BackgroundThe Cry6 family of proteins from Bacillus thuringiensis represents a group of powerful toxins with great potential for use in the control of coleopteran insects and of nematode parasites of importance to agriculture. These proteins are unrelated to other insecticidal toxins at the level of their primary sequences and the structure and function of these proteins has been poorly studied to date. This has inhibited our understanding of these toxins and their mode of action, along with our ability to manipulate the proteins to alter their activity to our advantage. To increase our understanding of their mode of action and to facilitate further development of these proteins we have determined the structure of Cry6Aa in protoxin and trypsin-activated forms and demonstrated a pore-forming mechanism of action.ResultsThe two forms of the toxin were resolved to 2.7 Å and 2.0 Å respectively and showed very similar structures. Cry6Aa shows structural homology to a known class of pore-forming toxins including hemolysin E from Escherichia coli and two Bacillus cereus proteins: the hemolytic toxin HblB and the NheA component of the non-hemolytic toxin (pfam05791). Cry6Aa also shows atypical features compared to other members of this family, including internal repeat sequences and small loop regions within major alpha helices. Trypsin processing was found to result in the loss of some internal sequences while the C-terminal region remains disulfide-linked to the main core of the toxin. Based on the structural similarity of Cry6Aa to other toxins, the mechanism of action of the toxin was probed and its ability to form pores in vivo in Caenorhabditis elegans was demonstrated. A non-toxic mutant was also produced, consistent with the proposed pore-forming mode of action.ConclusionsCry6 proteins are members of the alpha helical pore-forming toxins – a structural class not previously recognized among the Cry toxins of B. thuringiensis and representing a new paradigm for nematocidal and insecticidal proteins. Elucidation of both the structure and the pore-forming mechanism of action of Cry6Aa now opens the way to more detailed analysis of toxin specificity and the development of new toxin variants with novel activities.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-016-0295-9) contains supplementary material, which is available to authorized users.

Western corn rootworm (WCR), Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae), is a serious insect pest in the major corn growing areas of North America and in parts of Europe. WCR populations with resistance to Bacillus thuringiensis (Bt) toxins utilized in commercial transgenic traits have been reported, raising concerns over their continued efficacy in WCR management. Understanding the modes of action of Bt toxins is important for WCR control and resistance management. Although different classes of proteins have been identified as Bt receptors for lepidopteran insects, identification of receptors in WCR has been limited with no reports of functional validation. Our results demonstrate that heterologous expression of DvABCB1 in Sf9 and HEK293 cells conferred sensitivity to the cytotoxic effects of Cry3A toxins. The result was further validated using knockdown of DvABCB1 by RNAi which rendered WCR larvae insensitive to a Cry3A toxin. However, silencing of DvABCB2 which is highly homologous to DvABCB1 at the amino acid level, did not reduce the sensitivity of WCR larvae to a Cry3A toxin. Furthermore, our functional studies corroborate different mode-of-actions for other insecticidal proteins including Cry34Ab1/35Ab1, Cry6Aa1, and IPD072Aa against WCR. Finally, reduced expression and alternatively spliced transcripts of DvABCB1 were identified in a mCry3A-resistant strain of WCR. Our results provide the first clear demonstration of a functional receptor in the molecular mechanism of Cry3A toxicity in WCR and confirmed its role in the mechanism of resistance in a mCry3A resistant strain of WCR.

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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.

The defensive mucus produced by the slug Arion subfuscus is tough and remarkably sticky. It spreads rapidly and adheres strongly to a wide range of surfaces. The adhesion is equally strong on wettable (glass) and non-wettable (plastic) surfaces. The adhesion appears to depend on a group of proteins that adsorb equally well to a wide range of different natural and artificial surfaces. Prominent among these proteins were those that distinguish the adhesive secretion from the non-adhesive mucus. The adhesive proteins were not washed off by non-ionic detergent, nor was the adhesion of the glue as a whole affected by this treatment. In contrast, high salt concentrations washed the most abundant adhesive proteins off the surfaces, and correspondingly weakened the glue’s attachment. The most abundant of the adhesive proteins were C-lectins, which appear to form heterotrimers. These and other lectin-like proteins in slug glue have a high proportion of aromatic amino acids at conserved locations, and are relatively small and often basic. The aromatic and cationic side chains may provide a powerful combination promoting and maintaining surface adhesion.