Expression of Brown and Southern Black Widow Spider (Araneae: Theridiidae) Latrotoxins Is Tissue- and Life Stage-Specific for α-Latroinsectotoxins and δ-Latroinsectotoxins and Is Ubiquitous for α-Latrotoxins.

Widow spiders are widely known for their potent venom toxins that make them among the few spiders of medical concern. The latrotoxins are the most well-studied widow toxins and include both the vertebrate-specific latrotoxins and the insect-specific latroinsectotoxins (LITs). Previous studies have shown that toxins are not limited to expression in the venom glands of adult spiders; however, gaps exist in latrotoxin screening across all life stages for brown widows, Latrodectus geometricus and southern black widows, Latrodectus mactans. In this study, we screened male and female venom gland, cephalothorax, and abdomen tissues, spiderling cephalothorax and abdomen tissues, and eggs of both L. geometricus and L. mactans, for the presence of three latrotoxins: α-latrotoxin (α-LTX), and α- and δ-latroinsectotoxins (α/δ-LITs). Widows were locally collected. Extracted RNA was used to prepare cDNA that was analyzed by PCR for the presence or absence of latrotoxin expression. Results show that expression profiles between the two species are very similar but not identical. Expression of α-LTX was found in all life stages in all tissues examined for both species. For both species, no LIT expression was detected in eggs and variable patterns of α-LIT expression were detected in spiderlings and adults. Notably, δ-LIT could only be detected in females for both species. Our results show that latrotoxin expression profiles differ within and between widow species. Data on their expression distribution provide further insight into the specific latrotoxins that contribute to toxicity profiles for each life stage in each species and their specific role in widow biology.

Molecular architecture of black widow spider neurotoxins.

Latrotoxins (LaTXs) are presynaptic pore-forming neurotoxins found in the venom of Latrodectus spiders. The venom contains a toxic cocktail of seven LaTXs, with one of them targeting vertebrates (α-latrotoxin (α-LTX)), five specialized on insects (α, ß, γ, δ, ε- latroinsectotoxins (LITs), and one on crustaceans (α-latrocrustatoxin (α-LCT)). LaTXs bind to specific receptors on the surface of neuronal cells, inducing the release of neurotransmitters either by directly stimulating exocytosis or by forming Ca2+-conductive tetrameric pores in the membrane. Despite extensive studies in the past decades, a high-resolution structure of a LaTX is not yet available and the precise mechanism of LaTX action remains unclear. Here, we report cryoEM structures of the α-LCT monomer and the δ-LIT dimer. The structures reveal that LaTXs are organized in four domains. A C-terminal domain of ankyrin-like repeats shields a central membrane insertion domain of six parallel α-helices. Both domains are flexibly linked via an N-terminal α-helical domain and a small ß-sheet domain. A comparison between the structures suggests that oligomerization involves major conformational changes in LaTXs with longer C-terminal domains. Based on our data we propose a cyclic mechanism of oligomerization, taking place prior membrane insertion. Both recombinant α-LCT and δ-LIT form channels in artificial membrane bilayers, that are stabilized by Ca2+ ions and allow calcium flux at negative membrane potentials. Our comparative analysis between α-LCT and δ-LIT provides first crucial insights towards understanding the molecular mechanism of the LaTX family.

Latrodectus Facies After Latrodectus Hesperus Envenomation in a Pediatric Patient.

BACKGROUND: Black widow spider (Latrodectus spp) envenomation represents the most medically significant spider envenomation in the United States, prompting more than 2500 calls to poison centers annually. The female spider, which is responsible for symptomatic envenomations, is classically described as a shiny black spider with a red hourglass-shaped marking on the ventral abdomen. Clinical features of envenomation include painful muscle cramping, abdominal pain, and autonomic disturbances, such as tachycardia, hypertension, and diaphoresis. "Latrodectus facies" or "facies latrodectismica" is an additional distinctive but rarely described clinical finding characterized by periorbital edema, lacrimation, and blepharospasm. CASE REPORT: A 6-year-old female developed the typical clinical features of Latrodectus envenomation after being found in bed with a Western black widow spider (Latrodectus hesperus) with no ventral marking. She initially improved with opioid analgesia, but 6 h later her symptoms worsened again, and concurrent with this worsening she developed Latrodectus facies. She received additional opioid analgesia and all her symptoms resolved within 24 h. Her mother provided informed and written consent for the acquisition and publication of the facial photographs presented. WHY SHOULD AN EMERGENCY PHYSICIAN BE AWARE OF THIS?: A high degree of clinical suspicion is necessary to correctly diagnose Latrodectus envenomation, especially when the spider escapes unnoticed or in young children in whom the bite is not witnessed. To our knowledge, Latrodectus facies has not been reported previously in a young child, and recognition of this finding will aid clinicians in limiting unnecessary interventions and administering appropriate therapy.

