<p>The purpose of this study was to investigate the occurrence<strong> </strong>of Verotoxin-encoding bacteriophages in mussels, cultured downstream the sewage treatment plant<strong> </strong>in Lysekil.</p><p>Mussels were collected in three growing areas from April 2008 to March 2009. Real-time PCR was performed for detection of <em>vtx1</em> and <em>vtx2</em> genes and enrichment of bacteriophages on non Verotoxin-producing <em>Escherichia coli</em> O157: H7 was carried out. All samples in real-time PCR analysis were negative; no presence of Verotoxin-encoding phages was shown. No plaque was formed on blood agar base plates, indicating that no bacteriophages had been taken up by <em>E. coli</em> bacteria</p><p>The levels of Verotoxin-encoding phages and <em>E.coli</em> outside the sewage treatment plant in Lysekil were not high enough to be able to form VTEC in mussels, indicating that the faecal contamination was low. This does not exclude the presence of other more common pathogens such as norovirus and campylobacter.</p><p> </p><p> </p>

The purpose of this study was to investigate the occurrence of Verotoxin-encoding bacteriophages in mussels, cultured downstream the sewage treatment plant in Lysekil. Mussels were collected in three growing areas from April 2008 to March 2009. Real-time PCR was performed for detection of vtx1 and vtx2 genes and enrichment of bacteriophages on non Verotoxin-producing Escherichia coli O157: H7 was carried out. All samples in real-time PCR analysis were negative; no presence of Verotoxin-encoding phages was shown. No plaque was formed on blood agar base plates, indicating that no bacteriophages had been taken up by E. coli bacteria The levels of Verotoxin-encoding phages and E.coli outside the sewage treatment plant in Lysekil were not high enough to be able to form VTEC in mussels, indicating that the faecal contamination was low. This does not exclude the presence of other more common pathogens such as norovirus and campylobacter.

No description available.

Verotoxin produced by Enterohemorrhagic E. coli is comprised of a catalytic A subunit and a receptor Gb3 binding B subunit pentamer. VT causes protein synthesis inhibition by ribosomal inactivation in Gb3 positive cells via receptor mediated endocytosis and retrograde transport to the ER. We propose that verotoxin is a novel inhibitor for HIV-1 infection. Experiments conducted using VT treated Jurkat-C T cells and PHA/IL-2 activated human PBMCs reveal the anti-HIV-1 property of VT is receptor Gb3 independent since the catalytic A subunit alone is sufficient for inhibition. Possible mechanism of action involves mild inhibition of protein synthesis and cell proliferation. Recent findings in our lab suggest Gb3 is a natural resistance factor for HIV-1 infection, which was further investigated by selecting a Gb3 low subpopulation in THP-1 cells using VT treatment. Selected THP-1 cells were completely resistant to HIV-1 infection, however decreased surface CXCR4 expression may be a cause.

Verotoxin produced by Enterohemorrhagic E. coli is comprised of a catalytic A subunit and a receptor Gb3 binding B subunit pentamer. VT causes protein synthesis inhibition by ribosomal inactivation in Gb3 positive cells via receptor mediated endocytosis and retrograde transport to the ER. We propose that verotoxin is a novel inhibitor for HIV-1 infection. Experiments conducted using VT treated Jurkat-C T cells and PHA/IL-2 activated human PBMCs reveal the anti-HIV-1 property of VT is receptor Gb3 independent since the catalytic A subunit alone is sufficient for inhibition. Possible mechanism of action involves mild inhibition of protein synthesis and cell proliferation. Recent findings in our lab suggest Gb3 is a natural resistance factor for HIV-1 infection, which was further investigated by selecting a Gb3 low subpopulation in THP-1 cells using VT treatment. Selected THP-1 cells were completely resistant to HIV-1 infection, however decreased surface CXCR4 expression may be a cause.

No description available.

Even though anti‐cancer chemotherapy has been continuously improved during the last decades. problems with adverse effects and drug resistance still constitutes a considerable obstacle and sets a demand for new effective treatment options. Tissue homeostasis in multi‐cellular organisms is maintained through intrinsic cell death, apoptosis, which removes unwanted or damaged cells. Disrupted apoptosis is an important factor in tumorgenesis and drug resistance, therefore induction or restoration of apoptotic pathways is also important for the treatment of cancer. Several naturally occurring bacterial toxins have the ability to induce apoptosis and could thus be candidates to complement or improve the therapeutic effect of other anticancer drugs. The bacterial toxins, adenylate cyclase (AC) toxin from Bordetella pertussis, α‐toxin from Staphylococcus aureus and verotoxin‐1 (VT‐1) from Escherichia coli were investigated for their ability to induce apoptosis in different tumor cell lines. Toxin induction of cell death was investigated by cell viability assays, end‐stage apoptosis induction by DNA‐fregmentation (TUNEL) assay. Toxin receptor expression and signal transduction pathways to apoptosis were investigated by flow cytometry, caspase enzyme activity assays and western blot. Immunohistochemistry was used for identification of toxin receptor expression in tumor tissue samples. AC‐toxin was cytotoxic and induced apoptosis in cultured malignant plural mesothelioma (MPM) and small‐cell lung cancer (SCLC) cells. Low‐toxic concentrations of AC‐toxin enhanced cisplatin cytotoxicity and apoptosis in both cell lines. MPM‐cells with acquired cisplatin resistance were more sensitive to α‐toxin than the less resistant parental MPM cell line. A low‐toxic concentration of α‐toxin re‐sensitized resistant MPM cells to cisplatin cytotoxicity by apoptosis induced through the mitochondrial pathway without detectable activation of common up‐stream apoptosis signalling proteins. VT‐1 was highly cytotoxic and induced apoptosis in globotriosylceramide (Gb3) ‐expressing glioma, breast cancer and non‐small‐cell lung cancer (NSCLC) cells but was not cytotoxic to non‐Gb3‐expressing cells. PPMP, an inhibitor of glucosylceramide synthesis which makes exposed cells unable to synthesize Gb3 rendered Gb3‐expressing cells resistant to VT‐1. MPM cells with acquired‐cisplatin resistance expressed Gb3 in contrast to the absent of expression in the less resistant parental cell line. Gb3, could however be up‐regulated by cisplatin in Gb3‐negative MPM‐cells. Presence of a low‐toxic concentration of VT‐1 potentiated cisplatin‐induced cytotoxicity and apoptosis in the cisplatin‐resistance MPM cell line. VT‐1 was a potent inducer of apoptosis, probably via stress‐induced Mitogen‐activated protein kinase (MAPK)‐signaling involving c‐Jun N‐terminal kinase (JNK) and p38, leading to disruption of the mitochondrial membrane integrety, activation of caspase‐9 and ‐3, and ultimately DNA fragmentation and cell death. Gb3 expression was demonstrated in clinical specimens of glioblastoma and breast cancer making these tumor types interesting for further VT‐1 studies. We conclude that bacterial toxins may be used to induce apoptosis in several types of cancer cells. Low concentrations of verotoxin‐1 and α‐toxin may potentially be used to overcome acquired cisplatin‐resistance in cancer patients.

