An insecticidal peptide from the theraposid Brachypelma smithi spider venom reveals common molecular features among spider species from different genera

The soluble venom of the Mexican theraposid spider Brachypelma smithi was screened for insecticidal peptides based on toxicity to house crickets. An insecticidal peptide, named Bs1 (which stands for Brachypelma smithi toxin 1) was obtained in homogeneous form after the soluble venom was fractionated using reverse-phase and cation-exchange chromatography. It contains 41 amino acids cross-linked by three disulfide bridges. Its sequence is similar to an insecticidal peptide isolated from the theraposid spider Ornithoctonus huwena from China, and another from the hexathelid spider Macrothelegigas from Japan, indicating that they are phylogenetically related. A cDNA library was prepared from the venomous glands of B. smithi and the gene that code for Bs1 was cloned. Sequence analysis of the nucleotides of Bs1 showed similarities to that of the hexathelid spider from Japan proving additional evidence for close genetic relationship between these spider peptides. The mRNAs of these toxins code for signal peptides that are processed at the segment rich in acidic and basic residues. Their C-terminal amino acids are amidated. However, they contain only a glycine residue at the most C-terminal position, without the presence of additional basic amino acid residues, normally required for post-translation processing of other toxins reported in the literature. The possible mechanism of action of Bs1 was investigated using several ion channels as putative receptors. Bs1 had minor, but significant effects on the Para/tipE insect ion channel, which could indirectly correlate with the observed lethal activity to crickets.     

Identification and characterization of a novel antimicrobial peptide from the venom of the ant Tetramorium bicarinatum

A novel antimicrobial peptide, named Bicarinalin, has been isolated from the venom of the ant Tetramorium bicarinatum. Its amino acid sequence has been determined by de novo sequencing using mass spectrometry and by Edman degradation. Bicarinalin contained 20 amino acid residues and was C-terminally amidated as the majority of antimicrobial peptides isolated to date from insect venoms. Interestingly, this peptide had a linear structure and exhibited no meaningful similarity with any known peptides. Antibacterial activities against Staphylococcus aureus and S. xylosus strains were evaluated using a synthetic replicate. Bicarinalin had a potent and broad antibacterial activity of the same magnitude as Melittin and other hymenopteran antimicrobial peptides such as Pilosulin or Defensin. Moreover, this antimicrobial peptide has a weak hemolytic activity compared to Melittin on erythrocytes, suggesting potential for development into an anti-infective agent for use against emerging antibiotic-resistant pathogens.     

Purification and Amino Acid Sequence of MP-III 4R D49 Phospholipase A2 from Bothrops pirajai Snake Venom, a Toxin with Moderate PLA2 and Anticoagulant Activities and High Myotoxic Activity

MP-III 4R PLA₂ was purified from the venom of Bothrops pirajai venom (Bahia's jararacussu) after three chromatographic steps which started with RP-HPLC. The complete amino acid sequence of MP-III 4R PLA₂ from Bothrops pirajai was determined by amino acid sequencing of reduced and carboxymethylated MP-III 4R and the isolated peptides from clostripain and protease V8 digestion. MP-III 4R is a D49 PLA₂ with 121 amino acid residues and has a molecular weight estimated at 13,800 Da, with 14 half-cysteines. This protein showed moderate PLA₂ and anticoagulant activity. This PLA₂ does not have a high degree of homology with other bothropic PLA₂-like myotoxins (~75%) and nonbothropic myotoxins (~60%). MP-III 4R is a new PLA₂, which was isolated using exclusively analytical and preparative HPLC methods. Based on the N-terminal sequence and biological activities, MP-III 4R was identified as similar to piratoxin-III (PrTX-III), which was isolated by conventional chromatography based on molecular exclusion ion exchange chromatography. Clinical manifestations indicate that at the site of toxin injection, there may be pain of variable intensity, because animals continue to lick the limb. No clinical sign indicating general toxicity was noticed. Myotoxicity was observed in gastrocnemius muscle cells after exposure to MP-III 4R, with a high frequency (70%) of affected muscle fibers.     

