Shiga-like Toxin Type II Research Articles

Down-regulation of cytochrome P450 proteins and its activities by Shiga-like toxin II from Escherichia coli O157:H7

Escherichia coli O157:H7 infection frequently induces clinical complications such as hemolytic uremic syndromes and intestinal dysfunctions. These changes could alter the disposition of drugs, consequently changing their efficacy. However, the possible changes of drug-metabolizing activities by E. coli O157:H7 infection have not been addressed. Thus, we have investigated the effect of Shiga-like toxin type II (SLT-II), derived from E. coli O157:H7, on the hepatic cytochrome P450 (CYP) content and its activity in rats. SLT-II (2 μg per animal, i.v.) time-dependently decreased total CYP content and the contents of CYP2C11 and CYP3A2 in hepatic microsomal preparations up to 24 hr following injection. Consistently, SLT-II time-dependently decreased CYP activity in vivo, as represented by systemic clearance of antipyrine. An inhibitor of inducible nitric oxide synthase, S-methylisothiourea, restored the decreased systemic clearance of antipyrine by SLT-II, suggesting the involvement of the overproduction of nitric oxide by SLT-II. Moreover, dexamethasone restored the decreased systemic clearance of antipyrine by SLT-II. In the hepatic microsomal preparation, dexamethasone restored the SLT-II-induced decrease of CYP3A2 whereas S-methylisothiourea did not affect both CYP subtypes. Taken together, these results suggest that SLT-II might alter hepatic drug-metabolizing function during E. coli O157 infection and that more than one cytokines induced by SLT-II, including nitric oxide, might make a critical contribution to the decrease of CYP content and its activity.

Genes for tRNA(Arg) located in the upstream region of the Shiga toxin II operon in enterohemorrhagic Escherichia coli O157:H7.

We found two genes for tRNA(Arg) in the region upstream of genes for Shiga-like toxin type II (SLT-II) in Escherichia coli O157:H7. The two encoded forms of tRNA(Arg) recognize rare codons in E. coli K12 but these rare codons occur in the toxin genes at high frequency.

Evidence that Escherichia coli isolated from the intestine of healthy pigs hybridize with LT-II, ST-Ib and SLT-II DNA probes.

A DNA probe specific for genes coding for the heat-labile toxin type II (LT-II), heat-stable toxin type Ib (ST-Ib) and Shiga-like toxin type II (SLT-II), were used to examine 118 fecal Escherichia coli strains isolated from healthy pigs. Fifty-six (47.4%) of the isolated were hybrid ized with the LT-II probe. Thirty-nine strains (33%) possessed ST-Ib genes and five of the 118 isolates (4.2%) showed homology with the SLT-II DNA probe. E. coli that possessed unusual toxin genes for pigs were isolated with a high frequency, which indicates the importance of toxigenic clones found in nature. Uncommon virulence factors should be examined in order to improve the efficiency of diagnosis and control procedures.

Virulence of enterohemorrhagic Escherichia coli O91:H21 clinical isolates in an orally infected mouse model.

Escherichia coli K-12 strains producing high levels of Shiga-like toxin type II (SLT-II) but not SLT-I were previously shown to be virulent in an orally infected, streptomycin-treated mouse model. In this investigation, we tested the virulence of several SLT-II-producing enterohemorrhagic E. coli (EHEC) isolates from patients with hemorrhagic colitis or hemolytic uremic syndrome. All of the strains tested were able to colonize the mouse intestine. However, only two strains were consistently virulent for mice: O91:H21 strain B2F1 (Strr), which was previously shown to carry two copies of slt-II-related toxins, and O91:H21 strain H414-36/89 (Strr), which was found in this study to contain three genes from the slt-II group. The oral 50% lethal doses of strains B2F1 (Strr) and H414-36/89 (Strr) when fed to streptomycin-treated mice were less than 10 bacteria. Histological sections from moribund mice fed the O91:H21 strains demonstrated extensive renal tubular necrosis; however, hematological results were not consistent with a diagnosis of hemolytic uremic syndrome. The central role of SLT in the virulence of the O91:H21 EHEC strains was supported by the finding that streptomycin-treated mice preinoculated with monoclonal antibody specific for SLT-II survived oral challenge with either B2F1 (Strr) or H414-36/89 (Strr). The basis for the variation in virulence among the SLT-II-producing EHEC strains tested was not determined. However, a correlation between the capacity of an EHEC strain to grow in small intestinal mucus and lethality in the streptomycin-treated mice was observed.

