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

Abstract

During sporulation, Bacillus thuringiensis produces crystalline inclusions comprised of a mixture of delta-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 alpha1. 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 (alpha5) 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 alpha5 mutant toxin bound very well to membranes, but no oligomers were detected. Toxins with mutations in the loop connecting helices alpha2 and alpha3, 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.

FIG. 1

Digestion of the Cry1Ac toxin either in solution (lanes 1 to 4) or bound to M. sexta BBMV (lanes 5 to 8) with various concentrations of protease K at 37°C for 45 min. Following incubation, the samples were boiled in loading buffer for 3 min. Fractionation was achieved by SDS–10% PAGE, and staining was done with Coomassie blue. Lanes 1 and 5, treatment with 0.1 μg; lanes 2 and 6, treatment with 0.2 μg; lanes 3 and 7, treatment with 0.5 μg; lanes 4 and 8, no treatment. The stained bands were quantitated in a General Dynamics ImageQuant with the following percentages of control values: lane 1, 80%; lane 2, 28%; lane 3, 25%; and lanes 5 to 7, 95 to 100%.

FIG. 2

Immunoblot of toxin antigens extracted from M. sexta BBMV (see Materials and Methods). Lane 1, extraction of the Cry1Ab antigen from BBMV; lane 2, 50 ng of Cry1Ab; lanes 3 and 6, extraction of the Cry1Ac S170C toxin from BBMV; lane 4, extraction of the Cry1Ac A164D toxin from BBMV; lane 5, extraction of the Cry1Ac W210C toxin from BBMV; lane 7, standards of β-galactosidase and bovine serum albumin; lane 8 (from top to bottom), myosin, β-galactosidase, phosphorylase b, and bovine serum albumin standards.

FIG. 3

Immunoblot of toxin antigens extracted from M. sexta BBMV resolved by SDS–6% PAGE. Lane 1, extraction of the Cry1Ac antigen from BBMV; lane 2, 50 ng of Cry1Ac toxin; lane 3, extraction of the Cry1Ac A164P toxin from BBMV; lane 4, 30 ng of Cry1Ac A164P toxin; lane 5, extraction of the Cry1Ac L167F toxin from BBMV; lane 6, 50 ng of Cry1Ac L167F toxin; lane 7, extraction of the Cry1Ac A92D toxin from BBMV; lane 8, extraction of the Cry1Ac R93F toxin from BBMV; lane 9, extraction of the Cry1Ab toxin from BBMV; lane 10, 50 ng of Cry1Ab toxin.

FIG. 4

Immunoblot of Cry1Ac and H168R, following incubation with M. sexta BBMV. (A) Lanes 1 to 3, 20 μg of BBMV incubated with 20, 40, and 60 ng of Cry1Ac toxin, respectively; lane 4, 60 ng of Cry1Ac toxin; lanes 5 to 7, 20 μg of BBMV incubated with 20, 40, and 60 ng of H168R toxin, respectively; lane 8, 60 ng of H168R toxin; lane 9, standards as in Fig. 2 plus ovalbumin (45 kDa) and carbonic anhydrase (29 kDa). (B) Toxin samples (20 ng) were incubated with 20 μg of BBMV and then extracted for different times at 65°C. Lane 1, Cry1Ac toxin extracted at 65°C for 15 min; lane 2, Cry1Ac toxin extracted at 65°C for 5 min; lane 3, 20 ng of Cry1Ac toxin; lane 4, H168R toxin extracted at 65°C for 15 min; lane 5, H168R toxin extracted at 65°C for 5 min; lane 6, 20 ng of H168R toxin. Markers on the right indicate (from top to bottom) positions of myosin, β-galactosidase, and bovine serum albumin.

FIG. 5

Immunoblot of toxin antigens and aminopeptidase N extracted from M. sexta BBMV and resolved by SDS–6% PAGE. Lane 1, incubation of 50 ng of Cry1Ac toxin with BBMV; lane 2, 50 ng of Cry1Ac toxin; lane 3, incubation of the Cry1Ac W210C toxin with BBMV; lane 4, 50 ng of Cry1Ac W210C toxin; lane 5, 50 ng of Cry1C toxin; lane 6, same as lane 1 but treated with aminopeptidase N antibody; lane 7, M. sexta BBMV extract treated with aminopeptidase N antibody; lane 8 (from top to bottom), thyroglobulin, myosin, β-galactosidase, and bovine serum albumin standards.

FIG. 6

Immunoblot of the Cry1Ac toxin antigen extracted from BBMV prepared from M. sexta and H. virescens. Lane 1, 10 μg of M. sexta BBMV plus 50 ng of Cry1Ac toxin; lane 2, 20 μg of M. sexta BBMV plus 50 ng of Cry1Ac toxin; lane 3, 10 μg of H. virescens BBMV plus 50 ng of Cry1Ac toxin; lane 4, 20 μg of H. virescens BBMV plus 50 ng of Cry1Ac toxin; lane 5, 50 ng of Cry1Ac toxin.