Primary Human Colon Epithelial Cells (pHCoEpiCs) Do Express the Shiga Toxin (Stx) Receptor Glycosphingolipids Gb3Cer and Gb4Cer and Are Largely Refractory but Not Resistant towards Stx.

Shiga toxin (Stx) is released by enterohemorrhagic Escherichia coli (EHEC) into the human intestinal lumen and transferred across the colon epithelium to the circulation. Stx-mediated damage of human kidney and brain endothelial cells and renal epithelial cells is a renowned feature, while the sensitivity of the human colon epithelium towards Stx and the decoration with the Stx receptor glycosphingolipids (GSLs) globotriaosylceramide (Gb3Cer, Galα1-4Galß1-4Glcß1-1Cer) and globotetraosylceramide (Gb4Cer, GalNAcß1-3Galα1-4Galß1-4Glcß1-1Cer) is a matter of debate. Structural analysis of the globo-series GSLs of serum-free cultivated primary human colon epithelial cells (pHCoEpiCs) revealed Gb4Cer as the major neutral GSL with Cer (d18:1, C16:0), Cer (d18:1, C22:1/C22:0) and Cer (d18:1, C24:2/C24:1) accompanied by minor Gb3Cer with Cer (d18:1, C16:0) and Cer (d18:1, C24:1) as the dominant lipoforms. Gb3Cer and Gb4Cer co-distributed with cholesterol and sphingomyelin to detergent-resistant membranes (DRMs) used as microdomain analogs. Exposure to increasing Stx concentrations indicated only a slight cell-damaging effect at the highest toxin concentration of 1 µg/mL for Stx1a and Stx2a, whereas a significant effect was detected for Stx2e. Considerable Stx refractiveness of pHCoEpiCs that correlated with the rather low cellular content of the high-affinity Stx-receptor Gb3Cer renders the human colon epithelium questionable as a major target of Stx1a and Stx2a.

Real-time interaction analysis of Shiga toxins and membrane microdomains of primary human brain microvascular endothelial cells.

Infections of the human intestinal tract with enterohemorrhagic Escherichia coli (EHEC) result in massive extraintestinal complications due to translocation of EHEC-released Shiga toxins (Stxs) from the gut into the circulation. Stx-mediated damage of the cerebral microvasculature raises serious brain dysfunction being the most frequent cause of acute mortality in patients suffering from severe EHEC infections. Stx2a and Stx2e are associated with heavy and mild course of infection, respectively. Stx2a preferentially binds to globotriaosylceramide (Gb3Cer, Galα1-4Galß1-4Glcß1-1Cer), while Stx2e prefers globotetraosylceramide (Gb4Cer, GalNAcß1-3Galα1-4Galß1-4Glcß1-1Cer). Both glycosphingolipids (GSLs) were detected in detergent-resistant membranes (DRMs) of primary human brain microvascular endothelial cells (pHBMECs) resembling microdomains of the plasma membrane. In this study, we show that Gb3Cer and Gb4Cer of pHBMECs with saturated C16:0, C22:0, and C24:0 fatty acids dominated in DRMs, corresponding to the liquid-ordered membrane phase, whereas lipoforms carrying unsaturated C24:1 and C24:2 fatty acids prevailed in the non-DRM fractions, which correspond to the liquid-disordered membrane phase. Similarly, a shift of the phospholipids from saturated lipoforms in the DRM to unsaturated species in the non-DRM fractions was observed. Real-time biomolecular interaction analysis using affinity-purified Stx2a and Stx2e, recorded with a surface acoustic wave (SAW) biosensor, evidenced high binding strength of both toxins toward DRMs and failure in interaction with non-DRMs. These results support the hypothesis of preferential binding of Stxs toward microdomains harboring GSL receptors carrying saturated fatty acids in their lipid anchors. Collectively, unraveling the precise mechanisms of Stx-microdomain interaction may help to develop antiadhesive compounds to combat Stx-mediated cellular injury.

Dissemination of the blaCTX-M-15 gene among Enterobacteriaceae via outer membrane vesicles.

