Purification and characterization of a shiga-like toxin from escherichia coli strain 0157 of human origin implicated in haemolytic uremic syndrome

ABSTRACT

A Shiga-like toxin [SLT] from enterohemorrhagic Escherichia coli strain 0157 isolated from the stools of patients with haemolytic uremic syndrome [HUS] was produced, partially purified and the biological properties have been characterized. The production of the toxin was done on Tryptone Soy Broth containing mitomycin C. The purification scheme involved polymyxin beta extraction of the toxin from bacterial cell, differential [NH[4]]2 SO[4]. Precipitation and desalting by passage through a Sephadex G50 column chromatography equilibrated with 10 mM. Tris hydrochloride [pH 7.6]. The purification scheme resulted in high recovery of SLT activity in comparison to crude culture supernatant and the recovered cytotoxicity to Vero cells were 4 x 10[4] CD[50]/mg protein for crude culture supernatant, 1.8 x 10[5] CD[50]/mg for polymyxin B extract and 5 x 10[5] for the material obtained by [NH[4][2] SO[4]. fractionation and gel filtration. The partially purified SLT or Verotoxin was [I] Paralytic-lethal to mice when injiected intraperitoneally and the 50% lethal dose was [16.000] verotoxin units [ii] enterotoxic to rabbit ileal loop, at a very high dose [256, 000] verotoxoin unit, The purified toxin preparation was Partially homogenous as judged by Sodium dodecyl sulphate-polyacrylamide gel electrophoresis [SDS-PAGE]. The SLT preparation, under non reducing condition, exhibited 2 bands on SDS-PAGE corresponding to a molecolar mass of 33,000 and 7,500 kb together with other protein band, These 2 protein bands were consistent with the expected MWs of A and B subunits of SLT respectively