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

Alexey Dementiev,Colin Berry,Marianne M Lee,Nick X Wang,Kenneth E Narva,Michael K Chan,Jason Board,Xiaoping Xu,Anand Sitaram,Alice Wegener,Yan Hu,David Mccaskill,Samantha Griffin,Shao-Ching Hung,Raffi V Aroian,T D Hey ,Jessica M Hughes ,José Cristian Vidal-Quist ,Vimbai M Chikwana ,M.s Kelker

doi:10.1186/s12915-016-0295-9

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.

Highlights

The 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
Initial analysis The Cry6Aa protein is active in the gut of nematodes such as C. elegans, which is an acidic environment with a pH ranging from ~3.6 to 6.0 [33] or Coleoptera such as Diabrotica virgifera virgifera with pH ranging from 5.75 to 6.03 [34]
The Cry6Aa crystals produced in B. thuringiensis and purified by sucrose gradient centrifugation could be solubilized at pH 10.0; Cry6Aa derived from this source showed a tendency to form aggregates and to precipitate after solubilization at either pH 3.0 or 12.7, if this solubilization was followed by neutralization

Summary

Introduction

The Cry 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. The structures of the ß-sheet-rich toxins Cry (Parasporin4) [9], Cry (Parasporin 2) [10], and recently Cry51 [11] and the Cry34/Cry binary toxin [12] have been published but for many non-threedomain Cry proteins, neither structural data nor information on mechanism of action are available One such Cry protein is Cry6Aa [13], a protein with activity against Coleoptera such as the Western Corn Rootworm Diabrotica virgifera virgifera [14] and a range of nematodes, including both free-living (Caenorhabditis elegans and Panagrellus redivivus) and plant pathogenic (Heterodera glycines and Meloidogyne incognita) species [15,16,17,18] that cause large-scale losses to agriculture [19]

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Conclusion