Difference between revisions of "Part:BBa K5184042"

Latest revision as of 12:15, 2 October 2024

PpVP1-S

Essential Information

Abstract

In our project we employ venom peptides to act as insecticidal agents against spider mites. This part presents an original mite venom peptide with high potency against spider mites that is determined by in vivo testing against T. urticae. This mite venom peptide may also aid future iGEM teams in identifying homologous venom peptide genes in the acariforme superorder or even across the acarid subclass that is right now almost completely unexplored. The part collection includes: Parts expressing venom peptides that target various ion channels that leads to paralysis and death of spider mites: PpVP1S (BBa_K5184042), PpVP2S (BBa_K5184043), PpVP1F (BBa_K5184038), rCtx-4 (BBa_K5184021), HxTx-Hv1h (BBa_K5184033), Cs1A (BBa_K5184032); supplementary parts that allows extracellular secretion, facilitates venom peptide folding, and improves contact and oral toxicity (BBa_5184020, and BBa_K5184022); composite parts that guarantee correct folding of venom peptides exhibiting contact toxicity (BBa_K5184071-BBa_K5184076). Our part collection provides a feasible and efficient expression of a collection of venom peptides targeting a diverse range of molecular targets in spider mites. While any one of the venom peptides in the collection can achieve reasonable elimination efficiency, combination of multiple venom peptides with different molecular targets makes resistance development much harder. This can help and inspire future teams to design and perfect venom peptide-based pesticides that are capable of tackling many pests known for their prowess for developing resistance and, as template sequences for identification of other acariforme venom peptides.

Basic Characterization

PpVP1S is the modified version of the venom peptide we identified in genome of the predatory mite, Phytoseiulus persimilis, by genome mining. It shares a high degree of homology to venom peptides from the predatory mite species Neoseiulus barkeri, which is first identified in [1]. Via phylogenic analysis, we believe it likely achieves its paralyzing and insecticidal properties by interfering with voltage gated calcium channels whose affinity towards is then verified by computer modelling.

Sequences

Sequence and Features

Characterization

Molecular Structure

PpVP1S is the truncated version of the PpVP1F, composing of a total of 45 amino acids, with 8 cysteine residues that form 4 disulfide bridges, and also two antiparallel beta sheets. These motifs together contribute to the final highly compact folded conformation of the venom. Comparing to NbVP1S[1], it has the same set of cysteine network of C1xxxC2xxxC3C4xxxC5xC6xxxC7xC8, with disulfide bridges between C(1)C(4), C(2)C(5), C(3)C(8), and C(6)C(7)[Fig.1A], and therefore shares an almost congruent geometry [Fig.1B], differing by only 5 amino acids.

Genome Mining

Using identified mRNAs of the two N. barkeri venom peptides as query, we identified the two orthologous venom peptide-encoding loci in the P. persimilis genome (GenBank Assembly: GCA_037576195.1) using NCBI BLASTn [Fig.2]. Sequences from the BLAST result are then aligned with the two N. barkeri mRNAs to filter out the introns. Thus, the protein sequence of the P. persimilis orthologous venom peptides are obtained.

Truncation & Optimizations

Looking into molecular features of the sequences of the mite venom peptides: small, cysteine-rich and involves multiple disulfide bridges, we realized the mite venom peptides to be homologous to spider venom peptides, a class of versatile, widely-studied venom peptides found in spiders—phylogenically related to the acariformes. With some research into spider venom peptides, we realized that the cysteine-rich venom peptides’ toxicity is largely determined by a core venomous domain, and that other domains are largely irrelevant to the inhibitory effects the venom has on its molecular targets (illustrated dramatically by the fact that many recombinant SVPs retain only their core venomous domain, yet retains and usually achieves toxicity higher than that of their native forms). Since the mite venom peptide protein sequences are obtained directly from the genome of P. persimilis, the protein likely include various domains or structures that facilitates the protein’s expression and post-translational modifications and secretion in vivo. Since they are largely unnecessary for heterologous expression in unicellular chassis such as E. coli or P. Pastoris, such regions, could be truncated to improve expression efficiency and toxicity. We therefore, matching structural prediction results of the MVPs and a described, recombinant SVP (truncated down to only the central venomous domain), rCtx4 [2], identified the MVP’s core venom domain.

in silico Analysis

With further research into spider venom peptides’ molecular mechanisms for their paralyzing and lethal effects, we realized that they (and therefore MVPs due to high levels of homology between them) target neuronal ion channels or receptors. Their interference led to blockage of various receptors or ion channels or neurotransmitter receptors, leading to prevention of release or detection of neurotransmitters. This therefore interferes with normal conduction of neural impulses and achieves paralyzing and lethal effects. To analyze molecular action of venom peptides using computational approaches such as molecular docking, we constructed a phylogenic tree based on core venomous domains of various SVPs with known molecular targets [3], [4], [5], [6] along with the four MVPs, in an attempt to determine the MVPs' specific molecular targets. The results, in [Fig4], suggests that they likely interfere with voltage-gated calcium channels. We therefore tried to predict molecular effects of the venom peptides against the CaV channels, using both “conventional” docking tools such as HDock server, and “non-classical” tools such as AlphaFold (for its ability to accurately predict conformational changes in both receptor and a peptide ligand upon binding). The docking results from both AlphaFold and HDock suggests that the MVPs are likely to interfere with the alpha 2 subunit of the T. urticae CaV channels, blocking flow of ions and therefore release of neurotransmitters.

Toxicity Assay

The designed and optimized venom peptides are expressed using the expression system G1M5-His-SUMO-MVP-GNA, where G1M5-SUMO had been proved to facilitate correct folding and therefore soluble expression of the attached protein [7][Fig.5A], and GNA, which enhances the fusion protein’s oral and contact toxicity [8]. Plasmids pET28a-G1M5-His-SUMO-MVP-GNA [Fig.5B] are assembled using GoldenGate cloning and is transformed into BL21(DE3) strain. After IPTG induction and overnight incubation, the culture is harvested and underwent cell lysis; SDS-PAGE then ran for supernatant and precipitate. The supernatant is then treated with SUMO protease [Fig.5B].

Supernatant from cell lysis is treated with SUMO protease to, ideally, give MVP-GNA fusion proteins. For toxicity assay, Professor Huang from SCAU tested the contact toxicity of SUMO-protease-treated supernatant on 3 groups of 20 females of Tetranychus urticae, which is the most prominent specie in spider mites [14]. The treated supernatant is applied to the spider mites using a spraying method, with supernatant of induced BL21(DE3) that underwent the same treatments acting as control. The results reveal that PpVP1S shows very high toxicity against T. urticae [Fig.6C&D], achieving a death rate of 98.25% in the first day and 100% in the next.

Part Collection

Our part collection provides a comprehensive list of venom peptides with a diverse range of molecular targets, and all displays satisfactory elimination efficacy during our testings [Fig7A&B].