Purification and characterization of a Shigella dysenteriae 1-like toxin produced by Escherichia coli

Abstract

A toxin from an enteropathogenic strain of Escherichia coli (E. coli H30) was purified to apparent homogeneity from cell lysates. The steps used to isolate the E. coli H30 toxin included French pressure-cell disruption of bacteria grown in iron-depleted media. Affi-Gel Blue chromatography, chromatofocusing, and anti-Shiga toxin affinity chromatography. The mobilities of the subunits of radioiodinated E. coli H30 toxin and Shiga toxin observed after the two toxins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis were identical. In the absence of 2-mercaptoethanol, a narrow band was seen at Mr 31,500 (+/- 1,000), and a wide heavy band was observed between Mr 4,000 and 15,000. In the presence of 2-mercaptoethanol, bands were seen at Mr 31,500 (+/- 1,000), 27,000, and 4,000 to 15,000. Other similarities between purified E. coli H30 and Shiga 60R toxins included identical isoelectric points (7.03 +/- 0.02); comparable biological activities, i.e., cytotoxicity, lethality for mice, and enterotoxicity; and the same relative heat stabilities (up to 65 degrees C for 30 min). Nevertheless, the two toxins had apparently different molecular weights as determined by sucrose gradient analysis, by gel filtration, and by cross-linking experiments with dimethyl suberimidate. The Mr of native E. coli H30 toxin estimated from cross-linking studies was 48,000, whereas the estimated Mr of Shiga 60R toxin was 58,000. These results suggest that like the cholera-E. coli-heat-labile toxin family, a family of Shiga-like toxins exists.

Purification and biological properties of Escherichia coli verocytotoxin

“…The reasons for this discrepancy are unclear. The value of 39 kDa would be consistent with a subunit composition of 1 A and 1 or more B subunits [2].…”

“…The purification process described above was used to produce a VT preparation of high biologi-67 cal activity which, by gel filtration was 39 kDa in size; on SDS-PAGE analysis under reducing conditions, it contained a major protein with an estimated size of 32 kDa, and a polypeptide of less than 14 kDa which migrated with the buffer front. These correspond to the A and B subunits of the VT or Shiga-like toxin reported previously [2]. In that study, the A and B subunits were demonstrated using radiolabelled toxin, since sufficient material for visualization of unlabelled toxin by silver staining, as shown by the present study, was not available.…”

“…In that study, the A and B subunits were demonstrated using radiolabelled toxin, since sufficient material for visualization of unlabelled toxin by silver staining, as shown by the present study, was not available. The simplified procedure reported here was made possible by the more selective release of VT from the periplasmic space of the cells by polymyxin B [6] instead of the French press, which lyses the whole cell and results in a cruder starting VT preparation [2]. The specific activity of the final product was higher than reported in the above study, but this could be due to the different methods used for the determination of toxicity and protein concentration.…”

“…The partially purified VT of the human isolate H 30, had isoelectric points at pH 7.2 and 6.8 and an estimated size of 28 kDa by membrane filtration [1]. More recently, in a comparative study, O'Brien and LaVeck showed H 30 VT to be closely related to the Shiga toxin [2]. Both toxins were found to have a pI of 7.03, showed complete serological cross-neutralization, had a similar subunit structure, induced fluid secretion in rabbit ileal loops, were cytotoxic to HeEa cells, and caused paralysis and death in mice.…”

Prevalence and molecular typing of attaching and effacing Escherichia coli among calf populations in Belgium

“…Verotoxins are E. coli cytotoxins which are lethal for cultured Vero cells (Konowalchuck et al, 1977). These toxins share a number of properties with Shiga toxin produced by Shigella dysenteriae and are therefore also called Shiga-like toxins (O'Brien and La Veck, 1983). Verotoxins consist of two groups, with VT1 (SLT-I) constituting one group and VT2 (SLT-II) and antigenically related toxins constituting a second group (Newland et al, 1988).…”