edema.

Conclusions

We have showed that a combination of Stx-2 and LPS induced apoptosis of glial cells recently. Glial cells are indispensable for cerebral homeostasis; therefore, their dysfunction and death impairs cerebral homeostasis and results in encephalopathy. We postulate that the onset of encephalopathy in STEC infections occurs when Stx-2 attacks vascular endothelial cells of the blood–brain barrier, inducing their death. Stx-2 and LPS then attack the exposed glial cells that are no longer in contact with the endothelial cells. AQP4 is overexpressed in glial cells, resulting in their swelling and adversely affecting cerebral homeostasis. Once cerebral homeostasis is affected in such a way, encephalopathy is the likely result in STEC patients.

Electronic supplementary material

The online version of this article (doi:10.1186/s12929-015-0184-5) contains supplementary material, which is available to authorized users.     

Heparin blocks the adhesion of E. coli O157:H7 to human colonic epithelial cells

Adhesion of Shiga toxin-producing Enterohemorrhagic Escherichia coli (EHEC) O157:H7 to human colonic epithelium is a critical step for infection by this type of bacteria. Here, we demonstrate that adherence of EHEC O157:H7 to cultured human colonic T84 epithelial monolayers can be blocked by heparin and heparan sulfate in a dose-dependent fashion. In doing this, heparin and heparan sulfate also prevent dysfunction of the T84 barrier and disorganization of epithelial tight junction protein ZO-1 caused by EHEC O157:H7. This inhibition by heparin and heparan sulfate seems to result from a block in the binding interactions of bacteria intimin with epithelial β₁ integrins. This study provides evidence, for the first time, that heparin and heparan sulfate can serve as novel effective blockers in preventing EHEC O157:H7 infection.     

Role of H(abc) domain in membrane trafficking and targeting of syntaxin 1A

Membrane syntaxin plays essential roles in exocytosis in eukaryotic cells. The conservative H(abc) domain in plasma membrane syntaxins implies important roles for syntaxin targeting and function. Our previous study showed H(abc) domain was necessary for the trafficking and cluster distribution of syntaxin 1A on the plasma membrane. Here we identified which of the three domains (H(a), H(b) and H(c)) was essential for Stx1A trafficking and clustering. We found that, in INS-1 cells, the mutant truncated with either H(a), H(b) or H(c) domain could be sorted to the cell surface by a different mechanism compared to that of whole H(abc) truncated mutant. In contrast to wild type Stx1A, none of the mutants showed cluster distribution at the functional sites, suggesting that the physiological localization of Stx1A relies on intact H(abc) domain. Furthermore Munc18-1 is found not to be essential for Stx1A cluster distribution, despite important role in stabilizing membrane delivery of Stx1A.     

An efficient co-expression and purification system for the complex of Stx4 and C-terminal domain of Synip

Synip and Stx4 complex plays a key role in GLUT4 vesicle trafficking and fusion with plasma membrane. The interaction of Synip with Stx4 prevents interaction of VAMP2 located in GLUT4 vesicle with Stx4 in basal state. Insulin induces the dissociation of the Synip and Stx4 complex, and then triggers VAMP2 to interact with Stx4 to form the SNARE complex, thus promoting the vesicle fusion. In this report, we adopt a novel system for co-expression of the Synip and Stx4 by using two common vectors pGEX6p-1 and pET28a(+) to investigate their expression, purification, and interaction. Through this co-expression system, we successfully co-expressed the Synip and Stx4 complex with high yield, and co-purified at an approximate 1:1 molar ratio with high purity (95%). We also demonstrate that the 1-28 residues of Stx4 are dispensable for interaction with Synip using this co-expression system.     

