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Appl Environ Microbiol. 2005 Jan; 71(1): 558–561.
PMCID: PMC544208

Transcriptional Analysis of Genes Encoding Shiga Toxin 2 and Its Variants in Escherichia coli

Abstract

Six of 37 non-O157 Escherichia coli strains possessing Shiga toxin (Stx) 2 gene variant stx2d or stx2e secreted no detectable Stx. These isolates produced significantly less stx mRNA than Stx2d, Stx2e, Stx2c, or Stx2 secretors did. Standard screening procedures miss a significant subset of E. coli harboring stx2 variants.

Shiga toxin (Stx)-producing Escherichia coli strains (STEC) cause diarrhea, hemolytic uremic syndrome (HUS), or asymptomatic infections. Stx1 and Stx2 are two major Stx types, and each has variants (5, 8, 9, 12, 16, 17, 22). Whereas STEC containing stx2 or stx2c cause HUS (3), strains possessing stx2d (12) or stx2e are less virulent (3). Also, STEC from HUS patients produce significantly higher levels of Stx1 or Stx2 than bovine STEC do (14). In this study, we applied Vero cell cytotoxicity assays (6), enzyme immunoassays (EIA), latex agglutination (LA), and colony immunoblot assays to characterize Stx production in non-O157 E. coli clinical isolates that possess an stx2 variant as their sole stx gene.

Broth cultures of 37 non-O157 E. coli strains were investigated (Table (Table1)1) using Vero cell cytotoxicity assays, EIA (Ridascreen Verotoxin; R-Biopharm GmbH, Darmstadt, Germany), verotoxin-producing E. coli reverse passive LA (VTEC-RPLA; Denka Seiken Co., Tokyo, Japan), and colony immunoblot assays (Shigatoxin [Verotoxin] Colony Immunoblot; Sifin GmbH, Berlin, Germany) (3, 6, 19). Selected polymyxin B (5,000 U/ml; Sigma, Taufkirchen, Germany) bacterial extracts were also tested for Stx. Complete stx2d and stx2e genes were amplified with primer pairs SD-a (5′-TTCTAAGCAATCGGTCACT-3′)-SD-b (5′-GTAACTACATTGCTGCACAC-3′) and SE-a (5′-GAGCAGACGACACGATAACA-3′)-SE-b (5′-AACAGCATCCACAACACTA-3′), respectively, purified (PCR purification kit; QIAGEN, Hilden, Germany), sequenced, analyzed (DNASIS program; Hitachi Software), and compared with sequences from the National Center for Biotechnology Information database. A one-step quantitative reverse transcription (RT)-PCR was performed with the LightCycler system (Roche, Mannheim, Germany) and the QuantiTect SYBR green RT-PCR kit (QIAGEN) to compare stx2c, stx2d, and stx2e mRNA levels; two stx2+ E. coli strains served as positive controls. Total RNA was isolated (RNeasy Mini kit; QIAGEN) without mitomycin C and after mitomycin C (0.5 μg/ml; Sigma) induction. PCR was performed in microcapillary tubes in 10-μl volumes containing 100 ng of total RNA, 2× QuantiTect SYBR green RT-PCR master mix (5 μl), QuantiTect RT mix (0.1 μl), MgCl2 (4 mM), and a 0.5 μM concentration of each primer. The primers RT-stx2F (5′-CGACCCCTCTTGAACATA-3′) and RT-stx2R (5′-TAGACATCAAGCCCTCGTAT-3′), GK5 and GK6 (15), stx2dB-1 (5′-AAGAAGATATTTGTAGCGG-3′) and stx2dB-2 (5′-CGTCATTCCTGTTAACTGTGCG-3′), FK1 and FK2 (2), and icdA-F and icdA-R (21) were used to amplify stxA2, stxA2c, stxA2d, and stxA2e; stxB2 and stxB2c; stxB2d; stxB2e; and the housekeeping gene icdA, respectively. RT-PCR included reverse transcription (50°C, 30 min) and preliminary denaturation (95°C, 15 min) steps and 40 cycles of denaturation (94°C, 10 s), annealing (50°C, 20 s), and extension (72°C, 20 s). Melting curve analysis was performed with continuous fluorescence reading from 60 to 95°C. For each mRNA assayed, relative quantification was performed using an external standard (purified icd and the respective stx2 variant PCR products). Data were analyzed by the fit-point method with LightCycler version 3.5 software. stx mRNAs were normalized to icdA mRNA. Control reactions were performed without reverse transcriptase to confirm that the target detected was RNA.

TABLE 1.
Comparison of Stx detection in supernatants of 37 E. coli strainsa using the Vero cell cytotoxicity assay, enzyme immunoassay, and latex agglutination assay

Detection of Stx.

Five of 16 stx2d- and 1 of 12 stx2e-containing isolates, respectively, did not release Stx that was detectable by any assay (Tables (Tables11 and and2).2). Polymyxin extracted minute amounts of Stx from the periplasmic space of each of the five stx2d-harboring strains but not from the stx2e-containing strain (Table (Table22).

TABLE 2.
Detection of Stx in polymyxin B extracts of the E. coli strains which secreted no detectable Stx

Sequence analysis of stx genes.

