Bacterial Exotoxin B is a family of proteins functioning as the binding (B) component within the AB Toxin System. The family comprises pore-forming proteins, predominantly from Gram positive bacteria, that bind to the cell surface and perforate the cells, allowing the transport of the enzymatically active (A) component into the cell to confer its toxicity. While most family members are toxic to humans and livestock (Anthrax toxin, PA and Clostridial toxins; CdtB and Iota toxin), the Vpb4 subclass, from Bacillus thurengiensis, was discovered to exhibit larvicidal activity against the economically important maize pests Western Corn Rootworm and Two-spotted Leaf Beetle. Importantly, the Vpb4 proteins can function in the absence of any A component, making it an unusual member of the family. However, the precise molecular mechanism regarding how Vpb4 targets and kills insects without the need to deliver a second toxic protein remains unknown.
We elucidated the first pore structure of Vpb4 at 3.12 Å resolution (using cryo-EM). The structure reveals the absence of the highly conserved molecular bottleneck typically of the binary toxins. Electrophysiology studies revealed that the Vpb4 pore conducts ions at a greater flux than the archetypal family member, Anthrax (PA), across lipid bilayers. These results suggest that the Vpb4 subclass functions as pore alone.
Phylogenetic analysis identified that Vpb4 belongs to a clade that evolved early in the evolution of the family and is independent of the clades that contain the characterised binary toxins. We also identified several Vpb4-like proteins that we predict share this unique molecular mechanism. Our findings have significant implications for understanding the overall evolution and function of the Bacterial Exotoxin B family. Furthermore, this holistic study will facilitate the design of Vpb4 and its implementation in pest control, as well as the identification of similar independent pores as candidates for novel biopesticides.