BACKGROUND: Bacterial outer membrane vesicles (OMVs) are an emerging source of antibiotic resistance transfer but their role in the spread of the blaCTX-M-15 gene encoding the most frequent CTX-M ESBL in Enterobacteriaceae is unknown. OBJECTIVES: To determine the presence of blaCTX-M-15 and other antibiotic resistance genes in OMVs of the CTX-M-15-producing MDR Escherichia coli O104:H4 outbreak strain and the ability of these OMVs to spread these genes among Enterobacteriaceae under different conditions. METHODS: OMV-borne antibiotic resistance genes were detected by PCR; OMV-mediated transfer of blaCTX-M-15 and the associated blaTEM-1 was quantified under laboratory conditions, simulated intraintestinal conditions and under ciprofloxacin stress; resistance to antibiotics and the ESBL phenotype were determined by the CLSI disc diffusion methods and the presence of pESBL by plasmid profiling and Southern blot hybridization. RESULTS: E. coli O104:H4 OMVs carried blaCTX-M-15 and blaTEM-1 located on the pESBL plasmid, but not chromosomal antibiotic resistance genes. The OMVs transferred blaCTX-M-15, blaTEM-1 and the associated pESBL into Enterobacteriaceae of different species. The frequencies of the OMV-mediated transfer were significantly increased under simulated intraintestinal conditions and under ciprofloxacin stress when compared with laboratory conditions. The 'vesiculants' (i.e. recipients that received the blaCTX-M-15- and blaTEM-1-harbouring pESBL via OMVs) acquired resistance to cefotaxime, ceftazidime and cefpodoxime and expressed the ESBL phenotype. They were able to further spread pESBL and the blaCTX-M-15 and blaTEM-1 genes via OMVs. CONCLUSIONS: OMVs are efficient vehicles for dissemination of the blaCTX-M-15 gene among Enterobacteriaceae and may contribute to blaCTX-M-15 transfer in the human intestine.

PapG subtype-specific binding characteristics of Escherichia coli towards globo-series glycosphingolipids of human kidney and bladder uroepithelial cells.

Uropathogenic Escherichia coli (UPEC) are the primary cause of urinary tract infections (UTIs) in humans. P-fimbriae are key players for bacterial adherence to the uroepithelium through the Galα1-4Gal-binding PapG adhesin. The three identified classes I, II and III of PapG are supposed to adhere differently to host cell glycosphingolipids (GSLs) of the uroepithelial tract harboring a distal or internal Galα1-4Gal sequence. In this study, GSL binding characteristics were obtained in a nonradioactive adhesion assay using biotinylated E. coli UTI and urine isolates combined with enzyme-linked NeutrAvidin for detection. Initial experiments with reference globotriaosylceramide (Gb3Cer, Galα1-4Galß1-4Glcß1-1Cer), globotetraosylceramide (Gb4Cer, GalNAcß1-3Galα1-4Galß1-4Glcß1-1Cer) and Forssman GSL (GalNAcα1-3GalNAcß1-3Galα1-4Galß1-4Glcß1-1Cer) revealed balanced adhesion toward the three GSLs for PapG I-mediated attachment. In contrast, E. coli carrying PapG II or PapG III increasingly adhered to growing oligosaccharide chain lengths of Gb3Cer, Gb4Cer and Forssman GSL. Binding studies with GSLs from human A498 kidney and human T24 bladder epithelial cells, both being negative for the Forssman GSL, revealed the less abundant Gb4Cer vs. Gb3Cer as the prevalent receptor in A498 cells of E. coli expressing PapG II or PapG III. On the other hand, T24 cells exhibited a higher relative content of Gb4Cer vs. Gb3Cer alongside dominant binding of PapG II- or PapG III-harboring E. coli toward Gb4Cer and vastly lowered attachment to minor Gb3Cer. Further studies on PapG-mediated interaction with cell surface-exposed GSLs will improve our knowledge on the molecular mechanisms of P-fimbriae-mediated adhesion and may contribute to the development of antiadhesion therapeutics to combat UTIs.

Host cell interactions of outer membrane vesicle-associated virulence factors of enterohemorrhagic Escherichia coli O157: Intracellular delivery, trafficking and mechanisms of cell injury.

