The pesticidal Cry6Aa toxin from Bacillus thuringiensis is structurally similar to HlyE-family alpha pore-forming toxins

Abstract

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.

Bacillus thuringiensis targets the host intestinal epithelial junctions for successful infection of Caenorhabditis elegans

“…It is well‐documented that in insects and nematodes the midgut cell lysis can be caused by Bt Cry toxins alone through pore formation (Bravo et al ., ; Deist et al ., ; Dementiev et al ., ; Peng et al ., ). However, Cry toxins alone did not effectively disrupt the midgut epithelial junctions in C. elegans (Fig.…”

Structure and properties of AB21, a novel Agaricus bisporus protein with structural relation to bacterial pore‐forming toxins

“…The closest structural homologs belong to the ClyA 44–46 superfamily of bacterial pore‐forming toxins (Figure ). The best matches were two enterotoxins secreted by Bacillus cereus —HBL‐B (subunit B of hemolysin BL) and NheA (subunit A of Nhe toxin), aligned with a Z ‐score of 10.1 and 8.9, respectively, and the insecticidal Cry6Aa toxin from Bacillus thuringiensis , aligned with a Z ‐score of 9.4. The toxins consist of two sub‐domains: the tail domain made of a bundle of helices and the head domain.…”

“…To assess the relationship at the structural level, the structures of the encoded proteins were predicted by homology modelling in Phyre2 . Some of them were modeled according to the structure of Cry6Aa (as the most preferred template), even though the sequence identity with Cry6Aa was only ∼17%‐19%. The results showed that several genes coding for toxin‐like proteins are co‐localized with the ab21 gene in the genome (Figure ).…”

“…Enterotoxin subunits HBL‐B and NheA secreted by B. cereus and the insecticidal Cry6Aa toxin from B. thuringiensis were identified as the closest structural homologs to AB21. Despite the evident structural similarity of AB21 to the tail domain of bacterial toxins, AB21 lacks the functionally important head domain.…”

“…(subunit B of hemolysin BL) 47 and NheA (subunit A of Nhe toxin), 48 aligned with a Z-score of 10.1 and 8.9, respectively, and the insecticidal Cry6Aa toxin 49 However, the a3 and a4 helices of AB21 are significantly shorter than the corresponding helices in the toxins and the head domain, indicated as a region that interacts with the target membranes, 44 is absent in AB21. The expected inability of AB21 to interact with the membranes is in agreement with the experimental data.…”