Thorsten Kuczius

Escherichia coli harboring Shiga toxin 2 gene variants: frequency and association with clinical symptoms

Shiga toxin (Stx)-producing Escherichia coli (STEC) from patients with hemolytic-uremic syndrome (HUS), patients with diarrhea without HUS, or asymptomatic subjects were genotyped to assess associations between stx2 variants and clinical manifestations of infection. Neither stx2d nor stx2e was found in 268 STEC isolates from patients with HUS. Of 262 STEC isolates from patients with diarrhea, stx(2d) was found in 41 (15.6%; P<.000001), and stx2e was found in 12 (4.6%; P=.0004). The stx2c genotype frequency was similar among isolates from patients with HUS (3.7%) and diarrhea (5.0%). The frequencies of stx2c, stx2d, and stx2e among 96 STEC isolates from asymptomatic subjects were comparable to those among isolates from patients with diarrhea. None of the 626 STEC isolates contained stx2f. All stx2d-positive or stx2e-positive STEC isolates were eae negative and originated from subjects older than those with STEC isolates with stx2c. stx2c-positive STEC isolates can cause HUS, but the presence of stx2d or stx2e may predict a milder disease with a minimal risk of HUS.

Identification, Characterization, and Distribution of a Shiga Toxin 1 Gene Variant (stx1c) in Escherichia coli Strains Isolated from Humans

ABSTRACT By using sequence analysis of Shiga toxin 1 (Stx 1) genes from human and ovine Stx-producing Escherichia coli (STEC) strains, we identified an Stx1 variant in STEC of human origin that was identical to the Stx1 variant from ovine STEC, but demonstrated only 97.1 and 96.6% amino acid sequence identity in its A and B subunits, respectively, to the Stx1 encoded by bacteriophage 933J. We designated this variant “Stx1c” and developed stxB1 restriction fragment length polymorphism and stx1c-specific PCR strategies to determine the frequency and distribution of stx1c among 212 STEC strains isolated from humans. stx1c was identified in 36 (17.0%) of 212 STEC strains, 19 of which originated from asymptomatic subjects and 16 of which were from patients with uncomplicated diarrhea. stx1c was most frequently (in 23 STEC strains [63.9%]) associated with stx2d, but 12 (33.3%) of the 36 STEC strains possessed stx1c only. A single STEC strain possessed stx1c together with stx2 and was isolated from a patient with hemolytic-uremic syndrome. All 36 stx1c-positive STEC strains were eae negative and belonged to 10 different serogroups, none of which was O157, O26, O103, O111, or O145. Stx1c was produced by all stx1c-containing STEC strains, but reacted weakly with a commercial immunoassay. We conclude that STEC strains harboring the stx1c variant account for a significant proportion of human STEC isolates. The procedures developed in this study now allow the determination of the frequency of STEC strains harboring stx1c among clinical STEC isolates and their association with human disease in prospective studies.

Cytolethal distending toxin from Shiga toxin-producing Escherichia coli O157 causes irreversible G2/M arrest, inhibition of proliferation, and death of human endothelial cells.

ABSTRACT Recently, cytolethal distending toxin V (CDT-V), a new member of the CDT family, was identified in Shiga toxin-producing Escherichia coli (STEC) O157 and particular non-O157 serotypes. Here we investigated the biological effects of CDT-V from STEC O157:H− (strain 493/89) on human endothelial cells, which are believed to be major pathogenetic targets in severe STEC-mediated diseases. CDT-V caused dose-dependent G2/M cell cycle arrest leading to distension, inhibition of proliferation, and death in primary human umbilical vein endothelial cells (HUVEC) and two endothelial cell lines, EA.hy 926 cells (HUVEC derived) and human brain microvascular endothelial cells (HBMEC). The cell cycle effects of CDT-V were cell type specific. In HUVEC and EA.hy 926 cells, CDT-V caused a slowly developing but persistent G2/M block which resulted in delayed nonapoptotic cell death. In contrast, in HBMEC, CDT-V induced a rapidly evolving but transient G2/M block which was followed by progressive, mostly apoptotic cell death. In both HBMEC and EA.hy 926 cells, G2/M arrest was preceded by the early accumulation of a phosphorylated inactive form of cdc2 kinase. Significant G2/M arrest and inhibition of proliferation in both HUVEC and each of the endothelial cell lines were induced by 2 to 15 min of exposure to CDT-V, indicating that the effects of the toxin are irreversible. CDT-V-treated HBMEC and EA.hy 926 cells displayed fragmented nuclei and expressed phosphorylated histone protein H2AX, indicative of DNA damage followed by a DNA repair response. Our data demonstrate that CDT-V causes irreversible damage to human endothelial cells and thus may contribute to the pathogenesis of STEC-mediated diseases.