Outer membrane vesicles (OMVs) are important tools in bacterial virulence but their role in the pathogenesis of infections caused by enterohemorrhagic Escherichia coli (EHEC) O157, the leading cause of life-threatening hemolytic uremic syndrome, is poorly understood. Using proteomics, electron and confocal laser scanning microscopy, immunoblotting, and bioassays, we investigated OMVs secreted by EHEC O157 clinical isolates for virulence factors cargoes, interactions with pathogenetically relevant human cells, and mechanisms of cell injury. We demonstrate that O157 OMVs carry a cocktail of key virulence factors of EHEC O157 including Shiga toxin 2a (Stx2a), cytolethal distending toxin V (CdtV), EHEC hemolysin, and flagellin. The toxins are internalized by cells via dynamin-dependent endocytosis of OMVs and differentially separate from vesicles during intracellular trafficking. Stx2a and CdtV-B, the DNase-like CdtV subunit, separate from OMVs in early endosomes. Stx2a is trafficked, in association with its receptor globotriaosylceramide within detergent-resistant membranes, to the Golgi complex and the endoplasmic reticulum from where the catalytic Stx2a A1 fragment is translocated to the cytosol. CdtV-B is, after its retrograde transport to the endoplasmic reticulum, translocated to the nucleus to reach DNA. CdtV-A and CdtV-C subunits remain OMV-associated and are sorted with OMVs to lysosomes. EHEC hemolysin separates from OMVs in lysosomes and targets mitochondria. The OMV-delivered CdtV-B causes cellular DNA damage, which activates DNA damage responses leading to G2 cell cycle arrest. The arrested cells ultimately die of apoptosis induced by Stx2a and CdtV via caspase-9 activation. By demonstrating that naturally secreted EHEC O157 OMVs carry and deliver into cells a cocktail of biologically active virulence factors, thereby causing cell death, and by performing first comprehensive analysis of intracellular trafficking of OMVs and OMV-delivered virulence factors, we provide new insights into the pathogenesis of EHEC O157 infections. Our data have implications for considering O157 OMVs as vaccine candidates.

Metabolomic analysis of Shiga toxin 2a-induced injury in conditionally immortalized glomerular endothelial cells.

INTRODUCTION: Shiga toxin 2a (Stx2a) induces hemolytic uremic syndrome (STEC HUS) by targeting glomerular endothelial cells (GEC). OBJECTIVES: We investigated in a metabolomic analysis the response of a conditionally immortalized, stable glomerular endothelial cell line (ciGEnC) to Stx2a stimulation as a cell culture model for STEC HUS. METHODS: CiGEnC were treated with tumor necrosis factor-(TNF)α, Stx2a or sequentially with TNFα and Stx2a. We performed a metabolomic high-throughput screening by lipid- or gas chromatography and subsequent mass spectrometry. Metabolite fold changes in stimulated ciGEnC compared to untreated cells were calculated. RESULTS: 320 metabolites were identified and investigated. In response to TNFα + Stx2a, there was a predominant increase in intracellular free fatty acids and amino acids. Furthermore, lipid- and protein derived pro-inflammatory mediators, oxidative stress and an augmented intracellular energy turnover were increased in ciGEnC. Levels of most biochemicals related to carbohydrate metabolism remained unchanged. CONCLUSION: Stimulation of ciGEnC with TNFα + Stx2a is associated with profound metabolic changes indicative of increased inflammation, oxidative stress and energy turnover.

The 2011 German Enterohemorrhagic Escherichia Coli O104:H4 Outbreak-The Danger Is Still Out There.

Enterohemorrhagic Escherichia coli (EHEC) are Shiga toxin (Stx) producing bacteria causing a disease characterized by bloody (or non-bloody) diarrhea, which might progress to hemolytic uremic syndrome (HUS). EHEC O104:H4 caused the largest ever recorded EHEC outbreak in Germany in 2011, which in addition showed the so far highest incidence rate of EHEC-related HUS worldwide. The aggressive outbreak strain carries an unusual combination of virulence traits characteristic to both EHEC-a chromosomally integrated Stx-encoding bacteriophage, and enteroaggregative Escherichia coli-pAA plasmid-encoded aggregative adherence fimbriae mediating its tight adhesion to epithelia cells. There are currently still open questions regarding the 2011 EHEC outbreak, e.g., with respect to the exact molecular mechanisms resulting in the hypervirulence of the strain, the natural reservoir of EHEC O104:H4, and suitable therapeutic strategies. Nevertheless, our knowledge on these issues has substantially expanded since 2011. Here, we present an overview of the epidemiological, clinical, microbiological, and molecular biological data available on the 2011 German EHEC O104:H4 outbreak.

Membrane assembly of Shiga toxin glycosphingolipid receptors and toxin refractiveness of MDCK II epithelial cells.

