Showing papers by "Alison D. O'Brien published in 1992"

Shiga toxin: biochemistry, genetics, mode of action, and role in pathogenesis.

Abstract: Dysentery was well known and clearly described in many ancient texts and histories. The first step towards the description of the genus Shigella, however, was the identification of Entamoeba histolytica by Losch in 1875 and the separation of amebic from all other forms of dysentery (Losch, 1875). With this discovery, attention could be focused on the etiology of epidemic dysentery, and a partial description of the prototype Shigella sp., Shigella dysenteriae type 1, was published by Chantemesse and Widal in 1888. The definitive description of this organism was provided by Kiyoshi Shiga following an extensive dysentery epidemic in Japan in 1896 (Shiga 1898). It did not take long to determine that there was a potent toxic activity in this organism, and in 1900 Flexner reported that either living or killed cultures of Shiga’s bacillus injected into the peritoneal cavity of animals caused fever and diarrhea. Flexner concluded that shigellosis was due to a “toxic agent rather than to an infection per se”; however, the Observed effects were Probably due to endotoxin. The presence of a lethal toxin in extracts of heat-killed bacteria was shown independently by Neisser and Shiga (1903) and by Conradi (1903). Conradi (1903) also described the limb paralysis following parenteral inoculation of Shigella extracts in rabbits, characteristic of the so-called Shiga neurotoxin.

An enzymatic mutant of Shiga-like toxin II variant is a vaccine candidate for edema disease of swine.

Abstract: Edema disease (ED) of weanling pigs is caused by an infection with Escherichia coli that produces Shiga-like toxin II variant (SLT-IIv). Pathology identical to that caused by ED can be duplicated in pigs that are injected with less than 10 ng of purified SLT-IIv per kg of body weight. Therefore, SLT-IIv was mutated to create an immunoreactive form of the toxin that was significantly reduced in enzymatic activity. Initially, purified SLT-IIv was treated with formaldehyde which abrogated cytotoxic activity. Pigs were vaccinated with the toxoid (100 micrograms) to determine whether a toxoid was a viable vaccine candidate and whether young pigs were capable of mounting an immune response. Although the pigs developed a neutralizing antibody titer (1:128 to 1:512) 28 days postinjection, they also lost weight and developed ED lesions. The deleterious effect of the toxoid appeared to result from residual enzymatic activity or a reversion to a toxic form. An alternative method, site-directed mutagenesis, was employed to consistently reduce the enzymatic activity of SLT-IIv. Glutamate at position 167 of the mature A subunit was replaced by aspartate (E167D), and arginine at position 170 was replaced by lysine (R170K). These mutations reduced cytotoxic activity 10(4)-fold and 10-fold, respectively, while the enzymatic activities were decreased 400-fold and 5-fold, respectively. The activity of a toxin that contained both mutations (SLT-IIvE167D/R170K) closely resembled that of SLT-IIvE167D. When position 167 was replaced by glutamine (E167Q), the cytotoxic activity decreased 10(6)-fold and the enzymatic activity decreased approximately 1,500-fold. Pigs that were vaccinated with purified, mutant toxin designated SLT-IIvE167Q developed a neutralizing antibody titer of 1:512 21 days postinjection, and their tissues were free of ED lesions. These data suggest that SLT-IIvE167Q may represent an effective vaccine against ED.