Molecular Analysis of Bovine Spongiform Encephalopathy and Scrapie Strain Variation

Five mouse scrapie strains, a mouse-passaged scrapie isolate derived from a field case in sheep in Germany, and 2 mouse-passaged bovine spongiform encephalopathy (BSE) isolates were analyzed by immunoblot in regards to banding patterns of proteinase K-digested pathologic prion proteins (PrPres). To obtain reliable results, the photo-imager technique was used for measurement of staining band intensities. Distinct and reproducible profiles were observed for the different strains or isolates. A British and a German BSE isolate were similar, suggesting the same source of infection. The German scrapie isolate resembled scrapie strain ME7, which has frequently been isolated from sheep scrapie in the past. In selected strains or isolates, no influence of the mouse lines used was observed on PrPres profiles, nor were brain region-specific differences apparent. This investigation suggests that PrPres glycotyping can be an invaluable tool for the in vitro differentiation of BSE and scrapie isolates.

Enterohemorrhagic Escherichia coli hemolysin employs outer membrane vesicles to target mitochondria and cause endothelial and epithelial apoptosis.

Enterohemorrhagic Escherichia coli (EHEC) strains cause diarrhea and hemolytic uremic syndrome resulting from toxin-mediated microvascular endothelial injury. EHEC hemolysin (EHEC-Hly), a member of the RTX (repeats-in-toxin) family, is an EHEC virulence factor of increasingly recognized importance. The toxin exists as free EHEC-Hly and as EHEC-Hly associated with outer membrane vesicles (OMVs) released by EHEC during growth. Whereas the free toxin is lytic towards human endothelium, the biological effects of the OMV-associated EHEC-Hly on microvascular endothelial and intestinal epithelial cells, which are the major targets during EHEC infection, are unknown. Using microscopic, biochemical, flow cytometry and functional analyses of human brain microvascular endothelial cells (HBMEC) and Caco-2 cells we demonstrate that OMV-associated EHEC-Hly does not lyse the target cells but triggers their apoptosis. The OMV-associated toxin is internalized by HBMEC and Caco-2 cells via dynamin-dependent endocytosis of OMVs and trafficked with OMVs into endo-lysosomal compartments. Upon endosome acidification and subsequent pH drop, EHEC-Hly is separated from OMVs, escapes from the lysosomes, most probably via its pore-forming activity, and targets mitochondria. This results in decrease of the mitochondrial transmembrane potential and translocation of cytochrome c to the cytosol, indicating EHEC-Hly-mediated permeabilization of the mitochondrial membranes. Subsequent activation of caspase-9 and caspase-3 leads to apoptotic cell death as evidenced by DNA fragmentation and chromatin condensation in the intoxicated cells. The ability of OMV-associated EHEC-Hly to trigger the mitochondrial apoptotic pathway in human microvascular endothelial and intestinal epithelial cells indicates a novel mechanism of EHEC-Hly involvement in the pathogenesis of EHEC diseases. The OMV-mediated intracellular delivery represents a newly recognized mechanism for a bacterial toxin to enter host cells in order to target mitochondria.

New Immuno-PCR Assay for Detection of Low Concentrations of Shiga Toxin 2 and Its Variants

ABSTRACT Shiga toxin (Stx)-producing Escherichia coli (STEC) strains secrete toxins that are major virulence factors and diagnostic targets, but some STEC strains secrete Stx in amounts that cannot be detected using conventional cell cytotoxicity or immunological assays. Therefore, there is an urgent need for more-sensitive Stx detection methods. We describe the development of an assay that can detect low concentrations of Stx2 and its variants. An immuno-PCR Stx2 assay was developed based on an enzyme immunoassay (EIA) combining antibody capture and DNA amplification to increase the signal. The immuno-PCR assay detected 10 pg/ml of purified Stx2, compared to 1 ng/ml Stx2 detected by commercial EIA. Consequently, immuno-PCR detected Stx2 and its variants in STEC strains that produce the toxins at levels that are nondetectable by using the EIA, as well as the Stx2 in EIA-negative enriched stool cultures from patients. Our data demonstrate that the immuno-PCR developed here is a highly sensitive and specific method for the detection of trace amounts of Stx2 and Stx2 variants. It is therefore suitable for use by clinical microbiological laboratories to improve the toxin detection in clinical samples.