Treatment of chronic pain by designer cells controlled by spearmint aromatherapy.

Current treatment options for chronic pain are often associated with dose-limiting toxicities, or lead to drug tolerance or addiction. Here, we describe a pain management strategy, based on cell-engineering principles and inspired by synthetic biology, consisting of microencapsulated human designer cells that produce huwentoxin-IV (a safe and potent analgesic peptide that selectively inhibits the pain-triggering voltage-gated sodium channel NaV1.7) in response to volatile spearmint aroma and in a dose-dependent manner. Spearmint sensitivity was achieved by ectopic expression of the R-carvone-responsive olfactory receptor OR1A1 rewired via an artificial G-protein deflector to induce the expression of a secretion-engineered and stabilized huwentoxin-IV variant. In a model of chronic inflammatory and neuropathic pain, mice bearing the designer cells showed reduced pain-associated behaviour on oral intake or inhalation-based intake of spearmint essential oil, and absence of cardiovascular, immunogenic and behavioural side effects. Our proof-of-principle findings indicate that therapies based on engineered cells can achieve robust, tunable and on-demand analgesia for the long-term management of chronic pain.

Expression and immunological cross-reactivity of LALP3, a novel astacin-like metalloprotease from brown spider (Loxosceles intermedia) venom.

Loxosceles spiders' venom comprises a complex mixture of biologically active toxins, mostly consisting of low molecular mass components (2-40 kDa). Amongst, isoforms of astacin-like metalloproteases were identified through transcriptome and proteome analyses. Only LALP1 (Loxosceles Astacin-Like protease 1) has been characterized. Herein, we characterized LALP3 as a novel recombinant astacin-like metalloprotease isoform from Loxosceles intermedia venom. LALP3 cDNA was cloned in pET-SUMO vector, and its soluble heterologous expression was performed using a SUMO tag added to LALP3 to achieve solubility in Escherichia coli SHuffle T7 Express LysY cells, which express the disulfide bond isomerase DsbC. Protein purification was conducted by Ni-NTA Agarose resin and assayed for purity by SDS-PAGE under reducing conditions. Immunoblotting analyses were performed with specific antibodies recognizing LALP1 and whole venom. Western blotting showed linear epitopes from recombinant LALP3 that cross-reacted with LALP1, and dot blotting revealed conformational epitopes with native venom astacins. Mass spectrometry analysis revealed that the recombinant expressed protein is an astacin-like metalloprotease from L. intermedia venom. Furthermore, molecular modeling of LALP3 revealed that this isoform contains the zinc binding and Met-turn motifs, forming the active site, as has been observed in astacins. These data confirmed that LALP3, which was successfully obtained by heterologous expression using a prokaryote system, is a new astacin-like metalloprotease isoform present in L. intermedia venom.

Gene structure, regulatory control, and evolution of black widow venom latrotoxins.

Black widow venom contains α-latrotoxin, infamous for causing intense pain. Combining 33 kb of Latrodectus hesperus genomic DNA with RNA-Seq, we characterized the α-latrotoxin gene and discovered a paralog, 4.5 kb downstream. Both paralogs exhibit venom gland specific transcription, and may be regulated post-transcriptionally via musashi-like proteins. A 4 kb intron interrupts the α-latrotoxin coding sequence, while a 10 kb intron in the 3' UTR of the paralog may cause non-sense-mediated decay. Phylogenetic analysis confirms these divergent latrotoxins diversified through recent tandem gene duplications. Thus, latrotoxin genes have more complex structures, regulatory controls, and sequence diversity than previously proposed.