Le glycolipide Gb3/CD77 qui est fortement exprimé en surface des cellules de lymphome de Burkitt (LB) est le récepteur d’une toxine bactérienne la Vérotoxine-1 (VT-1). Notre équipe a montré précédemment que, dans les cellules de LB, la VT-1 induit une cascade apoptotique mettant en jeu les caspases et la mitochondrie. Mon travail a consisté à poursuivre l’analyse des bases moléculaires de ce processus, notamment en m’intéressant au rôle de la protéine pro-apoptotique Bid. Bid est un membre de la famille Bcl-2 qui est clivé par la caspase-8 au cours de l’apoptose et dont la forme tronquée (t-Bid) se relocalise à la mitochondrie. Grâce à l’utilisation de clones cellulaires de LB où Bid a été inhibée puis réexprimée sous forme non clivable et d’un inhibiteur de la caspase-8, nous avons montré que lors de l’apoptose induite par la VT1 : 1) la protéine entière Bid (full-length Bid ou FL-Bid) contrôle l’activation de protéines pro-apoptotiques Bax et de Bak ; 2) t-Bid et FL-Bid sont, toutes les deux, impliquées dans la libération des protéines pro-apoptotiques (cytochrome c et Smac/DIABLO) de l’espace intermembranaire de la mitochondrie vers le cytosol ; 3) FL-Bid contrôle l’homodimérisation de Bax et de Bak qui contribuerait à la libération initiale du cytochrome c et de Smac/DIABLO alors que t-Bid est nécessaire à l’hétérodimérisation de Bax et Bak qui permettrait l’amplification de cette libération. L’ensemble de ces résultats montre donc une coopération fonctionnelle entre Bax et Bak au cours de l’apoptose induite par la VT-1 et surtout met en évidence que l’activation de la voie caspase-8/t-Bid n’est absolument pas requise pour initier la mort cellulaire.Gb3/CD77 est aussi exprimé à la surface de certains lymphocytes B normaux, où il constitue un marqueur de différenciation mais sa fonction endogène reste encore indéterminée. Une deuxième partie de mon travail a consisté à essayer d’identifier le ligand physiologique de Gb3/CD77 pour comprendre son rôle biologique. Grâce à une analyse en spectrométrie de masse, nous avons identifié deux protéines potentiellement partenaires de Gb3/CD77 : la galectine-7 et la protéine S100A11. / The Gb3/CD77 glycolipid, which is strongly expressed in Burkitt's lymphoma (BL) cells, is a receptor for the bacterial toxin Verotoxin-1 (VT-1). Previously, our group has shown that VT-1 induces an apoptotic pathway in BL cells which is dependent on caspases and mitochondria. Here, we provide new insights into this pathway. A pro-apoptotic member in the Bcl-2 family, Bid is cleaved by caspase-8 and its truncated form t-Bid is translocated to mitochondria. Using LB cell clones where Bid was inhibited prior to being reexpressed as a non-cleavable mutated form (BID D59A) and a caspase-8 inhibitor to explore VT-1-induced apoptosis, we showed that 1) the full length Bid (FL-Bid) controls the activation of pro-apoptotic proteins Bax and Bak; 2) Both t-Bid and FL-Bid are involved in the release of pro-apoptotic proteins (cytochrome c and Smac/DIABLO) from the mitochondrial intermembrane space to the cytosol; 3) FL-Bid controls the homo-oligomerization of both Bax and Bak, likely contributing to the initial release of cytochrome c and Smac/DIABLO while t-Bid is needed for their hetero-oligomerization followed by amplification of the release. Together, these results reveal a functional cooperation between Bax and Bak during VT-1-induced apoptosis and, most importantly, that activation of caspase-8 and t-Bid is not required to induce the onset of cell death. Gb3/CD77 is also expressed in a proportion of normal B-lymphocytes where it constitutes a differentiation marker but whose function remains uncharacterized. In an effort to look for physiological ligands, we have used a biochemical approach followed by mass spectrometry analysis. Two proteins have been identified as potentially Gb3/CD77 partners, namely galectin-7 and protein S100A11.