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 ... -  《-》

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 ... -  《-》

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 ... -  《-》

A new pathogenicity island carrying an allelic variant of the Subtilase cytotoxin is common among Shiga toxin producing Escherichia coli of human and ovine origin.

Subtilase (SubAB) is a cytotoxin elaborated by some Shiga Toxin (Stx)-producing Escherichia coli (STEC) strains usually lacking the locus of enterocyte effacement (LEE). Two variants of SubAB coding genes have been described: subAB(1) , located on the plasmid of the STEC O113 98NK2 strain, and subAB(2) , located on a pathogenicity island (PAI) together with the tia gene, encoding an invasion determinant described in enterotoxigenic E. coli. In the present study, we determined the entire nucleotide sequence of the PAI containing the subAB(2) operon, termed Subtilase-Encoding PAI (SE-PAI), and identified its integration site in the pheV tRNA locus. In addition, a PCR strategy for discriminating the two subAB allelic variants was developed and used to investigate their presence in E. coli strains belonging to different pathotypes and in a large collection of LEE-negative STEC of human and ovine origin. The results confirmed that subAB genes are carried predominantly by STEC and showed their presence in 72% and 86% of the LEE-negative strains from human cases of diarrhoea and from healthy sheep respectively. Most of the subAB-positive strains (98%) identified possessed the subAB(2) allelic variant and were also positive for tia, suggesting the presence of SE-PAI. Altogether, our observations indicate that subAB(2) is the prevalent SubAB-coding operon in LEE-negative STEC circulating in European countries, and that sheep may represent an important reservoir for human infections with these strains. Further studies are needed to assess the role of tia and/or other genes carried by SE-PAI in the colonization of the host intestinal mucosa.

Michelacci V ，Tozzoli R ，Caprioli A ，Martínez R ，Scheutz F ，Grande L ，Sánchez S ，Morabito S ... -  《-》

Prevalence of subtilase cytotoxin-encoding subAB variants among Shiga toxin-producing Escherichia coli strains isolated from wild ruminants and sheep differs from that of cattle and pigs and is predominated by the new allelic variant subAB2-2.

Subtilase cytotoxin (SubAB) is an AB5 toxin produced by Shiga toxin (Stx)-producing Escherichia coli (STEC) strains usually lacking the eae gene product intimin. Three allelic variants of SubAB encoding genes have been described: subAB1, located on a plasmid, subAB2-1, located on the pathogenicity island SE-PAI and subAB2-2 located in an outer membrane efflux protein (OEP) region. SubAB is becoming increasingly recognized as a toxin potentially involved in human pathogenesis. Ruminants and cattle have been identified as reservoirs of subAB-positive STEC. The presence of the three subAB allelic variants was investigated by PCR for 152 STEC strains originating from chamois, ibex, red deer, roe deer, cattle, sheep and pigs. Overall, subAB genes were detected in 45.5% of the strains. Prevalence was highest for STEC originating from ibex (100%), chamois (92%) and sheep (65%). None of the STEC of bovine or of porcine origin tested positive for subAB. None of the strains tested positive for subAB1. The allelic variant subAB2-2 was detected the most commonly, with 51.4% possessing subAb2-1 together with subAB2-2. STEC of ovine origin, serotypes O91:H- and O128:H2, the saa gene, which encodes for the autoagglutinating adhesin and stx2b were significantly associated with subAB-positive STEC. Our results suggest that subAB2-1 and subAB2-2 is widespread among STEC from wild ruminants and sheep and may be important as virulence markers in STEC pathogenic to humans.

Nüesch-Inderbinen MT ，Funk J ，Cernela N ，Tasara T ，Klumpp J ，Schmidt H ，Stephan R ... -  《-》