Dramatic expansion of the black widow toxin arsenal uncovered by multi-tissue transcriptomics and venom proteomics.

BACKGROUND: Animal venoms attract enormous interest given their potential for pharmacological discovery and understanding the evolution of natural chemistries. Next-generation transcriptomics and proteomics provide unparalleled, but underexploited, capabilities for venom characterization. We combined multi-tissue RNA-Seq with mass spectrometry and bioinformatic analyses to determine venom gland specific transcripts and venom proteins from the Western black widow spider (Latrodectus hesperus) and investigated their evolution. RESULTS: We estimated expression of 97,217 L. hesperus transcripts in venom glands relative to silk and cephalothorax tissues. We identified 695 venom gland specific transcripts (VSTs), many of which BLAST and GO term analyses indicate may function as toxins or their delivery agents. ~38% of VSTs had BLAST hits, including latrotoxins, inhibitor cystine knot toxins, CRISPs, hyaluronidases, chitinase, and proteases, and 59% of VSTs had predicted protein domains. Latrotoxins are venom toxins that cause massive neurotransmitter release from vertebrate or invertebrate neurons. We discovered ≥ 20 divergent latrotoxin paralogs expressed in L. hesperus venom glands, significantly increasing this biomedically important family. Mass spectrometry of L. hesperus venom identified 49 proteins from VSTs, 24 of which BLAST to toxins. Phylogenetic analyses showed venom gland specific gene family expansions and shifts in tissue expression. CONCLUSIONS: Quantitative expression analyses comparing multiple tissues are necessary to identify venom gland specific transcripts. We present a black widow venom specific exome that uncovers a trove of diverse toxins and associated proteins, suggesting a dynamic evolutionary history. This justifies a reevaluation of the functional activities of black widow venom in light of its emerging complexity.

Novel biopesticide based on a spider venom peptide shows no adverse effects on honeybees.

Evidence is accumulating that commonly used pesticides are linked to decline of pollinator populations; adverse effects of three neonicotinoids on bees have led to bans on their use across the European Union. Developing insecticides that pose negligible risks to beneficial organisms such as honeybees is desirable and timely. One strategy is to use recombinant fusion proteins containing neuroactive peptides/proteins linked to a 'carrier' protein that confers oral toxicity. Hv1a/GNA (Galanthus nivalis agglutinin), containing an insect-specific spider venom calcium channel blocker (ω-hexatoxin-Hv1a) linked to snowdrop lectin (GNA) as a 'carrier', is an effective oral biopesticide towards various insect pests. Effects of Hv1a/GNA towards a non-target species, Apis mellifera, were assessed through a thorough early-tier risk assessment. Following feeding, honeybees internalized Hv1a/GNA, which reached the brain within 1 h after exposure. However, survival was only slightly affected by ingestion (LD50>100 µg bee(-1)) or injection of fusion protein. Bees fed acute (100 µg bee(-1)) or chronic (0.35 mg ml(-1)) doses of Hv1a/GNA and trained in an olfactory learning task had similar rates of learning and memory to no-pesticide controls. Larvae were unaffected, being able to degrade Hv1a/GNA. These tests suggest that Hv1a/GNA is unlikely to cause detrimental effects on honeybees, indicating that atracotoxins targeting calcium channels are potential alternatives to conventional pesticides.

Cysteine-rich toxins from Lachesana tarabaevi spider venom with amphiphilic C-terminal segments.

Venom of Lachesana tarabaevi (Zodariidae, "ant spiders") exhibits high insect toxicity and serves a rich source of potential insecticides. Five new peptide toxins active against insects were isolated from the venom by means of liquid chromatography and named latartoxins (LtTx). Complete amino acid sequences of LtTx (60-71 residues) were established by a combination of Edman degradation, mass spectrometry and selective proteolysis. Three toxins have eight cysteine residues that form four intramolecular disulfide bridges, and two other molecules contain an additional cystine; three LtTx are C-terminally amidated. Latartoxins can be allocated to two groups with members similar to CSTX and LSTX toxins from Cupiennius salei (Ctenidae) and Lycosa singoriensis (Lycosidae). The interesting feature of the new toxins is their modular organization: they contain an N-terminal cysteine-rich (knottin or ICK) region as in many neurotoxins from spider venoms and a C-terminal linear part alike some cytolytic peptides. The C-terminal fragment of one of the most abundant toxins LtTx-1a was synthesized and shown to possess membrane-binding activity. It was found to assume amphipathic α-helical conformation in membrane-mimicking environment and exert antimicrobial activity at micromolar concentrations. The tails endow latartoxins with the ability to bind and damage membranes; LtTx show cytolytic activity in fly larvae neuromuscular preparations. We suggest a membrane-dependent mode of action for latartoxins with their C-terminal linear modules acting as anchoring devices.

