Abstract
Cry6Aa1 is a Bacillus thuringiensis (Bt) toxin active against nematodes and corn rootworm insects. Its 3D molecular structure, which has been recently elucidated, is unique among those known for other Bt toxins. Typical three-domain Bt toxins permeabilize receptor-free planar lipid bilayers (PLBs) by forming pores at doses in the 1-50 μg/ml range. Solubilization and proteolytic activation are necessary steps for PLB permeabilization. In contrast to other Bt toxins, Cry6Aa1 formed pores in receptor-free bilayers at doses as low as 200 pg/ml in a wide range of pH (5.5-9.5) and without the need of protease treatment. When Cry6Aa1 was preincubated with Western corn rootworm (WCRW) midgut juice or trypsin, 100 fg/ml of the toxin was sufficient to form pores in PLBs. The overall biophysical properties of the pores were similar for all three forms of the toxin (native, midgut juice- and trypsin-treated), with conductances ranging from 28 to 689 pS, except for their ionic selectivity, which was slightly cationic for the native and midgut juice-treated Cry6Aa1, whereas dual selectivity (to cations or anions) was observed for the pores formed by the trypsin-treated toxin. Enrichment of PLBs with WCRW midgut brush-border membrane material resulted in a 2000-fold reduction of the amount of native Cry6Aa1 required to form pores and affected the biophysical properties of both the native and trypsin-treated forms of the toxin. These results indicate that, although Cry6Aa1 forms pores, the molecular determinants of its mode of action are significantly different from those reported for other Bt toxins.
Highlights
Cry6Aa1 is a Bacillus thuringiensis (Bt) toxin active against nematodes and corn rootworm insects
This study clearly shows, for the first time, that Cry6Aa1, a Bt toxin that is active against nematodes and coleopteran pests, is a pore-forming toxin
Attempts to characterize the mode of action of this toxin were limited to the demonstration that exposure of the nematode Caenorhabditis elegans to Cry6Aa allows its midgut epithelial cells to be labeled by propidium iodide, a well known viability probe [39, 61]
Summary
The Gram-positive bacterium Bacillus thuringiensis (Bt) forms crystal-like parasporal inclusions during sporulation [1, 2], which often comprise insecticidal proteins [3]. Known Bt toxins belong in majority to the Cry (crystal) family of proteins, whose most extensively studied members are insecticidal Because of their importance and long use in pest management programs, the elucidation of their mode of action has been the object of considerable work The Bt mode of action can be described as follows: crystal proteins are first ingested as protoxins, which are solubilized and proteolytically converted to smaller polypeptides in the insect midgut These activated toxins bind to specific receptors at the surface of midgut epithelial cells, allowing them to insert and form pores in the cell membrane [14, 15]. A clear and detailed knowledge of the susceptibility of the toxin to intestinal proteases, interaction with membrane receptors, and
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