Biopesticidal pore forming proteins derived from Bacillus thuringiensis (Bt) have long been used for the development and application of resistant genetically modified crops against major pests in the market, against moth larvae (eg Fall Army worm) and beetle larvae (eg Western Corn Rootworn). Bt uses pore forming proteins from over 15 different pore forming families. One system of interest is the Bt Vpb1/Vpa2 pore system which functions as an acid activated binary toxin pair in an A/B toxin delivery system in the same way that anthrax toxin (PA) delivers cytolytic factors (LF and EF). However, a paralogue Bt protein, Vpb4, functions to kill aphids without needing to deliver any cytotoxic factor and without the need for acidification. This is of value and interest to the agriculture industry.
Here we present various crystal, prepore and pore structures of the Vpb1/Vpa2 pore system together with the pore structure of Vpb4 to shed light on the assembly pathway and contrasting lytic mechanisms the two insecticidal pore forming proteins. The structure of Vpb4 revealed distinguishing features from other exotoxin B members, including the lack of a molecular bottleneck and an overall neutral charge of the pore lumen. Electrophysiology studies showed that the Vpb4 pore can conduct ions across lipid bilayers at a greater flux compared to PA. Collectively, these results suggest that Vpb4 subclass functions as bona fide pores to disrupt the insect guts through osmotic flux or leakage of the cellular content rather than relying on the delivery of cytotoxic factors.
Furthermore, through our phylogeny studies, we were able to predict several putative Vpb4-like proteins that could share this unique independent lytic mechanism. This holistic study of the Bacterial_Exotoxin_B family allows us to formulate the design principles for modulating the insect targeting specificity. It also allows for the identification of similar independently functioning pores with potential pest control activity.