A mechanism of Munc18b-syntaxin 3-SNAP25 complex assembly in regulated epithelial secretion

Syntaxin and Munc18 are essential for regulated exocytosis in all eukaryotes. It was shown that Munc18 inhibition of neuronal syntaxin 1 can be overcome by CDK5 phosphorylation, indicating that structural change disrupts the syntaxin-Munc18 interaction. Here, we show that this phosphorylation promotes the assembly of Munc18b-syntaxin 3-SNAP25 tripartite complex and membrane fusion machinery SNARE. Using siRNAs to screen for genes required for regulated epithelial secretion, we identified the requirements of CDK5 and Munc18b in cAMP-dependent gastric acid secretion. Biochemical characterization revealed that Munc18b bears a syntaxin 3-selective binding site located at its most C-terminal 53 amino acids. Significantly, the phosphorylation of Thr572 by CDK5 attenuates Munc18b-syntaxin 3 interaction and promotes formation of Munc18b-syntaxin 3-SNAP25 tripartite complex, leading to an assembly of functional Munc18b-syntaxin 3-SNAP25-VAMP2 membrane fusion machinery. Thus, our studies suggest a novel regulatory mechanism in which phosphorylation of Munc18b operates vesicle docking and fusion in regulated exocytosis.     

Functional characterization of fish neuroglobin: Zebrafish neuroglobin is highly expressed in amacrine cells after optic nerve injury and can translocate into ZF4 cells

Neuroglobin (Ngb) is a recently discovered vertebrate heme protein that is expressed in the brain and can reversibly bind oxygen. Mammalian Ngb is involved in neuroprotection under conditions of oxidative stress, such as ischemia and reperfusion. We previously found that zebrafish Ngb can penetrate the mammalian cell membrane. In the present study, we investigated the functional characteristics of fish Ngb by using the zebrafish cell line ZF4 and zebrafish retina. We found that zebrafish Ngb translocates into ZF4 cells, but cannot protect ZF4 cells against cell death induced by hydrogen peroxide. Furthermore, we demonstrated that a chimeric ZHHH Ngb protein, in which module M1 of human Ngb is replaced by that of zebrafish, is a cell-membrane-penetrating protein that can protect ZF4 cells against hydrogen peroxide exposure. Moreover, we investigated the localization of Ngb mRNA and protein in zebrafish retina and found that Ngb mRNA is expressed in amacrine cells in the inner nuclear layer and is significantly increased in amacrine cells 3 days after optic nerve injury. Immunohistochemical studies clarified that Ngb protein levels were increased in both amacrine cells and presynaptic regions in the inner plexiform layer after nerve injury. Taken together, we hypothesize that fish Ngb, whose expression is upregulated in amacrine cells after optic nerve injury, might be released from amacrine cells, translocate into neighboring ganglion cells, and function in the early stage of optic nerve regeneration. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.     

Simvastatin induces autophagic flux to restore cerulein-impaired phagosome-lysosome fusion in acute pancreatitis

BackgroundDuring pancreatitis, autophagy is activated, but lysosomal degradation of dysfunctional organelles including mitochondria is impaired, resulting in acinar cell death. Retrospective cohort analyses demonstrated an association between simvastatin use and decreased acute pancreatitis incidence.MethodsWe examined whether simvastatin can protect cell death induced by cerulein and the mechanisms involved during acute pancreatitis. Mice were pretreated with DMSO or simvastatin (20 mg/kg) for 24 h followed by 7 hourly cerulein injections and sacrificed 1 h after last injection to harvest blood and tissue for analysis.ResultsPancreatic histopathology revealed that simvastatin reduced necrotic cell death, inflammatory cell infiltration and edema. We found that cerulein triggered mitophagy with autophagosome formation in acinar cells. However, autophagosome-lysosome fusion was impaired due to altered levels of LAMP-1, AMPK and ULK-1, resulting in autophagosome accumulation (incomplete autophagy). Simvastatin abrogated these effects by upregulating LAMP-1 and activating AMPK which phosphorylated ULK-1, resulting in increased formation of functional autolysosomes. In contrast, autophagosomes accumulated in control group during pancreatitis. The effects of simvastatin to promote autophagic flux were inhibited by chloroquine. Mitochondria from simvastatin-treated mice were resistant to calcium overload compared to control, suggesting that simvastatin induced mitochondrial quality control to eliminate susceptible mitochondria. Clinical specimens showed a significant increase in cell-free mtDNA in plasma during pancreatitis compared to normal controls. Furthermore, genetic deletion of parkin abrogated the benefits of simvastatin.ConclusionOur findings reveal the novel role of simvastatin in enhancing autophagic flux to prevent pancreatic cell injury and pancreatitis.