Affiliation:
1. Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907
Abstract
ABSTRACT
During sporulation,
Bacillus thuringiensis
produces crystalline inclusions comprised of a mixture of δ-endotoxins. Following ingestion by insect larvae, these inclusion proteins are solubilized, and the protoxins are converted to toxins. These bind specifically to receptors on the surfaces of midgut apical cells and are then incorporated into the membrane to form ion channels. The steps required for toxin insertion into the membrane and possible oligomerization to form a channel have been examined. When bound to vesicles from the midguts of
Manduca sexta
larvae, the Cry1Ac toxin was largely resistant to digestion with protease K. Only about 60 amino acids were removed from the Cry1Ac amino terminus, which included primarily helix α1. Following incubation of the Cry1Ab or Cry1Ac toxins with vesicles, the preparations were solubilized by relatively mild conditions, and the toxin antigens were analyzed by immunoblotting. In both cases, most of the toxin formed a large, antigenic aggregate of ca. 200 kDa. These toxin aggregates did not include the toxin receptor aminopeptidase N, but interactions with other vesicle components were not excluded. No oligomerization occurred when inactive toxins with mutations in amphipathic helices (α5) and known to insert into the membrane were tested. Active toxins with other mutations in this helix did form oligomers. There was one exception; a very active helix α5 mutant toxin bound very well to membranes, but no oligomers were detected. Toxins with mutations in the loop connecting helices α2 and α3, which affected the irreversible binding to vesicles, also did not oligomerize. There was a greater extent of oligomerization of the Cry1Ac toxin with vesicles from the
Heliothis virescens
midgut than with those from the
M. sexta
midgut, which correlated with observed differences in toxicity. Tight binding of virtually the entire toxin molecule to the membrane and the subsequent oligomerization are both important steps in toxicity.
Publisher
American Society for Microbiology
Subject
Ecology,Applied Microbiology and Biotechnology,Food Science,Biotechnology
Cited by
64 articles.
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