Exploitation of small cysteine-rich spider protein toxins as bio-insecticides

Recombinant fusion protein technology allows specific insecticidal protein and peptide toxins to display activity in orally-delivered bio-pesticides. Here, some small cysteine-rich protein toxins were evaluated as insecticides, including δ-amaurobitoxin-Pl1a (Pl1a) from tangled nest spider (Pireneitega luctuosa), ω-atracotoxin-Hv1a (Hv1a) from funnel web spider (Hadronyche versuta) and κ-theraphotoxin-Ec2a (Ec2a) from Eucratoscelus constrictus, which target insect voltage-gated sodium channels, calcium activated potassium channels and voltage-regulated potassium channels, respectively. Recombinant proteins were produced using the yeast Pichia pastoris as expression host, by combining the coding sequences of the toxin with that of snowdrop lectin ("carrier"), that can deliver these toxins to the central nervous system of the target pest. Experimental results showed the toxins alone had limited or even no activities without being fused to the N-terminal of snowdrop lectin "carrier". Further, fusion of toxins to proteins other than snowdrop lectin also gave products with low or no biological activity. The absence of biological activity suggested that the toxin protein was not folding properly when expressed without fusion to the snowdrop lectin carrier, which meant GNA could not only direct transport of the toxins across the insect gut as a carrier, but also can help toxins to achieve correct folding. For example, the toxin Pl1a and a Pl1a/GNA fusion protein both caused mortality when injected into cabbage moth (Mamestra brassicae) larvae, but the Pl1a/GNA fusion protein was approximately 6 times as effective as recombinant Pl1a on a molar basis. Pl1a alone was not orally active against cabbage moth larvae, but a single 30 μg dose of the Pl1a/GNA fusion protein caused 100% larval mortality within 6 days when fed to 3rd instar larvae, and caused significant reductions in survival, growth and feeding in 4th - 6th instar larvae. To attempt to further improve the folding of recombinant fusion proteins, the predicted Pro-regions of toxins, between the signal peptide and the final mature sequence of the protein were examined. Inclusion of the Pro-region in the expression construct was hypothesised to result in improved folding of the toxin when expressed in P. pastoris. The results proved that the new type fusion protein (Pro-region/toxin/GNA) had much higher biological activity than toxins alone and higher activity than toxin/GNA fusion proteins. In addition, the Pro-region was successfully removed from the Pro-region/toxin/GNA proteins after expression. For example, the LD50 of Pro-Hv1a/GNA was decreased by 12 fold compared to Hv1a/GNA when injected into Mamestra brassicae larvae of different stages of development. Increased biological activity of Pro-Hv1a/GNA when compared to Hv1a/GNA was also observed when the proteins were injected into slugs. The increased biological activity of Pro-Hv1a/GNA on injection was also observed as increased oral toxicity of the fusion protein to insects. A single dose (20 μg) of fusion protein Hv1a/GNA caused no mortality to 5th instar larvae of M. brassicae, or 30% mortality to 3rd instar larvae; in contrast, 20 μg Pro-Hv1a/GNA caused 30% mortality to 5th instar larvae, and 90% mortality to 3rd instar larvae. Fusion proteins have the potential to be a new class of bio-pesticides for commercial application and have potential uses in complementing or replacing existing pesticides. Insecticide-resistant strains of peach potato aphid (Myzus persicae), designated "kdr", "super-kdr" and "kdr+super-kdr" contain mutations in the voltage-gated sodium channel (NaCh). Pl1a/GNA and Pro-Hv1a/GNA fusion proteins have the LC50 values of 0.35 and 0.19 mg ml-1 when fed to wild-type M. persicae. For insecticide-resistant aphids, the LC50 for the Pl1a/GNA fusion protein, which targets NaCh, was increased by 2-6 fold correlating with pyrethroid resistance (wild-type < kdr < super-kdr < kdr+super-kdr strains). In contrast, the LC50 for the Pro-Hv1a/GNA, which targets calcium channels, showed limited correlation with pyrethroid resistance. Therefore, mutations in the sodium channel in pyrethroid-resistant aphids also protect against a fusion protein containing a sodium channel-specific toxin, despite differences in ligard-channel interactions. This may be because changes to the spatial structure of domain II as a result of these mutations presumably also disturb the binding of Pl1a to receptor site 4, in domain II of sodium ion channel. However, mutations in the sodium channel do not confer resistance to a fusion protein targeting calcium channels. The use of fusion proteins with differing targets could delay resistance development in M. persicae.