Subtilase contributes to the cytotoxicity of a Shiga toxin-producing Escherichia coli strain encoding three different toxins

• Food-borne STEC strain expresses three toxin genes stx2, subAB1 and cdt-V.
• Differences of transcription with maximum for subAB1 and cdt-V after 3 h and stx2 after 6 h
• Relation of 1:26:100 for relative transcription levels of stx2:subAB1:cdt-V over the time curve
• Comparable cytotoxicity of wildtype O113:H21 strain TS18/08 and O157:H7 outbreak strain EDL933
• SubAB1 contributes to Vero cell cytotoxicity of STEC strain TS18/08.

ABSTRACTFood-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 3 h while stx2a transcription was highest after 6 h 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.