title: Cytotoxic and apoptotic effects of recombinant subtilase cytotoxin variants of shiga toxin-producing Escherichia coli.
abstract: In this study, the cytotoxicity of the recently described subtilase variant SubAB2-2 of Shiga toxin-producing Escherichia coli was determined and compared to the plasmid-encoded SubAB1 and the chromosome-encoded SubAB2-1 variant. The genes for the respective enzymatic active (A) subunits and binding (B) subunits of the subtilase toxins were amplified and cloned. The recombinant toxin subunits were expressed and purified. Their cytotoxicity on Vero cells was measured for the single A and B subunits, as well as for mixtures of both, to analyze whether hybrids with toxic activity can be identified. The results demonstrated that all three SubAB variants are toxic for Vero cells. However, the values for the 50% cytotoxic dose (CD50) differ for the individual variants. Highest cytotoxicity was shown for SubAB1. Moreover, hybrids of subunits from different subtilase toxins can be obtained which cause substantial cytotoxicity to Vero cells after mixing the A and B subunits prior to application to the cells, which is characteristic for binary toxins. Furthermore, higher concentrations of the enzymatic subunit SubA1 exhibited cytotoxic effects in the absence of the respective B1 subunit. A more detailed investigation in the human HeLa cell line revealed that SubA1 alone induced apoptosis, while the B1 subunit alone did not induce cell death.
authors.affiliation: Institute of Food Science and Biotechnology, Department of Food Microbiology and Hygiene, University of Hohenheim, Stuttgart, Germany.
authors.forename: J
authors.initials: J
authors.lastname: Funk
authors.affiliation: Institute of Pharmacology and Toxicology, University of Ulm Medical Center, Ulm, Germany.
authors.forename: N
authors.initials: N
authors.lastname: Biber
authors.affiliation: Institute of Food Science and Biotechnology, Department of Food Microbiology and Hygiene, University of Hohenheim, Stuttgart, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Schneider
authors.affiliation: Institute of Food Science and Biotechnology, Department of Food Microbiology and Hygiene, University of Hohenheim, Stuttgart, Germany.
authors.forename: E
authors.initials: E
authors.lastname: Hauser
authors.affiliation: Department of Pediatrics and Adolescent Medicine, University of Ulm Medical Center, Ulm, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Enzenmüller
authors.affiliation: Institute of Pharmacology and Toxicology, University of Ulm Medical Center, Ulm, Germany.
authors.forename: C
authors.initials: C
authors.lastname: Förtsch
authors.affiliation: Institute of Pharmacology and Toxicology, University of Ulm Medical Center, Ulm, Germany holger.barth@uni-ulm.de herbert.schmidt@uni-hohenheim.de.
authors.forename: H
authors.initials: H
authors.lastname: Barth
authors.affiliation: Institute of Food Science and Biotechnology, Department of Food Microbiology and Hygiene, University of Hohenheim, Stuttgart, Germany holger.barth@uni-ulm.de herbert.schmidt@uni-hohenheim.de.
authors.forename: H
authors.initials: H
authors.lastname: Schmidt
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D014644:Genetic Variation; D006367:HeLa Cells; D006801:Humans; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D054323:Shiga-Toxigenic Escherichia coli / Q000201:enzymology* / Q000235:genetics; D013381:Subtilisins / Q000235:genetics / Q000378:metabolism*; D014709:Vero Cells
chemical_list: D029968:Escherichia coli Proteins; D021122:Protein Subunits; D011994:Recombinant Proteins; D013381:Subtilisins; C527234:subtilase cytotoxin, E coli
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