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

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.

Hauser E ，Bruederle M ，Reich C ，Bruckbauer A ，Funk J ，Schmidt H ... -  《-》

Transcription of the Subtilase Cytotoxin Gene subAB(1) in Shiga Toxin-Producing Escherichia coli Is Dependent on hfq and hns.

Certain foodborne Shiga toxin-producing Escherichia coli (STEC) strains carry genes encoding the subtilase cytotoxin (SubAB). Although the mode of action of SubAB is under intensive investigation, information about the regulation of subAB gene expression is currently not available. In this study, we investigated the regulation of the chromosomal subAB1 gene in laboratory E. coli strain DH5α and STEC O113:H21 strain TS18/08 using a luciferase reporter gene assay. Special emphasis was given to the role of the global regulatory protein genes hfq and hns in subAB1 promoter activity. Subsequently, quantitative real-time PCR was performed to analyze the expression of Shiga toxin 2a (Stx2a), SubAB1, and cytolethal distending toxin V (Cdt-V) genes in STEC strain TS18/08 and its isogenic hfq and hns deletion mutants. The deletion of hfq led to a significant increase of up to 2-fold in subAB1 expression, especially in the late growth phase, in both strains. However, deletion of hns showed different effects on the promoter activity during the early and late exponential growth phases in both strains. Furthermore, upregulation of stx2a and cdt-V was demonstrated in hfq and hns deletion mutants in TS18/08. These data showed that the expression of subAB1, stx2a, and cdt-V is integrated in the regulatory network of global regulators Hfq and H-NS in Escherichia coliIMPORTANCE Shiga toxin-producing Escherichia coli (STEC) strains are responsible for outbreaks of foodborne diseases, such as hemorrhagic colitis and the hemolytic uremic syndrome. The pathogenicity of those strains can be attributed to, among other factors, the production of toxins. Recently, the subtilase cytotoxin was detected in locus of enterocyte effacement (LEE)-negative STEC, and it was confirmed that it contributes to the cytotoxicity of those STEC strains. Although the mode of action of SubAB1 is under intensive investigation, the regulation of gene expression is currently not known. The global regulatory proteins H-NS and Hfq have impact on many cellular processes and have been described to regulate virulence factors as well. Here, we investigate the role of hns and hfq in expression of subAB1 as well as stx2a and cdt-V in an E. coli laboratory strain as well as in wild-type STEC strain TS18/08.

Heinisch L ，Zoric K ，Krause M ，Schmidt H ... -  《-》

Virulence characteristics of Shiga toxin-producing Escherichia coli from raw meats and clinical samples.

Shiga toxin producing Escherichia coli (STEC) are dangerous foodborne pathogens. Foods are considered as important sources for STEC infection in human. In this study, STEC contamination of raw meats was investigated and the virulence factors of 120 clinical STEC strains characterized. STEC was detected in 4.4% of tested samples. Among 25 STEC strains from meats, five strains (20%) were positive for the eae gene, which encodes intimin, an important binding protein of pathogenic STEC. The remaining strains (80%) were eae-negative. However, 28% of them possessed the saa gene, which encodes STEC agglutinating adhesin. The ehxA gene encoding for enterohemolysin was found in 75% of the meat strains and the subAB gene, the product is of which subtilase cytotoxin, was found in 32% of these strains. The stx2a gene, a subtype of Shiga toxin gene (stx), was the most prevalent subtype among the identified meat STEC bacteria. None of the meat STEC was O157:H7 serotype. Nevertheless, 92% of them produced Shiga toxin (Stx). Among 120 clinical STEC strains, 30% and 70% strains harbored single and multiple stx subtypes, respectively. Most clinical STEC bacteria possessed eae (90.8%) and ehxA (96.7%) genes and 92.5% of them showed Stx productivity. Our study shows that some raw meat samples contain non-O157 STEC bacteria and some strains have virulence factors similar to those of clinical strains.

Hoang Minh S ，Kimura E ，Hoang Minh D ，Honjoh K ，Miyamoto T ... -  《-》

Identification of the newly identified subtilase cytotoxin-encoding gene (subAB2-2) among clinical Shiga toxin-producing Escherichia coli isolates.

Son HM ，Duc HM ，Honjoh K ，Miyamoto T ... -  《-》

The new allelic variant of the subtilase cytotoxin (subAB2) is common among Shiga toxin-producing Escherichia coli strains from large game animals and their meat and meat products.

Subtilase cytotoxin (SubAB) is an AB5 toxin produced by Shiga toxin (Stx)-producing Escherichia coli (STEC) strains usually lacking the eae gene product intimin. Two allelic variants of SubAB encoding genes have been described: subAB1, located on a plasmid, and subAB2, located on a pathogenicity island (PAI) together with tia gene. While subAB1 has been reported to be more frequent among bovine strains, subAB2 has been mainly associated with strains from small ruminants. We investigated the presence of the two variants of subAB among 59 eae-negative STEC from large game animals (deer and wild boar) and their meat and meat products in order to assess the role of other species in the epidemiology of subAB-positive, eae-negative STEC. For this approach, the strains were PCR-screened for the presence of subAB, including the specific detection of both allelic variants, for the presence of saa, tia and sab, and for stx subtyping. Overall, subAB genes were detected in 71.2% of the strains: 84.1% of the strains from deer and 33.3% of the strains from wild boar. Most of them (97.6%) possessed subAB2 and most of these subAB2-positive strains (92.7%) were also positive for tia and negative for saa, suggesting the presence of the subAB2-harbouring PAI. Subtype stx2b was present in most of the strains (67.8%) and a statistically significant association could be established between subAB2 and stx2b. Our results suggest that large game animals, mainly deer, may represent an important animal reservoir of subAB2-positive, eae-negative STEC, and also highlight the risk of human infection posed by the consumption of large game meat and meat products.

Sánchez S ，Díaz-Sánchez S ，Martínez R ，Llorente MT ，Herrera-León S ，Vidal D ... -  《-》