Shiga toxins (Stxs) are the major virulence factors of Stx-producing Escherichia coli (STEC), which cause hemorrhagic colitis and severe extraintestinal complications due to injury of renal endothelial cells, resulting in kidney failure. Since kidney epithelial cells are suggested additional targets for Stxs, we analyzed Madin-Darby canine kidney (MDCK) II epithelial cells for presence of Stx-binding glycosphingolipids (GSLs), determined their distribution to detergent-resistant membranes (DRMs), and ascertained the lipid composition of DRM and non-DRM preparations. Globotriaosylceramide and globotetraosylceramide, known as receptors for Stx1a, Stx2a, and Stx2e, and Forssman GSL as a specific receptor for Stx2e, were found to cooccur with SM and cholesterol in DRMs of MDCK II cells, which was shown using TLC overlay assay detection combined with mass spectrometry. The various lipoforms of GSLs were found to mainly harbor ceramide moieties composed of sphingosine (d18:1) and C24:1/C24:0 or C16:0 FA. The cells were highly refractory toward Stx1a, Stx2a, and Stx2e, most likely due to the absence of Stx-binding GSLs in the apical plasma membrane determined by immunofluorescence confocal laser scanning microscopy. The results suggest that the cellular content of Stx receptor GSLs and their biochemical detection in DRM preparations alone are inadequate to predict cellular sensitivity toward Stxs.

Attack of the clones: whole genome-based characterization of two closely related enterohemorrhagic Escherichia coli O26 epidemic lineages.

BACKGROUND: Enterohemorrhagic Escherichia coli (EHEC) O26:H11/H-, the most common non-O157 serotype causing hemolytic uremic syndrome worldwide, are evolutionarily highly dynamic with new pathogenic clones emerging rapidly. Here, we investigated the population structure of EHEC O26 isolated from patients in several European countries using whole genome sequencing, with emphasis on a detailed analysis of strains of the highly virulent new European clone (nEC) which has spread since 1990s. RESULTS: Genome-wide single nucleotide polymorphism (SNP)-based analysis of 32 EHEC O26 isolated in the Czech Republic, Germany, Austria and Italy demonstrated a split of the nEC (ST29C2 clonal group) into two distinct lineages, which we termed, based on their temporal emergence, as "early" nEC and "late" nEC. The evolutionary divergence of the early nEC and late nEC is marked by the presence of 59 and 70 lineage-specific SNPs (synapomorphic mutations) in the genomes of the respective lineages. In silico analyses of publicly available E. coli O26 genomic sequences identified the late nEC lineage worldwide. Using a PCR designed to target the late nEC synapomorphic mutation in the sen/ent gene, we identified the early nEC decline accompanied by the late nEC rise in Germany and the Czech Republic since 2004 and 2013, respectively. Most of the late nEC strains harbor one of two major types of Shiga toxin 2a (Stx2a)-encoding prophages. The type I stx2a-phage is virtually identical to stx2a-phage of EHEC O104:H4 outbreak strain, whereas the type II stx2a-phage is a hybrid of EHEC O104:H4 and EHEC O157:H7 stx2a-phages and carries a novel mutation in Stx2a. Strains harboring these two phage types do not differ by the amounts and biological activities of Stx2a produced. CONCLUSIONS: Using SNP-level analyses, we provide the evidence of the evolutionary split of EHEC O26:H11/H- nEC into two distinct lineages, and a recent replacement of the early nEC by the late nEC in Germany and the Czech Republic. PCR targeting the late nEC synapomorphic mutation in ent/sen enables the discrimination of early nEC strains and late nEC strains in clinical and environmental samples, thereby facilitating further investigations of their geographic distribution, prevalence, clinical significance and epidemiology.

Endothelial progenitor cells accelerate endothelial regeneration in an in vitro model of Shigatoxin-2a-induced injury via soluble growth factors.

Endothelial injury with consecutive microangiopathy and endothelial dysfunction plays a central role in the pathogenesis of the postenteropathic hemolytic uremic syndrome (D + HUS). To identify new treatment strategies, we examined the regenerative potential of endothelial progenitor cells (EPCs) in an in vitro model of Shiga toxin (Stx) 2a-induced glomerular endothelial injury present in D + HUS and the mechanisms of EPC-triggered endothelial regeneration. We simulated the proinflammatory milieu present in D + HUS by priming human renal glomerular endothelial cells (HRGECs) with tumor necrosis factor-α before stimulation with Stx2a. This measure led to a time- and concentration-dependent decrease of HRGEC viability of human renal glomerular endothelial cells as detected by a colorimetric assay. Coincubation with EPCs (104-105 cells/ml) under dynamic flow conditions led to a significant improvement of cell viability in comparison to untreated monolayers (0.45 ± 0.06 vs. 0.16 ± 0.04, P = 0.003). A comparable regenerative effect of EPCs was observed in a coculture model using cell culture inserts (0.41 ± 0.05 vs. 0.16 ± 0.04, P = 0.003) associated with increased concentrations of vascular endothelial growth factor, insulin-like growth factor I, fibroblast growth factor-2, and hepatocyte growth factor in the supernatant. Treatment of Stx2a-injured monolayers with a combination of these growth factors imitated this effect. EPCs did not show distinct sings of migration and angiogenic tube formation in functional assays. These data demonstrate that EPCs significantly improve endothelial viability after Stx2a-induced injury in vitro and that this effect is associated with the release of growth factors by EPCs.