Milking and partial characterization of venom from the Brazilian spider Vitalius dubius (Theraphosidae).

The theraphosid spider genus Vitalius contains several species found in southeastern Brazil. In this work, we used electrostimulation to obtain venom from Vitalius dubius and examined its general composition. Male spiders yielded significantly less (p < 0.05) venom (12.5 +/- 0.7 mg of liquid/spider, n = 16; mean +/- S.E.M.) than female spiders (25.5 +/- 2.0 mg of liquid/spider, n = 11). However, when corrected for spider weight, males yielded slightly more venom (2.89 +/- 0.16 mg/g vs. 2.45 +/- 0.76 mg/g for males and females, respectively, p < 0.05). Venom yield correlated linearly with spider weight for spiders weighing up to approximately 12-13 g, but decreased in very heavy females. There was a marked decrease in venom yield after the first milking. The protein concentration of pooled venom was 18.3 +/- 2.4 mg/ml (n = 4) and accounted for 16.6 +/- 4.7% of the dry venom weight. The venom contained high hyaluronidase activity (275 +/- 24 TRU/mg of protein, n = 4), with a molecular mass of approximately 45 kDa estimated by zymography. SDS-PAGE revealed a few proteins with molecular masses >14 kDa but showed two staining bands of peptides <14 kDa. The venom reacted in ELISA with affinity-purified IgG from commercial arachnidic antivenom. Immunoblotting with this IgG detected proteins of 30-140 kDa only. Fractionation of the venom by reverse-phase chromatography resulted in five major and eight minor peaks.

Insecticidal toxins from black widow spider venom.

The biological effects of Latrodectus spider venom are similar in animals from different phyla, but these symptoms are caused by distinct phylum-specific neurotoxins (collectively called latrotoxins) with molecular masses ranging from 110 to 140 kDa. To date, the venom has been found to contain five insecticidal toxins, termed alpha, beta, gamma, delta and epsilon-latroinsectotoxins (LITs). There is also a vertebrate-specific neurotoxin, alpha-latrotoxin (alpha-LTX), and one toxin affecting crustaceans, alpha-latrocrustatoxin (alpha-LCT). These toxins stimulate massive release of neurotransmitters from nerve terminals and act (1) by binding to specific receptors, some of which mediate an exocytotic signal, and (2) by inserting themselves into the membrane and forming ion-permeable pores. Specific receptors for LITs have yet to be identified, but all three classes of vertebrate receptors known to bind alpha-LTX are also present in insects. All LTXs whose structures have been elucidated (alpha-LIT, delta-LIT, alpha-LTX and alpha-LCT) are highly homologous and have a similar domain architecture, which consists of a unique N-terminal sequence and a large domain composed of 13-22 ankyrin repeats. Three-dimensional (3D) structure analysis, so far done for alpha-LTX only, has revealed its dimeric nature and an ability to form symmetrical tetramers, a feature probably common to all LTXs. Only tetramers have been observed to insert into membranes and form pores. A preliminary 3D reconstruction of a delta-LIT monomer demonstrates the spatial similarity of this toxin to the monomer of alpha-LTX.

A model genetic system for testing the in vivo function of peptide toxins.

We have developed a model genetic system for analyzing the function of peptide toxins from animal venoms. We engineered and propagated strains of Drosophila melanogaster expressing heat-inducible transgenes encoding either kappa-ACTX-Hv1c or omega-ACTX-Hv1a, two insect-specific neurotoxic peptides found in the venom of the Australian funnel-web spider Hadronyche versuta. Heat induction of transgene expression for 20 min was sufficient to kill all transgenic flies, indicating that the ion channels targeted by these toxins are viable insecticide targets. The unusual phenotype of flies induced to express omega-ACTX-Hv1a recapitulates that of a hypomorphic allele of the high-voltage-activated calcium channel Dmca1D, suggesting that this is likely to be the target of omega-ACTX-Hv1a.