A novel class of antimicrobial peptides from the scorpion Heterometrus spinifer

The venom peptides from the scorpion Heterometrus spinifer have been poorly characterized so far. Here, we identified a novel class of antimicrobial peptides from the venom gland of H. spinifer, which were referred to as HsAp, HsAp2, HsAp3 and HsAp4, respectively. Each of the four peptides consists of 29 amino acid residues, and is cationic and weakly amphipathic. They display no significant homology to any other known peptides, and thus represent a new family of venom peptides from scorpions. Antimicrobial assay showed that HsAp is able to inhibit the growth of both Gram-negative and Gram-positive bacteria with the MIC values of 11.8–51.2 μM. HsAp is also able to inhibit the growth of the tested fungus. Genomic analysis indicated that the genes of all the four peptides are intronless. Our studies expand the families of antimicrobial peptides from scorpions.     

Functional characterizations of residues Arg-158 and Tyr-170 of the mosquito-larvicidal Bacillus thuringiensis Cry4Ba

The insecticidal activity of Bacillus thuringiensis (Bt) Cry toxins involves toxin stabilization, oligomerization, passage across the peritrophic membrane (PM), binding to midgut receptors and pore-formation. The residues Arg-158 and Tyr-170 have been shown to be crucial for the toxicity of Bt Cry4Ba. We characterized the biological function of these residues. In mosquito larvae, the mutants R158A/E/Q (R158) could hardly penetrate the PM due to a significantly reduced ability to alter PM permeability; the mutant Y170A, however, could pass through the PM, but degraded in the space between the PM and the midgut epithelium. Further characterization by oligomerization demonstrated that Arg-158 mutants failed to form correctly sized high-molecular weight oligomers. This is the first report that Arg-158 plays a role in the formation of Cry4Ba oligomers, which are essential for toxin passage across the PM. Tyr-170, meanwhile, is involved in toxin stabilization in the toxic mechanism of Cry4Ba in mosquito larvae. [BMB Reports 2014; 47(10): 546-551]     

Molecular cloning and antibacterial activity of bombolitin isolated from the venom of a bumblebee, Bombus terrestris

Three major components of bumblebee venom are bombolitin, phospholipase A₂, and a serine protease, with bombolitin being the most abundant. Here, we describe the molecular cloning of bombolitin isolated from the venom of a bumblebee, Bombus terrestris, and demonstrate its antibacterial activity. The B. terrestris bombolitin gene consists of 2 exons encoding 56 amino acid residues. Comparative analysis shows that mature B. terrestris bombolitin consists of 18 amino acid residues, which are identical to those of B. ignitus bombolitin. B. terrestris bombolitin displayed antibacterial activity against both the Gram-negative bacterium Klebsiella pneumoniae and the Gram-positive bacterium Staphylococcus aureus, indicating that B. terrestris bombolitin may be a potential antimicrobial agent.     

Purification, characterization and cytokine release function of a novel Arg-49 phospholipase A(2) from the venom of Protobothrops mucrosquamatus

Group IIA phospholipase A(2) (PLA(2)) are major components in Viperidae/Crotalidae venom. In the present study, a novel PLA(2) named promutoxin with Arg at the site 49 has been purified from the venom of Protobothrops mucrosquamatus by chromatography. It consists of 122 amino acid residues with a molecular mass of 13,656 Da assessed by MALDI-TOF. It has the structural features of snake venom group IIA PLA(2)s, but has no PLA(2) enzymatic activity. Promutoxin shows higher amino acid sequence identity to the K49 PLA(2)s (72-95%) than to D49 PLA(2)s (52-58%). Promutoxin exhibits potent myotoxicity in the animal model with as little as 1 microg of promutoxin causing myonecrosis and myoedema in the gastrocnemius muscle of mice. Promutoxin is also able to stimulate the release of IL-12, TNFalpha, IL-6 and IL-1beta from human monocytes, and induce IL-2, TNFalpha and IL-6 release from T cells, indicating that this snake venom group IIA PLA(2) is actively involved in the inflammatory process in man caused by snake venom poisoning.     

