Aggregation of Bacillus thuringiensis Cry1A toxins upon binding to target insect larval midgut vesicles

During sporulation, Bacillus thuringiensis produces crystalline inclusions comprised of a mixture of delta-endotoxins. Following ingestion by insect l arvae, these inclusion proteins are solubilized, and the protoxins are conv erted to toxins. These bind specifically to receptors on the surfaces of mi dgut apical cells and are then incorporated into the membrane to form ion c hannels. The steps required for toxin insertion into the membrane and possi ble oligomerization to form a channel have been examined. When bound to ves icles from the midguts of Manduca sexta larvae, the Cry1Ac toxin,vas largel y resistant to digestion with protease K. Only about 60 amino acids were re moved from the Cry1Ac amino terminus, which included primarily helix alpha 1. Following incubation of the Cry1Ab or Cry1Ac toxins,vith vesicles, the p reparations were solubilized by relatively mild conditions, and the toxin a ntigens were analyzed by immunoblotting. In both cases, most of the toxin f ormed a large, antigenic aggregate of ca. 200 kDa. These toxin aggregates d id not include the toxin receptor aminopeptidase N, but interactions with o ther vesicle components were not excluded. No oligomerization occurred when inactive toxins with mutations in amphipathic helices (alpha 5) 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 h elix alpha 5 mutant toxin bound very well to membranes, but no oligomers we re detected. Toxins with mutations in the loop connecting helices alpha 2 a nd alpha 3, which affected the irreversible binding to vesicles, also did n ot oligomerize. There was a greater extent of oligomerization of the Cry1Ac toxin with vesicles from the Heliothis virescens midgut than with those fr om the M. sexta midgut, which correlated with observed differences in toxic ity. Tight binding of virtually the entire toxin molecule to the membrane a nd the subsequent oligomerization are both important steps in toxicity.