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H Karch - One of the best experts on this subject based on the ideXlab platform.
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molecular analysis of the plasmid encoded hemolysin of escherichia coli o157 h7 strain edl 933
Infection and Immunity, 1995Co-Authors: Herbert Schmidt, Lothar Beutin, H KarchAbstract:In this study, we determined the nucleotide sequence of the 5.4-kb SalI restriction fragment of the recombinant plasmid pEO40-1, cloned from the large plasmid of enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain EDL 933. This revealed two open reading frames which shared approximately 60% homology to the hlyC and hlyA genes of the E. coli alpha-hemolysin (alpha-hly) operon. We termed these genes EHEC-hlyA and EHEC-hlyC to distinguish them from the alpha-hly genes. Preliminary sequence analysis indicated that another open reading frame homolog to the hlyB gene is located close to the 3' end of EHEC-hlyA. The predicted molecular masses of the EHEC-hlyA and EHEC-hlyC gene products were 107 and 19.9 kDa, respectively. The EHEC hemolysin protein (EHEC-Hly) was not secreted into the culture supernatant by the strain EDL 933. However, hemolytic activity was found in the broth culture supernatant after transforming EDL 933 with the recombinant plasmid pRSC6 carrying the hlyB and hlyD genes from the E. coli alpha-hemolysin operon. The EHEC hemolysin was precipitated and used as an antigen for immunoblot analysis. This demonstrated that 19 of 20 reconvalescent-phase serum samples from patients with hemolytic uremic syndrome reacted specifically with the antigen; conversely, only 1 of 20 control serum samples demonstrated reactivity. To investigate the prevalence of EHEC hemolysin genes in diarrheagenic E. coli, a PCR was developed to specifically detect EHEC-hlyA. All Shiga-Like Toxin-producing O157 strains and 12 of 25 Shiga-Like Toxin-producing non-O157 strains were PCR positive; strains of other categories of diarrheagenic E. coli were PCR negative. All PCR-positive strains hybridized with the CVD 419 probe. We found the CVD 419 probe to be identical to the 3.4-kb HindIII fragment of plasmid pEO40 carrying most of the EHEC-hlyA gene and a part of the putative EHEC-hlyB gene. In this study, the newly discovered EHEC hemolysin was shown to be responsible for the enterohemolytic phenotype and demonstrated to be related but not identical to alpha-hemolysin. The EHEC hemolysin appears to have clinical importance because it occurs in all O157 strains tested and is reactive to sera of patients with hemolytic uremic syndrome.
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clonal structure and pathogenicity of shiga like Toxin producing sorbitol fermenting escherichia coli o157 h
Journal of Clinical Microbiology, 1993Co-Authors: H Karch, H Bohm, Herbert Schmidt, F Gunzer, S Aleksic, Jurgen HeesemannAbstract:We compared a collection of sorbitol-fermenting (SF) Escherichia coli O157:H- strains with SF E. coli O157:H45 and non-SF E. coli O157:H7 and E. coli O157:H- strains by pulsed-field gel electrophoresis. The SF E. coli O157:H- strains had identical or closely related XbaI patterns that differed markedly from those for the other E. coli O157 strains. Plasmid content and the presence of Shiga-Like Toxin-converting phages were determined for the SF E. coli O157:H- strains, indicating that these strains harbor a single 90-kb plasmid. They are lysogenized by Toxin-converting phages and harbor the eae gene. Nonmotile E. coli O157 strains were observed to adhere more efficiently to HEp-2 cells than the motile strains. From their phenotypic and genotypic features, the SF E. coli O157:H- strains may well represent a new clone with non-SF E. coli O157:H7 pathogenic characteristics. Images
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dna fingerprinting of escherichia coli o157 h7 strains by pulsed field gel electrophoresis
Journal of Clinical Microbiology, 1992Co-Authors: H Bohm, H KarchAbstract:Pulsed-field gel electrophoresis of genomic DNA was carried out on Escherichia coli O157:H7 strains from different geographic locations to determine its value in an epidemiological survey of O157 infections. Pulsed-field gel electrophoresis of XbaI-digested DNA fragments clearly separated E. coli O157:H7 strains from nontoxigenic E. coli O157:H19, O157:H43, and O157:H45 strains and from Shiga-Like-Toxin-producing E. coli strains of other serogroups. However, among the E. coli O157:H7 strains, the restriction patterns either were identical or differed only by a few fragment bands. In some cases, it was therefore impossible to distinguish among epidemiologically unrelated strains. Hybridization experiments with a DNA probe complementary to Shiga-Like Toxin II sequences revealed that the Shiga-Like Toxin II genes were located on DNA fragments of different lengths. Our data show that for a single highly conserved clone, such as E. coli O157:H7, other typing techniques may need to be performed in addition to DNA fingerprinting in epidemiological surveys.