Oxyopinins, large amphipathic peptides isolated from the venom of the wolf spider Oxyopes kitabensis with cytolytic properties and positive insecticidal cooperativity with spider neurotoxins

Five amphipathic peptides with antimicrobial, hemolytic, and insecticidal activity were isolated from the crude venom of the wolf spider Oxyopes kitabensis. The peptides, named oxyopinins, are the largest linear cationic amphipathic peptides from the venom of a spider that have been chemically characterized at present. According to their primary structure Oxyopinin 1 is composed of 48 amino acid residues showing extended sequence similarity to the ant insecticidal peptide ponericinL2 and to the frog antimicrobial peptide dermaseptin. Oxyopinins 2a, 2b, 2c, and 2d have highly similar sequences. At least 27 out of 37 amino acid residues are conserved. They also show a segment of sequence similar to ponericinL2. Circular dichroism analyses showed that the secondary structure of the five peptides is essentially alpha-helical. Oxyopinins showed disrupting activities toward both biological membranes and artificial vesicles, particularly to those rich in phosphatidylcholine. Electrophysiological recordings performed on insect cells (Sf9) showed that the oxyopinins produce a drastic reduction of cell membrane resistance by opening non-selective ion channels. Additionally, a new paralytic neurotoxin named Oxytoxin 1 was purified from the same spider venom. It contains 69 amino acid residue cross-linked by five disulfide bridges. Application of mixtures containing oxyopinins and Oxytoxin 1 to insect larvae showed a potentiation phenomenon, by which an increase lethality effect is observed. These results suggest that the linear amphipathic peptides in spider venoms and neuropeptides cooperate to capture insects efficiently.     

Isolation of an acidic phospholipase A2 from the venom of the snake Bothrops asper of Costa Rica: Biochemical and toxicological characterization

Phospholipases A₂ (PLA₂) are major components of snake venoms, exerting a variety of relevant toxic actions such as neurotoxicity and myotoxicity, among others. Since the majority of toxic PLA₂s are basic proteins, acidic isoforms and their possible roles in venoms are less understood. In this study, an acidic enzyme (BaspPLA₂-II) was isolated from the venom of Bothrops asper (Pacific region of Costa Rica) and characterized. BaspPLA₂-II is monomeric, with a mass of 14,212 ± 6 Da and a pI of 4.9. Its complete sequence of 124 amino acids was deduced through cDNA and protein sequencing, showing that it belongs to the Asp49 group of catalytically active enzymes. In vivo and in vitro assays demonstrated that BaspPLA₂-II, in contrast to the basic Asp49 counterparts present in the same venom, lacks myotoxic, cytotoxic, and anticoagulant activities. BaspPLA₂-II also differed from other acidic PLA₂s described in Bothrops spp. venoms, as it did not show hypotensive and anti-platelet aggregation activities. Furthermore, this enzyme was not lethal to mice at intravenous doses up to 100 μg (5.9 μg/g), indicating its lack of neurotoxic activity. The only toxic effect recorded in vivo was a moderate induction of local edema. Therefore, the toxicological characteristics of BaspPLA₂-II suggest that it does not play a key role in the pathophysiology of envenomings by B. asper, and that its purpose might be restricted to digestive functions. Immunochemical analyses using antibodies raised against BaspPLA₂-II revealed that acidic and basic PLA₂s form two different antigenic groups in B. asper venom.     

Isolation,enzymatic characterization and antiedematogenic activity of the first reported rattlesnake hyaluronidase from Crotalus durissus terrificus venom

A hyaluronidase (CdtHya1) from Crotalus durissus terrificus snake venom (CdtV) was isolated and showed to exhibit a high activity on hyaluronan cleavage. However, surveys on this enzyme are still limited. This study aimed at its isolation, functional/structural characterization and the evaluation of its effect on the spreading of crotoxin and phospholipase A₂ (PLA₂). The enzyme was purified through cation exchange, gel filtration and hydrophobic chromatography. After that, it was submitted to a reverse-phase fast protein liquid chromatography (RP-FPLC) and Edman degradation sequencing, which showed the first N-terminal 44 amino acid residues whose sequence evidenced identity with other snake venom hyaluronidases. CdtHya1 is a monomeric glycoprotein of 64.5 kDa estimated by SDS-PAGE under reducing conditions. It exhibited maximum activity in the presence of 0.2 M NaCl, at 37 °C, pH 5.5 and a specificity to hyaluronan higher than that to chondroitin-4-sulphate, chondroitin-6-sulphate or dermatan. Divalent cations (Ca²⁺ and Mg²⁺) and 1 M NaCl significantly reduced the enzyme activity. The specific activity of CdtHya1 was 5066 turbidity reducing units (TRU)/mg, against 145 TRU/mg for the soluble venom, representing a 34.9-fold purification. The pure enzyme increased the diffusion of crotoxin and PLA₂ through mice tissues. CdtHya1 (32 TRU/40 μL) potentiated crotoxin action, as evidenced by mice death, and it decreased the oedema caused by subplantar injections of buffer, crotoxin or PLA₂, thus evidencing the relevance of hyaluronidase in the crotalic envenoming. This work yielded a highly active antiedematogenic hyaluronidase from CdtV, the first one isolated from rattlesnake venoms.     