Transcriptional analysis of genes encoding Shiga toxin 2 and its variants in Escherichia coli

ABSTRACT Six of 37 non-O157 Escherichia coli strains possessing Shiga toxin (Stx) 2 gene variant stx2d or stx2e secreted no detectable Stx. These isolates produced significantly less stx mRNA than Stx2d, Stx2e, Stx2c, or Stx2 secretors did. Standard screening procedures miss a significant subset of E. coli harboring stx2 variants.

Regional and phenotype heterogeneity of cellular prion proteins in the human brain

Transmissible spongiform encephalopathies (TSEs) are neurological disorders that include genetic, infectious and sporadic forms of human Creutzfeldt‐Jakob disease (CJD). The pathogenic agent is the prion protein that is composed of an abnormal isoform (PrPSc) of a host‐encoded protein (PrPC). Analysis of the relative amounts of PrPSc glycoforms has been used to discriminate between various agents involved in TSE. The distribution and efficiency of conversion to PrPSc can be influenced by differences in the expression of PrPC. However, little attention has been given so far to the banding patterns of PrPC. Using four different antibodies recognizing amino‐ and carboxyl‐terminal PrP sequences we analysed the glycoforms of PrPC in seven regions of the human brain using brains obtained from six subjects. For determination of the staining intensities, signals were quantified by densitometry and reproducible patterns were accomplished by many repeated immunoblot analyses. When amino‐terminal binding antibodies were used for detection, PrPC in the frontal neocortex, nucleus lentiformis, thalamus, hippocampus and cerebellum displayed a glycotype with high staining of the diglycosylated isoforms. This was different from patterns in the pons and medulla oblongata, which showed a high intensity of the nonglycosylated isoform, and PrPC proteins, approximately 27 kDa in size, exhibited high staining using the carboxyl‐terminal binding antibodies. This intense staining followed from an overlay of full‐length and truncated PrPC isoforms. Furthermore, we found marked differences in the expression of PrPC. Variations in the processing of PrPC may lead to interregional differences in the glycoform composition of PrPSc in human brains.

Cellular prion protein acquires resistance to proteolytic degradation following copper ion binding

Abstract The conversion of cellular prion protein (PrPC) into its pathological isoform (PrPSc) conveys an increase in hydrophobicity and induces a partial resistance to proteinase K (PK). Interestingly, co-incubation with high copper ion concentrations also modifies the solubility of PrPC and induces a partial PK resistance which was reminiscent of PrPSc. However, concerns were raised whether this effect was not due to a copper-induced inhibition of the PK itself. We have therefore analyzed the kinetics of the formation of PK-resistant PrPC and excluded possible interference effects by removing unbound copper ions prior to the addition of PK by methanol precipitation or immobilization of PrPC followed by washing steps. We found that preincubation of PrPC with copper ions at concentrations as low as 50 µM indeed rendered these proteins completely PK resistant, while control substrates were proteolyzed. No other divalent cations induced a similar effect. However, in addition to this specific stabilizing effect on PrPC, higher copper ion concentrations insolution (> 200 µM) directly blocked the enzymatic activity of PK, possibly by replacing the Ca2+ ions in the active center of the enzyme. Therefore, as a result of this inhibition the proteolytic degradation of PrPC as well as PrPSc molecules was suppressed.

Molecular analysis of Irish sheep scrapie cases

Different strains of transmissible spongiform encephalopathies in humans and rodent models are associated with the accumulation of PrP(Sc) of distinct molecular characteristics. These characteristics include glycosylation profiles, fragment sizes and long-term resistance of PrP(Sc) to proteinase K. The first objective of this study was to determine the applicability of these criteria to characterize and differentiate sheep scrapie PrP(Sc) and bovine spongiform encephalopathy (BSE) PrP(Sc). PrP(Sc) in sheep scrapie samples from Ireland had clearly distinct molecular characteristics to PrP(Sc) in cattle BSE samples using a monoclonal antibody (MAb P4) directed to position 89-104 of ovine PrP using either brain homogenates or semi-purified scrapie-associated fibrils. Similar glycoprofiles were found when analysing scrapie PrP(Sc) in six different CNS regions (thoracic spinal cord, thalamus, basal ganglia, mediobasal hypothalamus, medulla oblongata and cortex). While the long-term resistance results using a different monoclonal antibody (raised to ruminant PrP positions 145-163; MAb L42) were similar to the results obtained with MAb P4, different glycotyping results were obtained. Given the variation in glycosylation patterns using different antibodies, we conclude that standardization of methodology and antibodies is crucial to the applicability of molecular analysis of ruminant BSE and scrapie samples.