Combining Mass Spectrometry, Surface Acoustic Wave Interaction Analysis, and Cell Viability Assays for Characterization of Shiga Toxin Subtypes of Pathogenic Escherichia coli Bacteria.

Shiga toxin (Stx)-producing Escherichia coli (STEC) and enterohemorrhagic E. coli (EHEC) as a human pathogenic subgroup of STEC are characterized by releasing Stx AB5-toxin as the major virulence factor. Worldwide disseminated EHEC strains cause sporadic infections and outbreaks in the human population and swine pathogenic STEC strains represent greatly feared pathogens in pig breeding and fattening plants. Among the various Stx subtypes, Stx1a and Stx2a are of eminent clinical importance in human infections being associated with life-threatening hemorrhagic colitis and hemolytic uremic syndrome, whereas Stx2e subtype is associated with porcine edema disease with a generalized fatal outcome for the animals. Binding toward the glycosphingolipid globotriaosylceramide (Gb3Cer) is a common feature of all Stx subtypes analyzed so far. Here, we report on the development of a matched strategy combining (i) miniaturized one-step affinity purification of native Stx subtypes from culture supernatant of bacterial wild-type strains using Gb3-functionalized magnetic beads, (ii) structural analysis and identification of Stx holotoxins by electrospray ionization ion mobility mass spectrometry (ESI MS), (iii) functional Stx-receptor real-time interaction analysis employing the surface acoustic wave (SAW) technology, and (iv) Vero cell culture assays for determining Stx-caused cytotoxic effects. Structural investigations revealed diagnostic tryptic peptide ions for purified Stx1a, Stx2a, and Stx2e, respectively, and functional analysis resulted in characteristic binding kinetics of each Stx subtype. Cytotoxicity studies revealed differing toxin-mediated cell damage ranked with Stx1a > Stx2a > Stx2e. Collectively, this matched procedure represents a promising clinical application for the characterization of life-endangering Stx subtypes at the protein level.

Enterohemorrhagic Escherichia coli O157 outer membrane vesicles induce interleukin 8 production in human intestinal epithelial cells by signaling via Toll-like receptors TLR4 and TLR5 and activation of the nuclear factor NF-κB.

Proinflammatory cytokines play important roles in the pathogenesis of diseases caused by enterohemorrhagic Escherichia coli (EHEC) O157, but the spectrum of bacterial components involved in the proinflammatory responses is not fully understood. Here, we investigated the abilities of outer membrane vesicles (OMVs), nanoparticles released by EHEC O157 during growth, to induce production of proinflammatory cytokines in human intestinal epithelial cells. OMVs from both EHEC O157:H7 and sorbitol-fermenting (SF) EHEC O157:H- induced production of interleukin-8 (IL-8) in Caco-2, HCT-8, and HT-29 intestinal epithelial cell lines. H7 flagellin was the key IL-8-inducing component of EHEC O157:H7 OMVs, whereas cytolethal distending toxin V and O157 lipopolysaccharide (LPS) largely contributed to IL-8 production elicited by flagellin-lacking OMVs from SF EHEC O157:H-. The H7 flagellin-mediated signaling via Toll-like receptor (TLR) 5, and O157 LPS-mediated signaling via TLR4/MD-2 complex, which were followed by activation of the nuclear factor NF-κB were major pathways underlying IL-8 production induced by EHEC O157 OMVs. The proinflammatory and immunomodulatory capacities of EHEC O157 OMVs have pathogenetic implications and support the OMVs as suitable vaccine candidates.

Microevolution of epidemiological highly relevant non-O157 enterohemorrhagic Escherichia coli of serogroups O26 and O111.

