Design of novel recombinant fusion proteins for use as bioinsecticides

Abstract

Insecticidal compounds play a pivotal role in effective crop protection strategies. The remarkable adaptability and resilience of herbivorous insect pests has led to widespread insecticidal resistance, rendering many traditional compounds ineffective. Combatting this issue requires effective modern pest control and insecticidal resistance management strategies, which can be realised through the diversification of our insecticidal repertoire. Nature itself offers a vast array of biochemistries suitable for this purpose, with millions of insectivorous species that produce potent, taxonomically selective, and biodegradable toxins.
This thesis presents a multifaceted approach to the initial evaluation, development, and design of innovative, recombinant fusion protein bioinsecticides. While investigating various design paradigms, the discussion centres around the inhibitor cysteine knot (ICK), a structural motif or protein scaffold adopted by neurotoxic peptides from a wide range of venomous and poisonous creatures. The repeated convergence upon this structure, particularly among insectivorous arachnids, demonstrates its flexibility and utility. This thesis thus demonstrates a multitude of promising biochemistries and insecticidal modes of action for consideration as lead compounds for bioinsecticide development, as well as the implementation of computationally assisted design systems, and novel bioengineering approaches. By these means, this research seeks to expand the arsenal of bioactive compounds available to combat insecticide resistance and avoid non-target effects on beneficial species.
Recombinant fusion of insecticidal venom toxins to the snowdrop lectin Galanthus nivalis agglutinin (GNA) has been shown to enhance their oral activity. GNA binds to proteins in the insect gut epithelium whereby it facilitates transport of the neurotoxin to the insect central nervous system. Expanding on this design concept, Chapter 3 discusses the design and production of recombinant fusion protein bioinsecticides where two venom ICK peptides are recombinantly fused to a single GNA component, thus doubling the molar toxin delivery capacity of the lectin. The double toxin fusion proteins HxTx-Hv1h(2)/GNA/His and GNA/Hv1a-Hv1h(2)/His were successfully expressed in the yeast Pichia pastoris. Both double toxin fusion proteins showed oral activity towards the two aphid species Myzus persicae and Arythosyphon pisum. When orally delivered to A. pisum, HxTx- Hv1h(2)/GNA/His and GNA/Hv1a-Hv1h(2)/His produced day 3 LC50 values of 13.8 and 15.8 μM respectively. These values are 2 X lower than those of the single toxin fusion protein counterparts, HxTx-Hv1h/GNA/His and GNA/Hv1a-Hv1h/His, with day 3 LC50 values of 33.2 μM and 26.2 μM respectively. These results indicate the successful delivery of two functional toxin components by the GNA carrier to the insect circulatory system, thus demonstrating an enhanced per mole insecticidal efficacy of the double toxin fusion protein bioinsecticides. Moreover, enhanced insecticidal activity was also observed following injection of HxTx-Hv1h(2)/GNA/His into the lepidopteran pest Mamestra brassicae. HxTx-Hv1h(2)/GNA/His produced an injection day 4 LD50 of 78.3 pmol/larva in M. brassicae larvae, a 10-fold increase compared to that of the single toxin HxTx-Hv1h/GNA/His, which produced a day 4 LD50 of 823 pmol/larva. Such efficacy is above what would be expected from the increase in toxin delivery, suggesting synergistic effects resulting from the localisation of two toxin components at the GNA delivery site.