Comparison of the relative toxicities of Shiga-like toxins type I and type II for mice

In earlier studies using a streptomycin-treated mouse model of infection caused by enterohemorrhagic Escherichia coli (EHEC), animals fed Shiga-like toxin type II (SLT-II)-producing strains developed acute renal cortical necrosis and died, while mice fed Shiga-like toxin type I (SLT-I)-producing clones did not die (E. A. Wadolkowski, L. M. Sung, J. A. Burris, J. E. Samuel, and A. D. O'Brien, Infect. Immun. 58:3959-3965, 1990). To examine the bases for the differences we noted between the two toxins in the murine infection model, we injected mice with purified toxins and carried out histopathological examinations. Despite the genetic and structural similarities between the two toxins, SLT-II had a 50% lethal dose (LD50) which was approximately 400 times lower than that of SLT-I when injected intravenously or intraperitoneally into mice. Histopathologic examination of toxin-injected mice revealed that detectable damage was limited to renal cortical tubule epithelial cells. Passive administration of anti-SLT-II antibodies protected mice from SLT-II-mediated kidney damage and death. Immunofluorescence staining of normal murine kidney sections incubated with purified SLT-I or SLT-II demonstrated that both toxins bound to cortical tubule and medullary duct epithelial cells. Compared with SLT-I, SLT-II was more heat and pH stable, suggesting that SLT-II is a relatively more stable macromolecule. Although both toxins bound to globotriaosylceramide, SLT-I bound with a higher affinity in a solid-phase binding assay. Differences in enzymatic activity between the two toxins were not detected. These data suggest that structural/functional differences between the two toxins, possibly involving holotoxin stability and/or receptor affinity, may contribute to the differential LD50s in mice.

Identification of Shiga-like toxin type II producing Escherichia coli using the polymerase chain reaction and a digoxigenin labelled DNA probe

Epidemiological studies have demonstrated that enterohaemorrhagic strains of Escherichia coli which cause the haemolytic uremic syndrome in humans and the oedema disease in pigs more frequently produce Shiga-like toxin type II (SLT-II) than any other member of the Shiga-like toxin family. A technique has been developed for the identification of SLT-II producing E. coli using the polymerase chain reaction (PCR) and a digoxigenin (DIG)-labelled DNA probe to facilitate the early detection and epidemiological analysis of these pathogens. Whole cell DNA liberated from isolated colonies during the denaturation step of PCR was amplified using a primer pair which is homologous to the slt-II gene sequences. The amplification products were transferred directly to a nitrocellulose membrane or following agarose gel eletrophoresis and DNA denaturation. A chemically labelled DNA probe, prepared using PCR with the incorporation of DIG, was used to identify the PCR products of strains which produced SLT-II or a variant of SLT-II.

Mapping the minimal contiguous gene segment that encodes functionally active Shiga-like toxin II

Shiga-like toxin type II (SLT-II) is one of two antigenically distinct cytotoxins produced by enterohemorrhagic Escherichia coli that are believed to play a central role in the pathogenesis of enterohemorrhagic E. coli-induced disease. SLT-II is a bipartite toxin with an enzymatically active A subunit that inhibits protein synthesis and an oligomeric B subunit that binds to the glycolipid globotriaosylceramide on eukaryotic cells. In this study, functional boundaries of the slt-II operon were mapped. Mutant proteins lacking the last four amino acids from the carboxy terminus of the 70-amino-acid mature SLT-II B polypeptide had no cytotoxic activity. However, when only two amino acids were removed from the carboxy terminus of the B subunit, the cytotoxic activity of the holotoxin was not altered drastically. Furthermore, a 21-amino-acid extension to the carboxy terminus of the SLT-II B polypeptide was tolerated with a minimum reduction in cytotoxic activity of the holotoxin. Deletion of the region coding for amino acids 3 through 18 of the 296-amino-acid mature SLT-II A polypeptide resulted in complete ablation of the cytotoxic activity of the holotoxin as well as abolition of the enzymatic activity of the A subunit. Thus, it appears that both 5'- and 3'-terminal coding sequences are essential for function of the slt-II operon.

