Pathogenesis of Shigella diarrhea. IX. Simplified high yield purification of Shigella toxin and characterization of subunit composition and function by the use of subunit-specific monoclonal and polyclonal antibodies.

Abstract

For over 80 years now, Shigella dysenteriae 1 has been known to produce one of the most potent of the lethal microbial toxins. It was originally called Shiga toxin (after the discoverer of the organism, K. Shiga) and classified as a neurotoxin because it results in a delayed-onset limb paralysis terminating in death when parenterally administered to sensitive animals (reviewed in reference 1). Shigella toxin is also cytotoxic to certain tissue culture cells, as well as enterotoxic (results in fluid secretion) when applied to intestinal mucosa (2-5). Biochemical and immunological evidence indicate that the three biological activities are the properties of the same molecule (3, 5). Its role in the pathogenesis of shigellosis has always been controversial, in part because other species of the genus could not be shown to produce the same toxin. This argument is no longer valid, for both S. flexneri and S. sonnei have been found to produce shigella toxin under appropriate in vitro conditions, and convalescent patients develop an antibody that neutralizes the dysenteriae 1 toxin (6-8). Pathogenic bacteria of other genera have also been found to produce a similar toxin that is neutralized by antibody to shigella toxin. These organisms include a variety ofE. coli serotypes including human enteropathogenic strains, the causative strain of human hemorrhagic colitis (Escherichia coli 0:155), the noninvasive rabbit pathogen RDEC-1, human Salmonella strains, and even Vibrio cholerae (9-11). The cross-reactive toxin has been dubbed "Shiga-like toxin." Since these E. coli strains do not produce the well-known LT or ST toxins and since a mutant strain of V. cholerae deleted of the gene for the production of the ADP-ribosyl transferase enzyme subunit A of cholera toxin (12) causes diarrhea in humans, the shigella (or Shiga-like) toxin may well be a critical virulence factor in diarrheal disease.Because of these observations, there is great interest in this toxin and the immunologically related products of other organisms. Shigeila toxin has recently (9-15) been purified and partially characterized by several laboratories. The

Maturational regulation of globotriaosylceramide, the Shiga-like toxin 1 receptor, in cultured human gut epithelial cells.

“…To accomplish this, we exposed three intestinal cell lines derived from human colon carcinomas (CaCo-2A, HT-29, and T-84) to the short chain fatty acid sodium butyrate, normally found in the intestinal lumen, which induces differentiation of many cells in culture (10)(11)(12)(13)(14)(15), and measured the effects on differentiation marker enzymes, the content of Gb3, and the binding and cytotoxic response to SLT-1. Toxin purification and labeling SLT-1 was purified from sonic lysates of E. coli HB101 lysogenized with bacteriophage H19B as reported previously (16) and was labeled with 251I by a modification of the chloramine T method, which does not alter its specific activity (17). Recombinant SLT-1 B was purified from Vibrio cholerae 0395 Nl (pSBC32) (18) and labeled with fluorescein isothiocyanate (FITC) by incubation of 2 mg/ml subunit in 0.1 M Na2CO3, pH 9.0, with 50 ul FITC (1 mg/ml in DMSO) overnight at 40C in the dark.…”

Characterization of Purified Shiga Toxin from Shigella dysenteriae 1

“…It consists of two subunits, A and B (9,10,12). The biological activities of Shiga toxin reported so far are neurotoxic activity in mice, rabbits and monkeys, with a lethal effect when injected intraperitoneally, cytotoxicity to various cultured cells, such as HeLa cells and Vero cells, and enterotoxic activity, causing induction of fluid accumulation in rabbit ligated ileal loops (3,8,9,11).…”

Physicochemical Characterization of A and B Subunits of Shiga Toxin and Reconstitution of Holotoxin from Isolated Subunits

“…As shown in Fig. 2, the materials in the first and second peaks each gave a single band staining for protein and were found to correspond to B and A subunit of the Shiga toxin, respectively, as reported by Olsnes et al (12) and Donohue-Rolfe et al (2) .…”

“…Furthermore, Olsnes et al (12) showed that the A subunit was nicked by either trypsin or a protease from Streptomyces griseus and cleaved into two fragments A1, and A2, of which Al inhibited protein synthesis. Donohue-Rolfe et al (2) reported the isolation of A and B subunits of Shiga toxin by treatment of the toxin with formic acid and then its chromatography on a Bio-gel P-60 column with 5% formic acid. Separation of these subunits was monitored by use of 125I-labeled Shiga toxin.…”