Purification and inflammatory edema induced by two PLA2 (Anch TX-I and Anch TX-II) from sea anemone Anthothoe chilensis (Actiniaria: Sagartiidae)

The Anch TX-I and II PLA₂ were purified from Anthothoe chilensis (Lesson, 1830) from the extract of the anemone after only two chromatographic step using molecular exclusion chromatography (Sephadex G-75) and reverse phase HPLC on μ-Bondapak C18 column. Both PLA₂ showed a molecular mass of ~ 14 kDa determined by MALDI-TOF mass spectrometry and showed a high catalytic activity (data not showed). Although homologous with mammalian or snake venom group I PLA₂s, Anch TX-I and II is sufficiently structurally different for the question of its placement into the existing PLA₂ classification scheme to arise. In addition, Anch TX-I and II, despite possessing many common structural features, also differ in some important structural properties. The amino acid sequence of both PLA₂ (Anch TX-I and III) showed high amino acid sequence identity with PLA₂Rhopilema nomadica and Bunodosoma caissarum Cnidaria and PLA₂ of group III protein isolated from the Mexican lizard Heloderma horridum horridum and Heloderma suspectum. In addition, Anch TX-I and Anch TX-II showed high amino acid sequence identity with PLA₂ from group III also showed significant overall homology to bee Apis dorsata, Bombus terrestris and Bombus pennsylvanicus and PLA₂. We also investigated the in vivo edematogenic activity of Anch TX-I and Anch TX-II in a model of paw and skin edema in rats and observed that both are able to induce dose-dependent edema.     

Insecticidal Toxic Proteins Produced by Photorhabdus luminescens akhurstii,a Symbiont of Heterorhabditis indica

We describe the isolation and characterization of an insect pathogenic bacterium from the entomopathogenic nematode Heterorhabditis indica (Karnataka strain), an isolate from the southern regions of India. The strain has been identified and characterized by phenotypic, biochemical tests and PCR-RFLP analysis of the 16S rRNA gene as Photorhabdus luminescens subsp. akhurstii. The insecticidal toxin complex produced by this bacterium has been purified through a series of steps including ultrafiltration, anion exchange chromatography, and gel filtration chromatography. The toxin consists of two protein complexes of approximately 1,000 kD and was active against the larvae of Spodoptera litura and Galleria mellonella.     

Evolutionary patchwork of an insecticidal toxin shared between plant-associated pseudomonads and the insect pathogens Photorhabdus and Xenorhabdus

Background

Root-colonizing fluorescent pseudomonads are known for their excellent abilities to protect plants against soil-borne fungal pathogens. Some of these bacteria produce an insecticidal toxin (Fit) suggesting that they may exploit insect hosts as a secondary niche. However, the ecological relevance of insect toxicity and the mechanisms driving the evolution of toxin production remain puzzling.

Results

Screening a large collection of plant-associated pseudomonads for insecticidal activity and presence of the Fit toxin revealed that Fit is highly indicative of insecticidal activity and predicts that Pseudomonas protegens and P. chlororaphis are exclusive Fit producers. A comparative evolutionary analysis of Fit toxin-producing Pseudomonas including the insect-pathogenic bacteria Photorhabdus and Xenorhadus, which produce the Fit related Mcf toxin, showed that fit genes are part of a dynamic genomic region with substantial presence/absence polymorphism and local variation in GC base composition. The patchy distribution and phylogenetic incongruence of fit genes indicate that the Fit cluster evolved via horizontal transfer, followed by functional integration of vertically transmitted genes, generating a unique Pseudomonas-specific insect toxin cluster.