Transcription of the Shiga-like toxin type II and Shiga-like toxin type II variant operons of Escherichia coli

Shiga-like toxin type II (SLT-II) and Shiga-like toxin type II variant (SLT-IIv) are cytotoxins produced by certain strains of Escherichia coli. Nucleotide sequence analyses had revealed that the structural genes for the A subunit and B subunit of SLT-II or SLT-IIv are arranged in an operon. Primer extension and S1 nuclease protection analyses identified a promoter for the slt-II operon 118 bases upstream of the slt-IIA gene. The slt-IIv promoter was demonstrated to be identical to the slt-II promoter. The slt-II and slt-IIv promoters differed significantly from the previously characterized Shiga toxin (stx) and Shiga-like toxin type 1 (slt-I) promoters. The transcriptional efficiencies of the stx and slt-II promoters were compared in fusions to the chloramphenicol acetyltransferase gene, and constitutive expression of the slt-II promoter was found to be equivalent to derepressed expression of the stx promoter. In contrast to the stx and slt-I promoters, the slt-II and slt-IIv promoters did not contain sequences for binding of the Fur repressor protein, and SLT-II production was not determined by iron levels in the media in various E. coli strains with wild-type or mutant ferric uptake regulation (fur) alleles. Northern (RNA) blot analysis demonstrated a single mRNA transcript for the slt-II operon, and further analysis of the slt-II operon by primer extension did not reveal an independent promoter for the B subunit gene. A putative rho-independent transcription terminator was identified 274 bases downstream of slt-IIB. These data indicated that the slt-II and slt-IIv operons differ from the stx/slt-I operon in regulation of their transcription by iron. Whether these regulatory differences enable the type I and type II groups of Shiga-like toxins to perform different roles in the pathogenesis of infectious diseases remains to be established.

Cloning and sequencing of a Shiga-like toxin type II variant from Escherichia coli strain responsible for edema disease of swine

A Shiga-like toxin type II variant (SLT-IIv) is produced by strains of Escherichia coli responsible for edema disease of swine and is antigenically related to Shiga-like toxin type II (SLT-II) of enterohemorrhagic E. coli. However, SLT-IIv is only active against Vero cells, whereas SLT-II is active against both Vero and HeLa cells. The structural genes for SLT-IIv were cloned from E. coli S1191, and the nucleotide sequence was determined and compared with those of other members of the Shiga toxin family. The A subunit genes for SLT-IIv and SLT-II were highly homologous (94%), whereas the B subunit genes were less homologous (79%). The SLT-IIv genes were more distantly related (55 to 60% overall homology) to the genes for Shiga toxin of Shigella dysenteriae type 1 and the nearly identical Shiga-like toxin type I (SLT-I) of enterohemorrhagic E. coli. (These toxins are referred to together as Shiga toxin/SLT-I.) The A subunit of SLT-IIv, like those of other members of this toxin family, had regions of homology with the plant lectin ricin. SLT-IIv did not bind to galactose-alpha 1-4-galactose conjugated to bovine serum albumin, which is an analog of the eucaryotic cell receptor for Shiga toxin/SLT-I and SLT-II. These findings support the hypothesis that SLT-IIv binds to a different cellular receptor than do other members of the Shiga toxin family but has a similar mode of intracellular action. The organization of the SLT-IIv operon was similar to that of other members of the Shiga toxin family. Iron did not suppress SLT-IIv or SLT-II production, in contrast with its effect on Shiga toxin/SLT-I. Therefore, the regulation of synthesis of SLT-IIv and SLT-II differs from that of Shiga toxin/SLT-I.

Nucleotide sequence analysis and comparison of the structural genes for Shiga-like toxin I and Shiga-like toxin II encoded by bacteriophages fromEscherichia coli933

The nucleotide sequence of the Shiga-like toxin type II (SLT-II) structural genes cloned from bacteriophage 933W of the enterohemorrhagic Escherichia coli O157:H7 strain 933 was determined. This sequence was compared with the published sequence for the structural genes of the antigenically distinct Shiga-like toxin type I (SLT-I) encoded by bacteriophage 933J. The SLT-I and SLT-II structural genes shared 58% overall nucleotide and 56% amino acid sequence homologies. The A and B subunits of SLT-I and SLT-II were nearly identical in size and had similar secondary structures and hydropathy plots. The regulation proposed for the SLT-II operon is similar to that previously proposed for SLT-I.