Brown recluse spider (Loxosceles reclusa) venom phospholipase D (PLD) generates lysophosphatidic acid (LPA).

Envenomation by the brown recluse spider (Loxosceles reclusa) may cause local dermonecrosis and, rarely, coagulopathies, kidney failure and death. A venom phospholipase, SMaseD (sphingomyelinase D), is responsible for the pathological manifestations of envenomation. Recently, the recombinant SMaseD from Loxosceles laeta was demonstrated to hydrolyse LPC (lysophosphatidylcholine) to produce LPA (lysophosphatidic acid) and choline. Therefore activation of LPA signalling pathways may be involved in some manifestations of Loxosceles envenomation. To begin investigating this idea, we cloned a full-length cDNA encoding L. reclusa SMaseD. The 305 amino acid sequence of the L. reclusa enzyme is 87, 85 and 60% identical with those of L. arizonica, L. intermedia and L. laeta respectively. The recombinant enzyme expressed in bacteria had broad substrate specificity. The lysophospholipids LPC, LPI (18:1-1-oleyol lysophosphatidylinositol), LPS, LPG (18:1-1-oleoyl-lysophosphatidylglycerol), LBPA (18:1-1-oleoyl-lysobisphosphatidic acid) (all with various acyl chains), lyso-platelet-activating factor (C16:0), cyclic phosphatidic acid and sphingomyelin were hydrolysed, whereas sphingosylphosphorylcholine, PC (phosphatidylcholine; C22:6, C20:4 and C6:0), oxidized PCs and PAF (platelet-activating factor; C16:0) were not hydrolysed. The PAF analogue, edelfosine, inhibited enzyme activity. Recombinant enzyme plus LPC (C18:1) induced the migration of A2058 melanoma cells, and this activity was blocked by the LPA receptor antagonist, VPC32183. The recombinant spider enzyme was haemolytic, but this activity was absent from catalytically inactive H37N (His37-->Asn) and H73N mutants. Our results demonstrate that Loxosceles phospholipase D hydrolyses a wider range of lysophospholipids than previously supposed, and thus the term 'SMaseD' is too limited in describing this enzyme.

Jingzhaotoxin-III, a novel spider toxin inhibiting activation of voltage-gated sodium channel in rat cardiac myocytes.

We have isolated a cardiotoxin, denoted jingzhaotoxin-III (JZTX-III), from the venom of the Chinese spider Chilobrachys jingzhao. The toxin contains 36 residues stabilized by three intracellular disulfide bridges (I-IV, II-V, and III-VI), assigned by a chemical strategy of partial reduction and sequence analysis. Cloned and sequenced using 3'-rapid amplification of cDNA ends and 5'-rapid amplification of cDNA ends, the full-length cDNA encoded a 63-residue precursor of JZTX-III. Different from other spider peptides, it contains an uncommon endoproteolytic site (-X-Ser-) anterior to mature protein and the intervening regions of 5 residues, which is the smallest in spider toxin cDNAs identified to date. Under whole cell recording, JZTX-III showed no effects on voltage-gated sodium channels (VGSCs) or calcium channels in dorsal root ganglion neurons, whereas it significantly inhibited tetrodotoxin-resistant VGSCs with an IC(50) value of 0.38 microm in rat cardiac myocytes. Different from scorpion beta-toxins, it caused a 10-mV depolarizing shift in the channel activation threshold. The binding site for JZTX-III on VGSCs is further suggested to be site 4 with a simple competitive assay, which at 10 microm eliminated the slowing currents induced by Buthus martensi Karsch I (BMK-I, scorpion alpha-like toxin) completely. JZTX-III shows higher selectivity for VGSC isoforms than other spider toxins affecting VGSCs, and the toxin hopefully represents an important ligand for discriminating cardiac VGSC subtype.

The multiple actions of black widow spider toxins and their selective use in neurosecretion studies.