Conclusions

Our findings suggest that multiple independent evolutionary events led to formation of at least three versions of the Mcf/Fit toxin highlighting the dynamic nature of insect toxin evolution.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1763-2) contains supplementary material, which is available to authorized users.     

Intraspecific variation of the crotamine and crotasin genes in Crotalus durissus rattlesnakes

Crotamine is a small basic myotoxin peptide of Crotalus durissus venom, with β-defensin scafold and variable concentration in individual venoms. The crotamine gene was mapped to the end of chromosome 2 and the signal intensity differed significantly between the two homologues. In contrast to crotamine, the paralogous crotasin gene is scarcely expressed in the venom glands. In this study, we analyzed the crotamine concentrations in the venoms of a total of 23 rattlesnakes from diverse Brazilian localities by ELISA as well as the copy number of both crotamine and crotasin genes by real-time PCR. Crotamine was found to constitute 5–29% of venom proteins varying greatly among individual animals. The crotamine gene exists from 1 to 32 copies per haploid genome, whereas the crotasin gene is present from 1 to 7 copies. Furthermore, we observed that the crotamine concentration and crotamine gene copy number are positively correlated (r² = 0.68), implying the variation of crotamine in venom results from the variation of the gene copy number. Sequencing of 50 independent copies of crotamine and crotasin genes from four different rattlesnakes revealed the presence of six crotasin isoforms with a single amino acid difference from the original crotasin sequence, whereas only two additional crotamine isoforms were observed. Taken together, our results suggested that after duplication from a common ancestor gene, crotamine and crotasin may have diverged in such a way that the crotamine gene underwent repetitive duplication to increase its copy number, whereas the crotasin gene diversified its sequence.     

Single concentration tests show synergism among Bacillus thuringiensis subsp. israelensis toxins against the malaria vector mosquito Anophelesalbimanus

Bioassays of insecticidal proteins from Bacillus thuringiensis subsp. israelensis with larvae of the malaria vector mosquito Anophelesalbimanus showed that the cytolytic protein Cyt1Aa was not toxic alone, but it increased the toxicity of the crystalline proteins Cry4Ba and Cry11Aa. Synergism also occurred between Cry4Ba and Cry11Aa toxins. Whereas many previous analyses of synergism have been based on a series of toxin concentrations leading to comparisons between expected and observed values for the concentration killing 50% of insects tested (LC₅₀), we describe and apply a method here that enables testing for synergism based on single concentrations of toxins.     

Three dimensional (3D) structure prediction and substrate-protein interaction study of the chitin binding protein CBP24 from B. thuringiensis

Bacillus thuringiensis is an insecticidal bacterium whose chitinolytic system has been exploited to improve insect resistance in crops. In the present study, we studied the CBP24 from B. thuringiensis using homology modeling and molecular docking. The primary and secondary structure analyses showed CBP24 is a positively charged protein and contains single domain that belongs to family CBM33. The 3D model after refinement was used to explore the chitin binding characteristics of CBP24 using AUTODOCK. The docking analyses have shown that the surface exposed hydrophilic amino acid residues Thr-103, Lys-112 and Ser-162 interact with substrate through H-bonding. While, the amino acids resides Glu-39, Tyr-46, Ser-104 and Asn-109 were shown to have polar interactions with the substrate. The binding energy values evaluation of docking depicts a stable intermolecular conformation of the docked complex. The functional characterization of the CBP24 will elucidate the substrate-interaction pathway of the protein in specific and the carbohydrate binding proteins in general leading towards the exploration and exploitation of the prokaryotic substrate utilization pathways.     

Co-refolding of two peptide fragments derived from Agrobacterium tumefaciens beta-glucosidase with catalytic activity

Four sites of the non-homologous region (coding amino acid residues of 347, 421, 466 and 533) of a gene were randomly selected for splitting to investigate the function of β-glucosidase from Agrobacterium tumefaciens in the co-refolding of peptides into the catalytically active enzyme. As a result of gene splitting, four N- and C-terminal domain peptides were obtained as insoluble inclusion bodies. No catalytic activity was observed when these fragments refolded individually. However, a considerable amount of activity was restored when the two fragments derived from N- and C- terminal peptides were co-refolded together. The deletion of amino acid residues in the non-homologous region resulted in a complete loss of enzyme activity, which suggests that truncation of amino acids in this region strongly affects the co-refolding ability of the enzyme to maintain activity.     

