Shigella dysenteriae 1 cytotoxin: periplasmic protein releasable by polymyxin B and osmotic shock

Treatment of Shigella dysenteriae 1 either with the antibiotic polymyxin B or by osmotic shock resulted in the release of 80 to 90% of the cytotoxin activity of the organism. Under the conditions employed, the release of toxin activity was accompanied by the appearance of a periplasmic enzyme, 5'-nucleotidase. There was no significant release of cytoplasmic contents, assessed by measurement of glucose-6-phosphate dehydrogenase activity. The release of cytotoxin and 5'-nucleotidase by polymyxin B were both dependent on the duration of incubation with, and the concentration of, the antibiotic. In terms of specific activity (cytotoxin activity per milligram of protein), the polymyxin B and osmotic shock extracts were 20- to 30-fold more active than crude toxin preparation derived from a whole-cell lysate. The data strongly support a periplasmic location for Shiga cytotoxin and the utility of the polymyxin B extraction to obtain starting material for toxin purification.