EHEC-Hämolysin: Molekulare und zelluläre Mechanismen der Wirtsschädigung
Zusammenfassung der Projektergebnisse
The hemolysin from enterohemorrhagic Escherichia coli (EHEC-Hly) is a pore-forming toxin of the RTX (repeats-in-toxin) family. It is present in most EHEC strains, which cause bloody diarrhea and severe extraintestinal complications such as renal failure and neurological disorders. We found that EHEC-Hly is associated with outer membrane vesicles (OMVs). This assembly stabilizes the toxin and substantially prolongs its hemolytic activity compared to its free form. A detailed analysis of EHEC OMVs revealed that they carry, besides EHEC-Hly, the major virulence factor of EHEC strains, i.e. Stx2a, as well as the CdtV holotoxin and flagellin. Immunogold-coupled anti-EHEC-Hly antibody exhibited an overall distribution of the toxin in the vesicle population, predominantly on the surface of the vesicles. Employing human brain microvascular endothelial cells (HBMECs) and human colon epithelial cells (Caco-2), the kinetics of fluorochrome-labeled OMVs uptake indicated the binding and internalization of EHEC-Hly-containing OMVs in a time-dependent manner. Furthermore, the endocytosis of EHEC-Hly-containing OMVs was found to be a dynamin-dependent and largely clathrin-mediated process. Confocal laser scanning microscopy studies of EHEC-Hly association with OMVs and EHEC-Hly trafficking into the endo-lysosomal compartments and mitochondria demonstrated the initial association of EHEC-Hly with OMVs followed by a gradual dissociation from OMVs, escape from the lysosomes and translocation to the mitochondria, which resulted in caspase-9-mediated apoptosis. Of note, lysosomal acidification is necessary for the separation and release of EHEC-Hly from OMVs within lysosomes. EHEC-Hly-harboring OMVs were found to induce production of proinflammatory cytokines in target cells. Interaction studies of OMVs revealed the presence of an OmpA- binding glycoprotein receptor on endothelial and epithelial cells. Knock-down experiments by means of RNA interference (RNAi) using small interfering RNAs (siRNAs) allowed for downregulation of the OmpA ligand. Deletions of the genes coding for additional OMV- associated outer membrane proteins including OmpF, OmpC, OmpA, OmpX and OmpW indicated that there is a direct correlation between the OmpA expression and the interaction of the EHEC-Hly with the OMVs. These data suggest that the OMV-OmpA is involved in EHEC-Hly association with and integration into OMVs as well as in the OMV uptake by the host cells.
Projektbezogene Publikationen (Auswahl)
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(2011) Enterohaemorrhagic Escherichia coli haemolysin is cleaved and inactivated by serine protease EspPα. Environ Microbiol 13:1327-1341
Brockmeyer J, Aldick T, Soltwisch J, Zhang W, Tarr PI, Weiss A, Dreisewerd K, Müthing J, Bielaszewska M, Karch H
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(2013) Enterohemorrhagic Escherichia coli hemolysin employs outer membrane vesicles to target mitochondria and cause endothelial and epithelial apoptosis. PLoS Pathog 9:e1003797
Bielaszewska M, Rüter C, Kunsmann L, Greune L, Bauwens A, Zhang W, Kuczius T, Kim KS, Mellmann A, Schmidt MA, Karch H
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(2013) Facing glycosphingolipid-Shiga toxin interaction: dire straits for endothelial cells of the human vasculature. Cell Mol Life Sci 70:425-457
Bauwens A, Betz J, Meisen I, Kemper B, Karch H, Müthing J
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(2014) Hemolysin of enterohemorrhagic Escherichia coli: structure, transport, biological activity and putative role in virulence. Int J Med Microbiol 304:521-529
Bielaszewska M, Aldick T, Bauwens A, Karch H
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(2017) Host cell interactions of outer membrane vesicle-associated virulence factors of enterohemorrhagic Escherichia coli O157: Intracellular delivery, trafficking and mechanisms of cell injury. PLoS Pathog 13:e1006159
Bielaszewska M, Rüter C, Bauwens A, Greune L, Jarosch KA, Steil D, Zhang W, He X, Lloubes R, Fruth A, Kim KS, Schmidt MA, Dobrindt U, Mellmann A, Karch H
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(2018) 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. Int J Med Microbiol 308:882-889
Bielaszewska M, Marejková M, Bauwens A, Kunsmann-Prokscha L, Mellmann, Karch H
