Cry6Aa1, a Bacillus thuringiensis nematocidal and insecticidal toxin, forms pores in planar lipid bilayers at extremely low concentrations and without the need of proteolytic processing

Abstract

Cry6Aa1 is a () toxin active against nematodes and corn rootworm insects. Its 3D molecular structure, which has been recently elucidated, is unique among those known for other toxins. Typical three-domain 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 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 toxins.

Functional validation of DvABCB1 as a receptor of Cry3 toxins in western corn rootworm, Diabrotica virgifera virgifera

“…We infected Sf9 cells in culture at a high titer with recombinant baculovirus expressing DvABCB1 to investigate and characterize the effect of subsequent addition of Cry toxins on Sf9 cell toxicity/viability. The toxins tested included Cry3Aa, Cry34Ab1/35Ab1, and Cry6Aa1, all of which were prepared and activated as previously described 51,52 . Cry6Aa1 is another Cry protein that is also highly active against corn rootworms 52 .…”

“…The toxins tested included Cry3Aa, Cry34Ab1/35Ab1, and Cry6Aa1, all of which were prepared and activated as previously described 51,52 . Cry6Aa1 is another Cry protein that is also highly active against corn rootworms 52 . The cells were evaluated for morphological changes by phase contrast light microscopy after overnight incubation with activated toxins.…”

The Caenorhabditis elegans CUB-like-domain containing protein RBT-1 functions as a receptor for Bacillus thuringiensis Cry6Aa toxin

“…It has been reported that Cry6Aa is a pore-forming toxin [15] and propidium iodide (PI) can enter cells through pores formed by Cry6Aa in C. elegans [14]. To determine whether RBT-1 is required for the pore-formation activity of Cry6Aa in nematodes, we conducted PI staining assays.…”

“…To date, nematicidal activity has been reported in several B. thuringiensis Cry protein families, such as Cry5 and Cry6 [11,12]. Interestingly, Cry6Aa, a novel nematicidal ClyA-type alpha-pore-forming toxin [13][14][15], does not kanamycin (50 μg/ml). C. elegans strains were maintained on nematode-growth media (NGM) plates seeded with E. coli OP50 at 20˚C using standard techniques [26].…”

Structural and Mechanistic Features of ClyA-Like α-Pore-Forming Toxins

“…Crystal structures of Cry6Aa confirmed that the protein belonged to the wider family of ClyA-like proteins [ 11 ], which are topologically closest to Hbl-B and NheA ( Figure 1 b). Current evidence points to a homooligomeric assembly state of Cry6Aa, and the pore formation has recently been verified [ 24 ]. It is curious that within the ClyA family, Cry6Aa holds greatest structural resemblance to Hbl-B and NheA (see Figure 1 a), which are components of the tripartite Hbl and Nhe toxins, respectively (see below).…”