Enterohemorrhagic Escherichia coli (EHEC) are a cause of bloody diarrhea, hemorrhagic colitis (HC) and the potentially fatal hemolytic uremic syndrome (HUS). While O157:H7 is the dominant EHEC serotype, non-O157 EHEC have emerged as serious causes of disease. In Germany, the most important non-O157 O-serogroups causing one third of EHEC infections, including diarrhea as well as HUS, are O26, O103, O111 and O145. Interestingly, we identified EHEC O-serogroups O26 and O111 in one single sequence type complex, STC29, that also harbours atypical enteropathogenic E. coli (aEPEC). aEPEC differ from typical EHEC merely in the absence of stx-genes. These findings inspired us to unravel a putative microevolutionary scenario of these non-O157 EHEC by whole genome analyses. Analysis of single nucleotide polymorphisms (SNPs) of the maximum common genome (MCG) of 20 aEPEC (11 human/ 9 bovine) and 79 EHEC (42 human/ 36 bovine/ 1 food source) of STC29 identified three distinct clusters: Cluster 1 harboured strains of O-serogroup O111, the central Cluster 2 harboured only O26 aEPEC strains, while the more heterogeneous Cluster 3 contained both EHEC and aEPEC strains of O-serogroup O26. Further combined analyses of accessory virulence associated genes (VAGs) and insertion sites for mobile genetic elements suggested a parallel evolution of the MCG and the acquisition of virulence genes. The resulting microevolutionary model suggests the development of two distinct EHEC lineages from one common aEPEC ancestor of ST29 by lysogenic conversion with stx-converting bacteriophages, independent of the host species the strains had been isolated from. In conclusion, our cumulative data indicate that EHEC of O-serogroups O26 and O111 of STC29 originate from a common aEPEC ancestor and are bona fide zoonotic agents. The role of aEPEC in the emergence of O26 and O111 EHEC should be considered for infection control measures to prevent possible lysogenic conversion with stx-converting bacteriophages as major vehicle driving the emergence of EHEC lineages with direct Public Health consequences.

Lysogenic conversion of atypical enteropathogenic Escherichia coli (aEPEC) from human, murine, and bovine origin with bacteriophage Φ3538 Δstx2::cat proves their enterohemorrhagic E. coli (EHEC) progeny.

Bacteriophages play an important role in the evolution of bacterial pathogens. A phage-mediated transfer of stx-genes to atypical enteropathogenic E. coli (aEPEC) which are prevalent in different hosts, would convert them to enterohemorrhagic E. coli (EHEC). We decided to confirm this hypothesis experimentally to provide conclusive evidence that aEPEC isolated from different mammalian hosts are indeed progenitors of typical EHEC which gain the ability to produce Shiga-Toxin by lysogeny with stx-converting bacteriophages, utilizing the model phage Φ3538 Δstx2::cat. We applied a modified in vitro plaque-assay, using a high titer of a bacteriophage carrying a deletion in the stx2 gene (Φ3538 Δstx2::cat) to increase the detection of lysogenic conversion events. Three wild-type aEPEC strains were chosen as acceptor strains: the murine aEPEC-strain IMT14505 (sequence type (ST)28, serotype Ont:H6), isolated from a striped field mouse (Apodemus agrarius) in the surrounding of a cattle shed, and the human aEPEC-strain 910#00 (ST28, Ont:H6). The close genomic relationship of both strains implies a high zoonotic potential. A third strain, the bovine aEPEC IMT19981, was of serotype O26:H11 and ST21 (STC29). All three aEPEC were successfully lysogenized with phage Φ3538 Δstx2::cat. Integration of the bacteriophage DNA into the aEPEC host genomes was confirmed by amplification of chloramphenicol transferase (cat) marker gene and by Southern-Blot hybridization. Analysis of the whole genome sequence of each of the three lysogens showed that the bacteriophage was integrated into the known tRNA integration site argW, which is highly variable among E. coli. In conclusion, the successful lysogenic conversion of aEPEC with a stx-phage in vitro underlines the important role of aEPEC as progenitors of EHEC. Given the high prevalence and the wide host range of aEPEC acceptors, their high risk of zoonotic transmission should be recognized in infection control measures.

Anti-Shiga toxin 2 antibodies in enterohemorrhagic Escherichia coli O104:H4 infected patients may predict hemolytic uremic syndrome.

BACKGROUND AND AIM: An outbreak of Shiga toxin 2 (Stx2) producing enterohemorrhagic and enteroaggregative Escherichia coli O104:H4 infection in May 2011 in Germany caused enterocolitis and an unprecedented high 22% rate of hemolytic uremic syndrome (HUS). We hypothesized that anti-Stx2 IgM or IgG titers might predict HUS development. METHODS: Thirty-two patients infected with enterohemorrhagic Escherichia coli O104:H4 (HUS: n = 23; non-HUS: n = 9) were retrospectively screened for anti-Stx2 IgM/IgG and matched with clinical data regarding HUS development, fever, superinfection, dialysis, neurological symptoms, intensive care, antibiotic treatment, and plasmapheresis. RESULTS: Only HUS patients showed a prominent Stx2-specific humoral response in the early acute phase. Despite a strong trend towards prediction of HUS development, statistical analysis revealed no significant correlation between high IgM/IgG titers and further key clinical parameters such as fever, superinfection, neurological symptoms, antibiotic treatment, and plasmapheresis. CONCLUSIONS: Anti-Stx2 antibodies seem to accompany or even precede HUS development.