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molecular detection of sorbitol fermenting escherichia coli o157 in patients with hemolytic uremic syndrome
Journal of Clinical Microbiology, 1992Co-Authors: F Gunzer, H Bohm, S Aleksic, Holger Russmann, Martin Bitzan, H KarchAbstract:Shiga-Like Toxin-producing Escherichia coli strains of serogroup O157 were identified in 26 of 104 patients with hemolytic-uremic syndrome and in 18 of 668 patients with diarrhea. All strains were identified by colony hybridization with DNA probes complementary to Shiga-Like Toxin I and Shiga-Like Toxin II gene sequences and characterized by biochemical tests and serotyping. Seventeen of these 44 patients had E. coli O157 strains which were unusual because they fermented sorbitol within 24 h of incubation and were positive for beta-glucuronidase activity. Culture filtrates of these sorbitol-fermenting strains were highly toxic to Vero cells in culture. Serological tests and DNA analysis performed by restriction endonuclease digestion of B-subunit Toxin genes revealed that all 17 isolates produced Shiga-Like Toxin II. Although by using molecular probes we established a high frequency of sorbitol-fermenting E. coli O157 strains in the patients we examined, further studies on the prevalence of such isolates in other areas of endemic disease are clearly warranted.
H Bohm - One of the best experts on this subject based on the ideXlab platform.
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clonal structure and pathogenicity of shiga like Toxin producing sorbitol fermenting escherichia coli o157 h
Journal of Clinical Microbiology, 1993Co-Authors: H Karch, H Bohm, Herbert Schmidt, F Gunzer, S Aleksic, Jurgen HeesemannAbstract:We compared a collection of sorbitol-fermenting (SF) Escherichia coli O157:H- strains with SF E. coli O157:H45 and non-SF E. coli O157:H7 and E. coli O157:H- strains by pulsed-field gel electrophoresis. The SF E. coli O157:H- strains had identical or closely related XbaI patterns that differed markedly from those for the other E. coli O157 strains. Plasmid content and the presence of Shiga-Like Toxin-converting phages were determined for the SF E. coli O157:H- strains, indicating that these strains harbor a single 90-kb plasmid. They are lysogenized by Toxin-converting phages and harbor the eae gene. Nonmotile E. coli O157 strains were observed to adhere more efficiently to HEp-2 cells than the motile strains. From their phenotypic and genotypic features, the SF E. coli O157:H- strains may well represent a new clone with non-SF E. coli O157:H7 pathogenic characteristics. Images
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dna fingerprinting of escherichia coli o157 h7 strains by pulsed field gel electrophoresis
Journal of Clinical Microbiology, 1992Co-Authors: H Bohm, H KarchAbstract:Pulsed-field gel electrophoresis of genomic DNA was carried out on Escherichia coli O157:H7 strains from different geographic locations to determine its value in an epidemiological survey of O157 infections. Pulsed-field gel electrophoresis of XbaI-digested DNA fragments clearly separated E. coli O157:H7 strains from nontoxigenic E. coli O157:H19, O157:H43, and O157:H45 strains and from Shiga-Like-Toxin-producing E. coli strains of other serogroups. However, among the E. coli O157:H7 strains, the restriction patterns either were identical or differed only by a few fragment bands. In some cases, it was therefore impossible to distinguish among epidemiologically unrelated strains. Hybridization experiments with a DNA probe complementary to Shiga-Like Toxin II sequences revealed that the Shiga-Like Toxin II genes were located on DNA fragments of different lengths. Our data show that for a single highly conserved clone, such as E. coli O157:H7, other typing techniques may need to be performed in addition to DNA fingerprinting in epidemiological surveys.
