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

Abstract

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 coli. IMPORTANCE 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.

Genetics, Toxicity, and Distribution of Enterohemorrhagic Escherichia coli Hemolysin

“…The authors assumed that the LEE-negative STEC strains used in the study might probably be the reason for the discrepancy to the study of Li et al, who examined the influence of H-NS in a LEE-positive EHEC O157:H7 strain [31]. This assumption is further supported by the results obtained in our group, in which a deletion of hns in the eae -negative STEC O113:H21 strain TS18/08 (first described in [32]) was constructed for the purpose of another research project concerning subtilase cytotoxin expression [33]. The hns deletion mutant showed a hyper-hemolytic phenotype on blood agar plates, whereas the complementation of the mutant with a recombinant hns gene on a medium-copy plasmid restored the characteristic wild-type turbid lysis zones.…”

Cytotoxic Effects of Recombinant StxA2-His in the Absence of Its Corresponding B-Subunit

“…The ligation was conducted at 22 °C for one hour. Subsequently, ligation batches were transformed to chemically competent E. coli DH5α as described previously [ 25 ].…”

“…To verify constructed plasmids, they were sequenced by Sanger sequencing using primers pET22-seq-for and pET22-seq-rev (see Table 2 ) as described earlier [ 51 ]. After confirmation of the correct insertion of the stxA2a and stxB2a genes in the pET22b(+) expression vector, plasmids were transformed to chemically competent E. coli C41 (DE3) or competent E. coli C43 (DE3) cells, respectively, as described earlier [ 25 ]. The expression plasmids for stxA2a and stxB2a were designated pKR09 and pKR10, respectively.…”

“…Quantitative real-time PCR (qRT-PCR) experiments showed that both genes were transcribed similarly during laboratory cultivation of this strain, having their transcription optimum after three hours of cultivation [ 24 ]. Furthermore, additional qRT-PCR data indicated the role of the global regulators Hfq and H-NS in the regulation of both stx 2a and subAB 1 [ 25 ].…”

Host response to the subtilase cytotoxin produced by locus of enterocyte effacement‐negative Shiga‐toxigenic Escherichia coli

“…Although the regulation of subAB gene expression is still unknown, the latest study showed that transcription of subAB 1 is regulated by hfq and hns in the STEC O113:H21 strain. 17 Generally, Shiga toxin (Stx) 2 expression in LEE-positive STEC O157:H7 is enhanced by treatment with H 2 O 2 or antibiotics. 18 However, our recent findings suggest that both Stx2 and SubAB expressions in LEE-negative STEC O113:H21 are not promoted by antibiotics, mitomycin C, or H 2 O 2 (unpublished data).…”