The stxB2d and stxB2e genes from the six Stx nonsecretors were 100% identical to stxB2d and stxB2e of prototype strains EH250 and 2771/97 (GenBank accession numbers AF043627 and AJ249351, respectively), which secrete the corresponding Stx (Table (Table2).2). stxA2d and stxA2e from these strains were 97.1 to 99.9% homologous to the respective subunits of strains EH250 and 2771/97. The deduced amino acid sequences of StxA subunits of these strains are compared in Fig. Fig.1.1. There was no frame shift, deletion, insertional inactivation, or truncation of stx in any nonproducing strain.

FIG. 1.
Comparison of deduced amino acid (aa) sequences of StxA subunits from stx2d- or stx2e-harboring strains which secrete no detectable Stx with those from Stx2d-producing strain EH250 (GenBank accession number ...

Quantitative RT-PCR analysis and mitomycin C effect.

The five Stx2d nonsecretors (Fig. (Fig.2,2, lanes 1, 3, 5, 7, and 9) and one Stx2e nonsecretor (lanes 15) had significantly less cognate stx mRNA (both subunits) than the Stx2d (lanes 11 and 13) and Stx2e (lanes 17) secretors. Mitomycin C did not significantly augment stxA (Fig. (Fig.2A,2A, lanes 2, 4, 6, 8, 10, and 16) or stxB (Fig. (Fig.2B,2B, lanes 2, 4, 6, 8, 10, and 16) transcription. In contrast, in Stx2d or Stx2e secretors, mitomycin C increased both stxA and stxB transcription approximately 100-fold (Fig. (Fig.2,2, lanes 12, 14, and 18).

FIG. 2.
Relative stxA2d, stxA2e, stxA2c, and stxA2 (A) and stxB2d, stxB2e, stxB2c, and stxB2 (B) mRNA expression levels in Stx nonsecretors and Stx secretors analyzed by quantitative RT-PCR. In lanes 1 to 26, the following strains are depicted (the stx2 allele, ...

Comparison of stx2 and stx2 variant transcription in Stx secretors.

stxA (Fig. (Fig.2A)2A) and stxB (Fig. (Fig.2B)2B) mRNA levels were higher in strains containing stx2e (Fig. (Fig.2,2, lanes 17), stx2c (lanes 19 and 21), or stx2 (lanes 23 and 25) than in those containing stx2d (lanes 11 and 13), but the differences reached statistical significance only for stxA. No significant differences in stx2d mRNA levels were detected between Stx2d secretors isolated from diarrhea (strain EH250 [Fig. [Fig.2,2, lanes 13]) and asymptomatic infection (strain 4797/97 [lanes 11]). Isolates from HUS (strains 4911/99 and 1309/99 [lanes 19 and 23]) and from diarrhea (strains 3032/03 and 3733/97 [lanes 21 and 25]) had similar stx2 or stx2c mRNA levels. However, mitomycin C significantly increased stx2c (Fig. (Fig.2,2, lanes 20 and 22) and stx2 (lanes 24 and 26) transcription in all secretors of the respective Stx.

These data suggest that Stx detection can be compromised by Stx nonsecretors and that molecular methods detecting stx might offer advantages (13, 18). The reason for Stx nonexpression is not the insertion sequence IS1203v within stxB (7), because stx genes in the Stx nonproducers are structurally intact. Stx production appears to be controlled transcriptionally, and its failure is not remedied by mitomycin C induction of the lytic cycle (11, 20), suggesting that stx genes of these strains are not phage borne or are located on defective phages. In contrast, mitomycin augments stx transcription in strain 2771/97 and the Stx2d secretors EH250 and 4797/97, in agreement with our previous report that stx2e in strain 2771/97 is located on an intact phage (10), which suggests that also stx2d genes in both Stx2d secretors are phage encoded. Notably, we found no association between the stx transcription level and clinical outcome of the infection within producers of Stx2c, Stx2d, or classical Stx2, in concordance with a recent North American study of E. coli O157:H7 infections (1). Interestingly, although Stxs are A1B5 toxins, the mRNA levels were similar for the A- and B-subunit genes (Fig. (Fig.2),2), suggesting that the stoichiometry of the subunits is not transcriptionally regulated. In summary, we have demonstrated discordance between stx genotype and Stx expression, for stx2d or stx2e. Expression appears to be controlled at the level of transcription. Phenotypic detection of Stx will miss STEC that poorly secrete Stx.

Nucleotide sequence accession numbers.

Nucleotide sequences for stx2d from E. coli strains 24196/97, 6451/98, and 5293/98 and stx2e from E. coli strain 26725/97 have been entered into the EMBL database under accession numbers AJ567995 to AJ567998, respectively.

Acknowledgments

This study was supported by a grant from the Bundesministerium für Bildung und Forschung (BMBF) Project Network of Competence Pathogenomics Alliance (“Functional genomic research on enterohaemorrhagic Escherichia coli,” number 119523).

We thank H. Tschäpe (Robert-Koch Institute, Wernigerode, Germany) for serotyping and P. I. Tarr (Washington University School of Medicine, St. Louis, Mo.) for critical reading of the manuscript and stimulating discussions. The technical assistance of O. Böhler is greatly appreciated.

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