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

Abstract

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-ll is active against both Vero and HeLa cells.The structural genes for SLT-llv 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-al-4-galactose coijugated to bovine serum albumin, which is an analog of the eucaryotic cell receptor for Shiga toxin/SLT-I and SLT-ll. 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-llv 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 toxinlSLT-I. Therefore, the regulation of synthesis of SLT-IIv and SLT-II differs from that of Shiga toxhi/SLT-I.Some Escherichia coli strains produce cytotoxins that are related to the Shiga toxin produced by Shigella dysenteriae type 1. These Shiga-like toxins (SLTs), which are also called verotoxins (19) Letter, Lancet i:702, 1983). Two antigenically distinct types of SLTs, SLT-I and SLT-II, that cause hemorrhagic colitis and/ or the hemolytic-uremic syndrome have been isolated from E. coli (39; S. M. Scotland, H. R. Smith, G. A. Willshaw, and B. Rowe, Letter, Lancet ii:216, 1983). SLT-I but not SLT-II is neutralized by polyclonal antisera to Shiga toxin (39). Shiga toxin, SLT-I, SLT-II, and the plant toxin ricin have the same mechanism of action (10, 11). These toxins are N-glycosidases that cleave a specific adenine residue in the 28S subunit of eucaryotic rRNA which in turn causes protein synthesis to cease. The A subunit of each of these toxins is responsible for the N-glycosidase activity. Hovde et al. recently demonstrated that amino acid 167 (glutamic acid) in the A subunit of SLT-I is critical for the enzymatic activity of the toxin (13). The A subunit of Shiga toxin, SLT-I, and presumably SLT-II is noncovalently linked to multiple copies of the B subunit (9). The eucaryotic receptor to which the B subunits of Shiga toxin and SLT-I bind is a galactose-al-4-galactose-containing glycolipid designated Gb3 (16,(21)(22)(23) STF-3, 19...

Comparison of Vero-cytotoxin-encoding Phages from Escherichia coli of Human and Bovine Origin

“…The failure to obtain VT phages from strains of porcine origin confirm the results of Marques et al (1987). Recently, the structural genes for a variant of SLTII, active on Vero but not HeLa cells, have been cloned from total cellular DNA of an E. coli strain causing oedema disease in pigs (Weinstein et al, 1988). These authors concluded that the toxin sequences were chromosomally located.…”

Alteration of the carbohydrate binding specificity of verotoxins from Gal alpha 1-4Gal to GalNAc beta 1-3Gal alpha 1-4Gal and vice versa by site-directed mutagenesis of the binding subunit.

“…VT-1, VT-2, and SLT-II have been shown to bind specifically to globotriaosylceramide (Gb3; Galal-4GalI31-4GlcCer, in which Cer is ceramide) (9,10). Although the B subunit of SLT-II is 84% identical to that of VTE (11,12), the latter was found to bind primarily to globotetraosylceramide (Gb4; GalNAcI31-3Galal-4Ga4j31-4GlcCer) (13, -14) and to a lesser extent to Gb3 (13,14). Because of the homology between the B subunits, we were able to target a limited number of codons in the B cistrons of VTE and VT-1 for site-directed mutagenesis.…”

VTEC enteropathogenicity

“…Why strains carry the genes for two forms of VT is not clear, and how VTl and VT2 each affect the pathogenesis of disease is not fully understood. VTEC associated with pig oedema express a variant of VT2 termed VT2ve (Weinstein et al 1988a). This form of VT is remarkable in that it is lethal for Vero cells but not cytotoxic for HeLa cells.…”