The black widow spider venom contains several large protein toxins--latrotoxins--that are selectively targeted against different classes of animals: vertebrates, insects, and crustaceans. These toxins are synthesised as large precursors that undergo proteolytic processing and activation in the lumen of the venom gland. The mature latrotoxins demonstrate strong functional structure conservation and contain multiple ankyrin repeats, which mediate toxin oligomerisation. The three-dimensional structure has been determined for alpha-latrotoxin (alphaLTX), a representative venom component toxic to vertebrates. This reconstruction explains the mechanism of alphaLTX pore formation by showing that it forms tetrameric complexes, harbouring a central channel, and that it is able to insert into lipid membranes. All latrotoxins cause massive release of neurotransmitters from nerve terminals of respective animals after binding to specific neuronal receptors. A G protein-coupled receptor latrophilin and a single-transmembrane receptor neurexin have been identified as major high-affinity receptors for alphaLTX. Latrotoxins act by several Ca(2+)-dependent and -independent mechanisms based on pore formation and activation of receptors. Mutant recombinant alphaLTX that does not form pores has been used to dissect the multiple actions of this toxin. As a result, important insights have been gained into the receptor signalling and the role of intracellular Ca(2+) stores in the effect of alphaLTX.

An overview of peptide toxins from the venom of the Chinese bird spider Selenocosmia huwena Wang [=Ornithoctonus huwena (Wang)].

The bird spider Selenocosmia huwena Wang [=Ornithoctonus huwena (Wang)] is one of the most venomous spiders in China. The venom of this spider contains a mixture of compounds with different types of biological activity. About 400 proteins and peptides from the venom can be separated and detected by 2D electrophoresis. Of these, 14 peptide toxins have been purified and characterized from the venom of this spider, with several peptide toxins exhibiting structural similarity but high functional diversity. Most of these huwentoxins (HWTX) contain 30-40 amino acids with three disulfide bonds and adopt an "inhibitor cystine-knot" (ICK) motif in their three dimensional structure, except for huwentoxin-II (HWTX-II) which adopts a novel scaffold different from the ICK motif. As a group, the toxins possess quite different biological activities including inhibition of voltage-gated calcium and sodium channels, insecticidal activity, lectin-like agglutination, and inhibition of trypsin. Eight cDNAs encoding seven toxins, HWTX-I, -II, -III, -IIIa, -IV -V, and, -VII and one lectin, S. huwena lectin-I (SHL-I), have been cloned and sequenced. Comparison of the cDNA sequences of the eight peptides from S. huwena indicates that they can be classified into two different superfamilies according to the "prepro" region of their cDNA sequences.

Subtype-selective reconstitution of synaptic transmission in sympathetic ganglion neurons by expression of exogenous calcium channels.

Fast cholinergic neurotransmission between superior cervical ganglion neurons (SCGNs) in cell culture is initiated by N-type Ca(2+) currents through Ca(v)2.2 channels. To test the ability of different Ca(2+)-channel subtypes to initiate synaptic transmission in these cells, SCGNs were injected with cDNAs encoding Ca(v)1.2 channels, which conduct L-type currents, Ca(v)2.1 channels, which conduct P/Q-type Ca(2+) currents, and Ca(v)2.3 channels, which conduct R-type Ca(2+) currents. Exogenously expressed Ca(v)2.1 channels were localized in nerve terminals, as assessed by immunocytochemistry with subtype-specific antibodies, and these channels effectively initiated synaptic transmission. Injection with cDNA encoding Ca(v)2.3 channels yielded a lower level of presynaptic labeling and synaptic transmission, whereas injection with cDNA encoding Ca(v)1.2 channels resulted in no presynaptic labeling and no synaptic transmission. Our results show that exogenously expressed Ca(2+) channels can mediate synaptic transmission in SCGNs and that the specificity of reconstitution of neurotransmission (Ca(v)2.1 > Ca(v)2.3 >> Ca(v)1.2) follows the same order as in neurons in vivo. The specificity of reconstitution of neurotransmission parallels the specificity of trafficking of these Ca(v) channels to nerve terminals.

The black widow's versatile venom.

The structure of a tetrameric form of the spider neurotoxin, alpha-latrotoxin, has been determined by single particle cryo-electron microscopy. It reveals a pore complex with extended arms that could bind receptors involved in synaptic vesicle exocytosis.