Shiga toxin glycosphingolipid receptors and their lipid membrane ensemble in primary human blood-brain barrier endothelial cells.

Shiga toxin (Stx)-mediated injury to microvascular endothelial cells in the brain significantly contributes to the pathogenesis of the hemolytic-uremic syndrome caused by enterohemorrhagic Escherichia coli (EHEC). Stxs are AB5 toxins and the B-pentamers of the two major Stx subtypes Stx1a and Stx2a preferentially bind to the glycosphingolipid (GSL) globotriaosylceramide (Gb3Cer) expressed by human endothelial cells. Here we report on comprehensive structural analysis of the different lipoforms of Gb3Cer (Galα4Galß4Glcß1Cer) and globotetraosylceramide (Gb4Cer, GalNAcß3Galα4Galß4Glcß1Cer, the less effective Stx receptor) of primary human brain microvascular endothelial cells and their association with lipid rafts. Detergent-resistant membranes (DRMs), obtained by sucrose density gradient ultracentrifugation, were used as lipid raft-analogous microdomains of the liquid-ordered phase and nonDRM fractions were employed as equivalents for the liquid-disordered phase of cell membranes. Structures of the prevalent lipoforms of Gb3Cer and Gb4Cer were those with Cer (d18:1, C16:0), Cer (d18:1, C22:0) and Cer (d18:1, C24:1/C24:0) determined by electrospray ionization mass spectrometry that was combined with thin-layer chromatography immunodetection using anti-Gb3Cer and anti-Gb4Cer antibodies as well as Stx1a and Stx2a subtypes. Association of Stx receptor GSLs was determined by co-localization with lipid raft-specific membrane protein flotillin-2 and canonical lipid raft marker sphingomyelin with Cer (d18:1, C16:0) and Cer (d18:1, C24:1/C24:0) in the liquid-ordered phase, whereas lyso-phosphatidylcholine was detectable exclusively in the liquid-disordered phase. Defining the precise microdomain structures of primary endothelial cells may help to unravel the initial mechanisms by which Stxs interact with their target cells and will help to develop novel preventive and therapeutic measures for EHEC-mediated diseases.

Colocalization of receptors for Shiga toxins with lipid rafts in primary human renal glomerular endothelial cells and influence of D-PDMP on synthesis and distribution of glycosphingolipid receptors.

Damage of human renal glomerular endothelial cells (HRGECs) of the kidney represents the linchpin in the pathogenesis of the hemolytic uremic syndrome caused by Shiga toxins of enterohemorrhagic Escherichia coli (EHEC). We performed a comprehensive structural analysis of the Stx-receptor glycosphingolipids (GSLs) globotriaosylceramide (Gb3Cer, Galα4Galß4Glcß1Cer) and globotetraosylceramide (Gb4Cer, GalNAcß3Galα4Galß4Glcß1Cer) and their distribution in lipid raft analog detergent-resistant membranes (DRMs) and nonDRMs prepared from primary HRGECs. Predominant receptor lipoforms were Gb3Cer and Gb4Cer with Cer (d18:1, C16:0), Cer (d18:1, C22:0) and Cer (d18:1, C24:1/C24:0). Stx-receptor GSLs co-distribute with sphingomyelin (SM) and cholesterol as well as flotillin-2 in DRMs, representing the liquid-ordered membrane phase and indicating lipid raft association. Lyso-phosphatidylcholine (lyso-PC) was identified as a nonDRM marker phospholipid of the liquid-disordered membrane phase. Exposure of primary HRGECs to the ceramide analogon d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP) reduced total Gb3Cer and Gb4Cer content, roughly calculated from two biological replicates, down to half and quarter of its primordial content, respectively, but strengthened their prevalence and cholesterol preponderance in DRMs. At the same time, the distribution of PC, SM and lyso-PC to subcellular membrane fractions remained unaffected by D-PDMP treatment. Defining the GSL composition and precise microdomain structures of primary HRGECs may help to develop novel therapeutic options to combat life-threatening EHEC infections.

Antibiotic-Mediated Modulations of Outer Membrane Vesicles in Enterohemorrhagic Escherichia coli O104:H4 and O157:H7.