Assessment of the nemertean mucosal toxin nemertide α-1 in Chapter 4 represents a departure from the more prominent focus on arachnid and scorpion venom peptides in bioinsecticide research, shedding light on the unique properties and potent insecticidal potential inherent in marine animal toxins. The nemertean ribbon worms have converged upon the ICK toxin scaffold, utilising it to immobilise invertebrate predators and prey which contact their mucosal secretion. As such, the native α-1 toxin displayed oral activity with day 2 LC50 values of 22.2 μM and 55.4 μM against A. pisum and M. persicae respectively. α-1 also produced oral activity against the lepidopteran pest M. brassicae producing 100 % mortality following ingestion of 3.2 nmol. This is less than 2 X the dose required to produce 60 % mortality by injection, a parity in oral and injection activity rarely observed in venom ICK peptides. Unfortunately, α-1 was also demonstrated to be significantly more insecticidal against two pollinator species, Apis mellifera and Bombus terrestris, than any other here tested venom ICK peptide. This broad action may be attributable to the molecular target of α-1, insect voltage gated sodium (Nav) channels, having undergone less evolutionary conservation than the target of all other ICK peptides discussed in this thesis, insect voltage gated calcium (Cav) channels. Thus, α-1 demonstrates the utility of non-venom ICK peptides in the search for natural insecticidal compounds, while highlighting the importance of the early assessment of off-target effects and appropriate molecular target selection.
ω-phylotoxin-Tbo-IT1, an ICK peptide from the venom of Tibellus oblongus is the first described ICK peptide from the understudied arachnid family Philodromidae. This inhibitor of insect Cav channels was demonstrated to possess oral activity against the two aphid species M. persicae and A. pisum with calculated day 4 LC50 values of 91.5 μM and 213.9 μM respectively. Tbo-IT1 also produced a unique phenotypic effect following injection into M. brassicae larvae. Injection doses of 3 nmol/larva resulted in disruption of rectilinear locomotion, localised posterior paralysis, and necrosis progressing from the posterior to anterior end. This effect suggests a more multifaceted mode of action of the toxin than previously understood and may provide valuable clues about the distribution and functionality of calcium channels in the larvae. Tbo-IT1 demonstrated high taxonomic selectivity, producing no observable phenotype, nor mortality following injection of 30 nmol into B. terrestris. Attempts to enhance the insecticidal activity of Tbo-IT1 via recombinantly fusion to GNA failed due to destabilisation of the ICK scaffold during expression. In Chapter 5, utilisation of the Artificial intelligence (AI) structural prediction software, AlphaFold 2, to validate in silico rational modifications made to the primary sequence of Tbo-IT1 GNA fusion proteins, successfully facilitated the design and production of modified ICK peptides amenable to recombinant fusion to GNA. Accurate structural predictions allowed for high-throughput iterative modifications to the linker region to be structurally assessed, with improvements to the expression profile and protein stability verified experimentally. Further modifications to Tbo-IT1 resulted in the recombinant expression of Hx-IT1, with amino acids non-native to Tbo-IT1 constituting 45 %, the first semi-natural ICK toxin. This chimeric ICK peptide maintained the oral activity of Tbo-IT1 towards aphids, with day 4 LC50 values of 148.1 μM and 248.1 μM against M. persicae and A. pisum, respectively, as well as its non-toxicity towards B terrestris at all doses tested. Loss of the paralytic and necrotic phenotype following injection into M. brassicae begins to elucidate the significance of the substituted residues in the effect of Tbo-IT1 towards this species. Recombinant fusion of Hx-IT1 to GNA (GNA/Hx-IT1/His) facilitated a significant increase in the chimeric toxin’s insecticidal activity, with day 4 LC50 values of 24.4 μM and 12.2 μM when orally delivered to M. persicae and A. pisum. This is a respective 6-fold and 20-fold increase in activity compared to Hx-IT1, and a respective 3-fold and 17-fold increase in activity compared to Tbo-IT1. The injection toxicity of Hx-IT1, with a day 4 LC50 of 23.7 nmol, was similarly enhanced 4-fold by recombinant fusion to GNA, with GNA/Hx-IT1 eliciting a day 4 LC50 of 5.3 nmol.
The novel insecticidal compounds, recombinant fusion technologies, and AI supplemented design techniques discussed in this thesis, each contribute towards the endeavour to enhance the number of insecticidal control measures. By assessing novel compounds, with potentially unique modes of action and specific target sites, and establishing rapid and accessible design techniques to enhance and modulate the insecticidal activity of natural neurotoxic compounds, the diversity of the repertoire of effective and ecologically safe bioinsecticides for use in the protection of crops is possibly limitless.