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molecular detection of sorbitol fermenting escherichia coli o157 in patients with hemolytic uremic syndrome
Journal of Clinical Microbiology, 1992Co-Authors: F Gunzer, H Bohm, S Aleksic, Holger Russmann, Martin Bitzan, H KarchAbstract:Shiga-Like Toxin-producing Escherichia coli strains of serogroup O157 were identified in 26 of 104 patients with hemolytic-uremic syndrome and in 18 of 668 patients with diarrhea. All strains were identified by colony hybridization with DNA probes complementary to Shiga-Like Toxin I and Shiga-Like Toxin II gene sequences and characterized by biochemical tests and serotyping. Seventeen of these 44 patients had E. coli O157 strains which were unusual because they fermented sorbitol within 24 h of incubation and were positive for beta-glucuronidase activity. Culture filtrates of these sorbitol-fermenting strains were highly toxic to Vero cells in culture. Serological tests and DNA analysis performed by restriction endonuclease digestion of B-subunit Toxin genes revealed that all 17 isolates produced Shiga-Like Toxin II. Although by using molecular probes we established a high frequency of sorbitol-fermenting E. coli O157 strains in the patients we examined, further studies on the prevalence of such isolates in other areas of endemic disease are clearly warranted.
M P Jackson - One of the best experts on this subject based on the ideXlab platform.
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evaluation of a technique for identification of shiga like Toxin producing escherichia coli by using polymerase chain reaction and digoxigenin labeled probes
Journal of Clinical Microbiology, 1993Co-Authors: D Begum, Nancy A Strockbine, E G Sowers, M P JacksonAbstract:A polymerase chain reaction (PCR) technique for the identification of Shiga-Like Toxin (SLT)-producing Escherichia coli was assessed by using 95 strains of SLT-producing E. coli and 5 Shigella dysenteriae type 1 strains. PCR was used for the amplification of slt gene sequences from whole bacterial colonies. A digoxigenin-labeled DNA probe was used for identification of the PCR products in a spot blot hybridization assay. Modifications were made to adapt this technique for the proper identification of 10 SLT-producing isolates which were refractory to the heat lysis step that was used to liberate whole-cell DNA for PCR and 6 isolates which gave nonspecific amplification products. The sensitivity and specificity of this assay were each 99% when compared with Toxin neutralization results by using SLT-specific monoclonal antibodies. These values indicate that this detection technique could be suitable for use in a clinical laboratory.
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detection of shiga Toxin producing shigella dysenteriae type 1 and escherichia coli by using polymerase chain reaction with incorporation of digoxigenin 11 dutp
Journal of Clinical Microbiology, 1991Co-Authors: M P JacksonAbstract:A technique has been developed for the detection of Shiga Toxin- and Shiga-Like Toxin type I (ShT/SLT-I)-producing Shigella dysenteriae type 1 and Escherichia coli by using the polymerase chain reaction with the incorporation of digoxigenin-11-dUTP. Target DNA liberated from whole cells was amplified, using primer pairs homologous to the A-subunit genes of ShT/SLT-I. The TTP analog digoxigenin-11-dUTP was incorporated into the reaction mixture, permitting nonradioactive labeling of the amplified DNA. The labeled polymerase chain reaction products were hybridized to specific gene sequences immobilized on a nitrocellulose membrane and detected by using an alkaline phosphatase-conjugated antibody to digoxigenin and the enzyme substrates. Toxin-producing strains of E. coli and S. dysenteriae type 1 were identified as colored spots on the membrane. Because this technique does not require DNA purification, gel electrophoresis, or radioactive DNA probes, it is suitable for the clinical detection of ShT/SLT-I-producing strains of S. dysenteriae type 1 and E. coli. Images
Jean Gariepy - One of the best experts on this subject based on the ideXlab platform.
