Part:BBa_K5184043

PpVP2-S

Essential Information

Abstract

This part presents an original, novel mite venom peptide with high potency against spider mites (Acariformes: Tetranychidae). Named PpVP2S, this part was obtained from genome mining results by our own team, and has the effects assessed both in silico and in vivo. Amongst the very first ever identified from the acariforme family, this part will also provide the foundation for identification of homologous venom peptides in the enormous, diverse family of acarids.

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 integrates these parts to guarantee correct folding of venom peptides with high levels of 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. Such a collection allows cooperative-combination of multiple venom peptides that together affect multiple molecular target sites, significantly reducing chance of resistance development. 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 rapidly developing resistance and, as template sequences for identification of other acariforme venom peptides.

Basic Characterization

PpVP2S 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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Characterization

Molecular Structure

PpVP2S is the truncated version of the PpVP2F, composing of a total of 55 amino acids, containing 8 cysteine residues that form 4 disulfide bridges, and two antiparallel beta sheets, together contributing to the final highly compact conformation of the venom. Comparing to NbVP2[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.

Fig.1: A. Disulfide network of the venomous domain of PpVP2S, involving 8 cysteine residues and 4 disulfide bridges B. Comparison of structural prediction results of core venomous domains of NbVP2F (transparent light gray) and PpVP2F (solid blue) from AlphaFold Server; the cysteine residues are colored orange

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.

Fig.2: BLAST results of two N. barkeri venom peptide mRNAs against the P. persimilis genome.

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 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 (illlustrated dramatically by the fact that many recombinant SVPs retain only their core venmous 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.<

Fig.3: Structural comparisons between AlphaFold Structural prediction results of PpVP2F (White and blue), and rCtx4 (transparent light grey); the identified core venomous domain of PpVP2F is colored in blue and the cysteine residues colored orange

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 their molecular targets. The results, in [Fig4], suggests that they likely interfere with voltage-gated calcium channels and potentially calcium-activated potassium channels.
We therefore tried to predict molecular effects of the venom peptides against CaV and KCa 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.

Fig.4: A. Phylogenic tree from core venomous domains of MVPs and several SVPs with known molecular targets (all ion channels, ion type identified at the end of each name) B. Docking results of PpVP2S against T. urticae voltage-gated calcium channel using AlphaFold

Safety

Venom peptides' ability to inhibit ion channels extremely efficiently originates from the fact that they can bind or associate closely to their receptors. Their paralyzing or even lethal activity is therefore highly dependent on conformations of the receptor (i.e. Ion channel or neurotransmitter receptor).
Given the fact that PpVP2S originates from a predatory mite specie who feeds completely on non-mammalian preys, the structure of the peptide will be highly selected for association with the prey (i.e. Spider mites) molecular targets, and therefore, ineffectual towards similar structures in species other than spider mites.
As suggested by [9], insect CaV channels vary greatly to mammalian calcium channels due to both a much smaller repertoire of subunits and low level of homology (<68%), and that varies to a moderate degree between insect orders. This means that PpVP2S, targetting voltage-gated-calcium channels, is highly unlikely to be poisonous towards mammals (as demonstrated by the fact that 10000 tine of some CaV-targetting venom peptides' lethal dose for insects may not display even slightest toxicity against mammals) and evolutionarily more distant beneficial insects.

Toxicity Assay

The designed and optimized venom peptides are expressed using the expression system G1M5-His-SUMO-MVP-GNA, where the G1M5-SUMO on the N-terminus is proved to facilitate correct folding and therefore soluble expression of the attached protein [7][Fig.5A], and the C-terminus GNA significantly enhances fusion protein’s oral and contact toxicity [8].
Plasmids pET28a-G1M5-His-SUMO-MVP-GNA are assembled using GoldenGate cloning, and after verification by colony PCR and sequencing[Fig6A&B], is transformed into BL21(DE3) strain. After IPTG induction and overnight incubation, the culture is harvested and underwent sonication cell lysis; SDS-PAGE was then ran for supernatant and precipitate samples, verifying soluble expression of the recombined PpVP2S using our current expression strategies.

Fig5: A. G1M5 signal peptide meditates extracellular secretion of the fusion protein B. Plasmid construct pET28a-G1M5-His-SUMO-PpVP2S-GNA-His C. SDS-PAGE of SUMO-digested supernatant of PpVP2S, with supernatant of BL21(DE3) as control; S: supernatant, SUMO: SUMO protease-treated supernatant Fig6: A. Colony PCR results of the pET28a-G1M5-His-SUMO-PpVP2S-GNA-His B. Alignment of sequencing results of colony PCR products C. SDS-PAGE for supernatant and SUMO-treated supernatant of PpVP2S, with supernatant of BL21(DE3) as control

Supernatant after cell lysis is then treated with SUMO protease remove the SUMO tag, leaving behind only MVP-GNA fusion proteins[Fig5B]. The treated supernatant is sent to Professor Huang of SCAU for toxicity bioassay, where he tested the contact toxicity of SUMO-protease-treated supernatant on 3 groups of 20 females of Tetranychus urticae, the most prominent species in spider mites [14].
The supernatant is applied to the spider mites using a spraying method once only on day 1, and the three groups of T. urticae mites' lethality is assessed at 24, 48, and 72 hours after spraying. For control, supernatant of BL21(DE3) is used. The results reveal that PpVP2S shows very high toxicity against T. urticae [Fig7C&D], achieving a death rate of 98.25% in the first day and 100% in the next.

Fig6: A. T. urticae before being sprayed with PpVP2S B. T. urticae after being sprayed with PpVP2S C. Survival plot of PpVP2S against female T. urticae using a spraying method, CK is induced liquid culture of BL21(DE3), of which acts as control D. Lethality data of PpVP2S over 24, 48, and 72 hours, CK is induced liquid culture of BL21(DE3), of which acts as control; data is the means of ± SD of three parallel replicate experiments

Part Collection

Our part collection provides a comprehensive list of venom peptides with a diverse range of molecular targets, which suppresses the development of drug resistance, and thus ensuring the efficacy of our acaricide. Through the incorporation of a G1M5-SUMO tag, all of the venom peptides are successfully expressed and digested by SUMO protease. According to the toxicity bioassay, all of the venom peptides display significant contact efficacy[Fig8A&B], especially PpVP2S, a novel MVP that we discovered by ourselves through genome mining. In future applications, we hope that our collection of venom peptides will not only be highly effective, but also solve the problem of drug resistance development that traditional pesticides always encounter.

Fig8: A. Survival plot of 6 venom peptides against female T. urticae using a spraying method, CK is induced liquid culture of BL21(DE3), of which acts as control D. Lethality data of 6 venom peptides over 24, 48, and 72 hours, CK is induced liquid culture of BL21(DE3), of which acts as control; data is the means of ± SD of three parallel replicate experiments

Our Part Collection

Current VP	Venom Name	Targeted Ion Channel	New?	Part Number	Original Specie
✳️	PpVP2S	Ca	New	BBa_K5184043	Phytoseiulus persimilis
	PpVP1S	Ca	New	BBa_K5184042	Phytoseiulus persimilis
	PpVP1F	Ca	New	BBa_K5184038	Phytoseiulus persimilis
	rCtx4	Na		BBa_K5184021	Phoneutria depilata
	Cs1A	Ca		BBa_K5184032	Calommata signata
	HxTx-Hv1h	Ca, K		BBa_K5184033	Hadronyche versuta