Ciprofloxacin, meropenem, fosfomycin, and polymyxin B strongly increase production of outer membrane vesicles (OMVs) in Escherichia coli O104:H4 and O157:H7. Ciprofloxacin also upregulates OMV-associated Shiga toxin 2a, the major virulence factor of these pathogens, whereas the other antibiotics increase OMV production without the toxin. These two effects might worsen the clinical outcome of infections caused by Shiga toxin-producing E. coli Our data support the existing recommendations to avoid antibiotics for treatment of these infections.

Sorbitol-Fermenting Enterohemorrhagic Escherichia coli O157:H- Isolates from Czech Patients with Novel Plasmid Composition Not Previously Seen in German Isolates.

Sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:H- strains, first identified in Germany, have emerged as important pathogens throughout Europe. Besides chromosomally encoded Shiga toxin 2a (the major virulence factor), several putative virulence loci, including the hly, etp, and sfp operons, encoding EHEC hemolysin, type II secretion system proteins, and Sfp fimbriae, respectively, are located on the 121-kb plasmid pSFO157 in German strains. Here we report novel SF EHEC O157:H- strains isolated from patients in the Czech Republic. These strains share the core genomes and chromosomal virulence loci encoding toxins (stx2a and the cdtV-ABC operon) and adhesins (eae-γ, efa1, lpfAO157OI-141, and lpfAO157OI-154) with German strains but differ essentially in their plasmids. In contrast to all previously detected SF EHEC O157:H- strains, the Czech strains carry two plasmids, of 79 kb and 86 kb. The 79-kb plasmid harbors the sfp operon, but neither of the plasmids contains the hly and etp operons. Sequence analyses demonstrated that the 79-kb plasmid (pSFO157 258/98-1) evolved from pSFO157 of German strains by deletion of a 41,534-bp region via homologous recombination, resulting in loss of the hly and etp operons. The 86-kb plasmid (pSFO157 258/98-2) displays 98% sequence similarity to a 92.7-kb plasmid of an extraintestinal pathogenic E. coli bloodstream isolate. Our finding of this novel plasmid composition in SF EHEC O157:H- strains extends the evolutionary history of EHEC O157 plasmids. Moreover, the unique molecular plasmid characteristics permit the identification of such strains, thereby facilitating further investigations of their geographic distribution, clinical significance, and epidemiology.IMPORTANCE Since their first identification in Germany in 1989, sorbitol-fermenting enterohemorrhagic Escherichia coli O157:H- (nonmotile) strains have emerged as important causes of the life-threatening disease hemolytic-uremic syndrome in Europe. They account for 10 to 20% of sporadic cases of this disease and have caused several large outbreaks. The strains isolated throughout Europe share conserved chromosomal and plasmid characteristics. Here we identified novel sorbitol-fermenting enterohemorrhagic E. coli O157:H- patient isolates in the Czech Republic which differ from all such strains reported previously by their unique plasmid characteristics, including plasmid number, composition of plasmid-carried virulence genes, and plasmid origins. Our findings contribute substantially to understanding the evolution of E. coli O157 strains and their plasmids. In practical terms, they enable the identification of strains with these novel plasmid characteristics in patient stool samples and thus the investigation of their roles as human pathogens in other geographic areas.

Shiga toxin of enterohaemorrhagic Escherichia coli directly injures developing human erythrocytes.

Haemolytic anaemia is one of the characteristics of life-threatening extraintestinal complications in humans during infection with enterohaemorrhagic Escherichia coli (EHEC). Shiga toxins (Stxs) of EHEC preferentially damage microvascular endothelial cells of the kidney and the brain, whereby occluded small blood vessels may elicit anaemia through mechanical erythrocyte disruption. Here we show for the first time that Stx2a, the major virulence factor of EHEC, is also capable of direct targeting developing human erythrocytes. We employed an ex vivo erythropoiesis model using mobilized CD34(+) haematopoietic stem/progenitor cells from human blood and monitored expression of Stx receptors and Stx2a-mediated cellular injury of developing erythrocytes. CD34(+) haematopoietic stem/progenitor cells were negative for Stx2a receptors and resistant towards the toxin. Expression of Stx2a-binding glycosphingolipids and toxin sensitivity was apparent immediately after initiation of erythropoietic differentiation, peaked for basophilic and polychromatic erythroblast stages and declined during maturation into orthochromatic erythroblasts and reticulocytes, which became highly refractory to Stx2a. The observed Stx-mediated toxicity towards erythroblasts during the course of erythropoiesis might contribute, although speculative at this stage of research, to the anaemia caused by Stx-producing pathogens.