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RAPID COMMUNICATION Shiga-Like Toxin Purges Human Lymphoma From Bone Marrow of Severe Combined Immunodeficient Mice
2016Co-Authors: C. Lacasse, Mazen T. Saleh, Bruce Patterson, Mark D. Minden, Jean GariepyAbstract:Shiga-Like Toxin-l (SLT-1) is a bacterial Toxin that kills cells by inhibiting protein synthesis. SLT-1 is composed of one cytotoxic A-subunit and five B-subunits that bind to CD77, a cell-surface glycolipid. In the human hematopoietic system, CD77 expression is restricted to a subset of activated B cells and derived cancers. Here we report that SLT-1 treatment of murine bone marrow ex vivo effectively cures severe com-bined immunodeficient mice of a human B-cell lymphoma xenograft while sparing normal hematopoietic precursor WENTY THOUSAND North Americans died of non-Hodgkin’s lymphomas (NHLs) in 1994 alone. A large proportion of NHLs are follicular (B-cell) lymphomas that are classified as low-grade lymphomas and for which no curative treatment exists. ’ Autologous bone marrow trans-plantation (ABMT) in patients with B-cell malignancy i
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an evolved ribosome inactivating protein targets and kills human melanoma cells in vitro and in vivo
Molecular Cancer, 2010Co-Authors: Jean Gariepy, Melissa Cheung, Leigh Revers, Subodini Perampalam, Xin Wei, Reza Kiarash, David Green, Aws AbdulwahidAbstract:Few treatment options exist for patients with metastatic melanoma, resulting in poor prognosis. One standard treatment, dacarbazine (DTIC), shows low response rates ranging from 15 to 25 percent with an 8-month median survival time. The development of targeted therapeutics with novel mechanisms of action may improve patient outcome. Ribosome-inactivating proteins (RIPs) such as Shiga-Like Toxin 1 (SLT-1) represent powerful scaffolds for developing selective anticancer agents. Here we report the discovery and properties of a single chain ribosome-inactivating protein (scRIP) derived from the cytotoxic A subunit of SLT-1 (SLT-1A), harboring the 7-amino acid peptide insertion IYSNKLM (termed SLT-1AIYSNKLM) allowing the Toxin variant to selectively target and kill human melanoma cells. SLT-1AIYSNKLM was able to kill 7 of 8 human melanoma cell lines. This scRIP binds to 518-A2 human melanoma cells with a dissociation constant of 18 nM, resulting in the blockage of protein synthesis and apoptosis in such cells. Biodistribution and imaging studies of radiolabeled SLT-1AIYSNKLM administered intravenously into SCID mice bearing a human melanoma xenograft indicate that SLT-1AIYSNKLM readily accumulates at the tumor site as opposed to non-target tissues. Furthermore, the co-administration of SLT-1AIYSNKLM with DTIC resulted in tumor regression and greatly increased survival in this mouse xenograft model in comparison to DTIC or SLT-1AIYSNKLM treatment alone (115 day median survival versus 46 and 47 days respectively; P values < 0.001). SLT-1AIYSNKLM is stable in serum and its intravenous administration resulted in modest immune responses following repeated injections in CD1 mice. These results demonstrate that the evolution of a scRIP template can lead to the discovery of novel cancer cell-targeted compounds and in the case of SLT-1AIYSNKLM can specifically kill human melanoma cells in vitro and in vivo.
Alison D Obrien - One of the best experts on this subject based on the ideXlab platform.
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the specific activities of shiga like Toxin type ii slt ii and slt ii related Toxins of enterohemorrhagic escherichia coli differ when measured by vero cell cytotoxicity but not by mouse lethality
Infection and Immunity, 1994Co-Authors: Susanne W Lindgren, James E Samuel, Clare K Schmitt, Alison D ObrienAbstract:Characteristically, enterohemorrhagic Escherichia coli (EHEC) strains produce Shiga-Like Toxin type I (SLT-I), SLT-II, or both of these immunologically distinct cytoToxins. No antigenic or receptor-binding variants of SLT-I have been identified, but a number of SLT-II-related Toxins have been described. Because EHEC O91:H21 strain B2F1, which produces two SLT-II-related Toxins, is exquisitely virulent in an orally infected, streptomycin-treated mouse model (oral 50% lethal dose [LD50],
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profile of escherichia coli o157 h7 pathogen responsible for hamburger borne outbreak of hemorrhagic colitis and hemolytic uremic syndrome in washington
Journal of Clinical Microbiology, 1993Co-Authors: Alison D Obrien, A R Melton, C K Schmitt, M L Mckee, M L Batts, D E GriffinAbstract:We analyzed Escherichia coli O157:H7 isolates from stool samples of five patients who had bloody diarrhea and were infected during a large food-borne outbreak of hemorrhagic colitis in Washington state. The isolates were assessed for Shiga-Like Toxin profile, adherence and plasmid traits, mouse virulence, capsule, and enterohemolysin production. The profiles of the five isolates were indistinguishable from each other and similar to that of E. coli O157:H7 strain EDL933, an organism responsible for a similar hamburger-associated food poisoning episode in 1982.