title: Subtilase contributes to the cytotoxicity of a Shiga toxin-producing Escherichia coli strain encoding three different toxins.
abstract: Food-borne Shiga toxin-producing Escherichia coli (STEC) O113:H21 strain TS18/08, that has previously been isolated from mixed minced meat, harbors the Shiga toxin (Stx) encoding allele stx2a, the plasmid-located subtilase cytotoxin encoding allele subAB1 and the cytolethal distending toxin type V encoding gene cdt-V. In the current study, it could be shown that each of these toxin genes was transcribed with different transcription levels at different time points by RT real time PCR under laboratory batch conditions in LB-broth. The transcription maximum for cdt-V and subAB1 was observed after 3h while stx2a transcription was highest after 6h of incubation. During this time the mean relationship of the amount of stx2a:subAB1:cdt-V transcripts was 1:26:100. Furthermore, isogenic stx2a and cdt-V chromosomal deletion mutants were constructed to measure the contribution of SubAB1 to the overall cytotoxicity of this strain. In this context, a further copy of stx2 was detected in this strain and was also deleted. Comparing the cytotoxicity of supernatants of the resulting mutant strains TS18/08-3 (Δstx2-1Δstx2-2Δcdt-V) and TS18/08-4 (Δstx2-1Δstx2-2Δcdt-VΔsubAB1) on Vero cells demonstrated a contribution of SubAB1 to the overall cytotoxic effect while the 4-fold isogenic deletion mutant did not show any cytotoxic effect and that was comparable to the non-toxic laboratory E. coli strain C600. The cytotoxic effect could be restored by complementation with the recombinant low copy plasmid pWSK29 harboring subAB1 under the control of its own promoter. In addition, the cytotoxicity of wild type strain TS18/08 to Vero cells was in the same range as the EHEC O157:H7 strain EDL933. Therefore, food-borne STEC O113:H21 strain TS18/08 can be considered as a putative human pathogen.
authors.affiliation: Institute of Food Science and Biotechnology, Department of Food Microbiology and Hygiene, Garbenstrasse 28, University of Hohenheim, 70599 Stuttgart, Germany.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Hauser
authors.affiliation: Institute of Food Science and Biotechnology, Department of Food Microbiology and Hygiene, Garbenstrasse 28, University of Hohenheim, 70599 Stuttgart, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Bruederle
authors.affiliation: Institute of Food Science and Biotechnology, Department of Food Microbiology and Hygiene, Garbenstrasse 28, University of Hohenheim, 70599 Stuttgart, Germany.
authors.forename: Carolin
authors.initials: C
authors.lastname: Reich
authors.affiliation: Institute of Food Science and Biotechnology, Department of Food Microbiology and Hygiene, Garbenstrasse 28, University of Hohenheim, 70599 Stuttgart, Germany.
authors.forename: Annette
authors.initials: A
authors.lastname: Bruckbauer
authors.affiliation: Institute of Food Science and Biotechnology, Department of Food Microbiology and Hygiene, Garbenstrasse 28, University of Hohenheim, 70599 Stuttgart, Germany.
authors.forename: Joschua
authors.initials: J
authors.lastname: Funk
authors.affiliation: Institute of Food Science and Biotechnology, Department of Food Microbiology and Hygiene, Garbenstrasse 28, University of Hohenheim, 70599 Stuttgart, Germany. Electronic address: herbert.schmidt@uni-hohenheim.de.
authors.forename: Herbert
authors.initials: H
authors.lastname: Schmidt
mesh_terms: D000818:Animals; D001427:Bacterial Toxins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002522:Chlorocebus aethiops; D002872:Chromosome Deletion; D004927:Escherichia coli Infections / Q000382:microbiology*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008460:Meat / Q000382:microbiology; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D022621:Shiga Toxin / Q000235:genetics*; D054323:Shiga-Toxigenic Escherichia coli / Q000235:genetics / Q000302:isolation & purification / Q000472:pathogenicity*; D013381:Subtilisins / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic / Q000235:genetics; D014709:Vero Cells
chemical_list: D001427:Bacterial Toxins; D029968:Escherichia coli Proteins; C067044:cytolethal distending toxin; D022621:Shiga Toxin; D013381:Subtilisins; C527234:subtilase cytotoxin, E coli
keywords: Cytotoxicity; Subtilase cytotoxin; Toxin deletion mutants; Toxin transcription; Vero cell assay; qRT-PCR