A novel phospholipase A2 from Agkistrodon blomhoffii ussurensis venom: Purification,proteomic, functional and structural characterizations

A phospholipase A₂ was isolated from the snake venom of Chinese Agkistrodon blomhoffii Ussurensis by column chromatography using DEAE Sephadex A-50 ion-exchange chromatography, Sephadex G-75 gel filtration chromatography and Mono Q ion-exchange chromatography, and designated as Akbu-PLA₂. It showed an average molecular mass of 13,980 ± 3 amu determined by MALDI TOF mass spectrometry. Protein identification results from HPLC-nESI-MS/MS analysis indicated that the Akbu-PLA₂ was a new snake venom acidic PLA₂. Seven peptides were sequenced from Akbu-PLA₂ by HPLC-nESI-MS/MS analysis. Sequencing alignment indicated that Akbu-PLA₂ shared homolog peptides of phospholipases A₂ from the venoms of Gloydius ussurensis, Gloydius halys, Gloydius halys (halys viper), Deinagkistrodon acutus and Agkistrodon halys Pallas. Akbu-PLA₂ has an optimum hydrolytic activity temperature of ∼45 °C. The intrinsic fluorescences of Tyr and Trp residues of Akbu-PLA₂ showed emission wavelengths red-shifted by 13.6 and 1.6 nm from those of free Tyr and Trp, respectively. Akbu-PLA₂ was shown to contain one Ca²⁺ per monomer by ICP-AES measurement. The Ca²⁺ ion was found to be critical for both the hydrolytic activity and the structure of Akbu-PLA₂. Ca²⁺ increased the emission fluorescence intensity and the hydrophobicity of the environment of Akbu-PLA₂. The hydrolytic activity of Akbu-PLA₂ was accelerated due to the addition of Ca²⁺ ion by enhancing the substrate binding. However, a protein component with the molecular weight two-fold relative to that of Akbu-PLA₂ was found to be difficult to eliminate for the purification of Akbu-PLA₂. HPLC-nESI-MS/MS detected the same peptides from it as from Abku-PLA₂, which indicated that it should be a homodimer of Akbu-PLA₂. A proteomic approach, 2D SDS-PAGE coupled to HPLC-nESI-MS/MS, supported the co-existence of the Akbu-PLA₂ monomer and dimer in the crude snake venom. Results from the combination of phosphoprotein and glycoprotein specific stains combined with the HPLC-nESI-MS/MS method indicated that both the Akbu-PLA₂ monomer and dimer were both phosphorylated and glycosylated. The addition of exogenous Ca²⁺ ion was found to be able to promote the dimer formation of Akbu-PLA₂. We conclude that a novel PLA₂ was successfully obtained. The systemically biochemical, proteomic, structural and functional characterization results from Akbu-PLA₂ reveal new threads and provide valuable inputs for the study of snake venom phospholipases A₂.     

Molecular cloning and characterization of a venom phospholipase A2 from the bumblebee Bombus ignitus

Phospholipase A₂ (PLA₂) is one of the main components of bee venom. Here, we identify a venom PLA₂ from the bumblebee, Bombus ignitus. Bumblebee venom PLA₂ (Bi-PLA₂) cDNA, which was identified by searching B. ignitus venom gland expressed sequence tags, encodes a 180 amino acid protein. Comparison of the genomic sequence with the cDNA sequence revealed the presence of four exons and three introns in the Bi-PLA₂ gene. Bi-PLA₂ is an 18-kDa glycoprotein. It is expressed in the venom gland, cleaved between the residues Arg44 and Ile45, and then stored in the venom sac. Comparative analysis revealed that the mature Bi-PLA₂ (136 amino acids) possesses features consistent with other bee PLA₂s, including ten conserved cysteine residues, as well as a highly conserved Ca²⁺-binding site and active site. Phylogenetic analysis of bee PLA₂s separated the bumblebee and honeybee PLA₂ proteins into two groups. The mature Bi-PLA₂ purified from the venom of B. ignitus worker bees hydrolyzed DBPC, a known substrate of PLA₂. Immunofluorescence staining of Bi-PLA₂-treated insect Sf9 cells revealed that Bi-PLA₂ binds at the cell membrane and induces apoptotic cell death.