The omega-atracotoxins: selective blockers of insect M-LVA and HVA calcium channels.

The omega-atracotoxins (omega-ACTX) are a family of arthropod-selective peptide neurotoxins from Australian funnel-web spider venoms (Hexathelidae: Atracinae) that are candidates for development as biopesticides. We isolated a 37-residue insect-selective neurotoxin, omega-ACTX-Ar1a, from the venom of the Sydney funnel-web spider Atrax robustus, with high homology to several previously characterized members of the omega-ACTX-1 family. The peptide induced potent excitatory symptoms, followed by flaccid paralysis leading to death, in acute toxicity tests in house crickets. Using isolated smooth and skeletal nerve-muscle preparations, the toxin was shown to lack overt vertebrate toxicity at concentrations up to 1 microM. To further characterize the target of the omega-ACTXs, voltage-clamp analysis using the whole-cell patch-clamp technique was undertaken using cockroach dorsal unpaired median neurons. It is shown here for the first time that omega-ACTX-Ar1a, and its homolog omega-ACTX-Hv1a from Hadronyche versuta, reversibly block both mid-low- (M-LVA) and high-voltage-activated (HVA) insect calcium channel (Ca(v)) currents. This block occurred in the absence of alterations in the voltage-dependence of Ca(v) channel activation, and was voltage-independent, suggesting that omega-ACTX-1 family toxins are pore blockers rather than gating modifiers. At a concentration of 1 microM omega-ACTX-Ar1a failed to significantly affect global K(v) channel currents. However, 1 microM omega-ACTX-Ar1a caused a modest 18% block of insect Na(v) channel currents, similar to the minor block of Na(v) channels reported for other insect Ca(v) channel blockers such as omega-agatoxin IVA. These findings validate both M-LVA and HVA Ca(v) channels as potential targets for insecticides.

Arachnid toxinology in Australia: from clinical toxicology to potential applications.

The unique geographic isolation of Australia has resulted in the evolution of a distinctive range of Australian arachnid fauna. Through the pioneering work of a number of Australian arachnologists, toxinologists, and clinicians, the taxonomy and distribution of new species, the effective clinical treatment of envenomation, and the isolation and characterisation of the many distinctive neurotoxins, has been achieved. In particular, work has focussed on several Australian arachnids, including red-back and funnel-web spiders, paralysis ticks, and buthid scorpions that contain neurotoxins capable of causing death or serious systemic envenomation. In the case of spiders, species-specific antivenoms have been developed to treat envenomed patients that show considerable cross-reactivity. Both in vitro and clinical case studies have shown they are particularly efficacious in the treatment of envenomation by spiders even from unrelated families. Despite their notorious reputation, the high selectivity and potency of a unique range of toxins from the venom of Australian arachnids will make them invaluable molecular tools for studies of neurotransmitter release and vesicle exocytosis as well as ion channel structure and function. The venoms of funnel-web spiders, and more recently Australian scorpions, have also provided a previously untapped rich source of insect-selective neurotoxins for the future development of biopesticides and the characterisation of previously unvalidated insecticide targets. This review provides a historical viewpoint of the work of many toxinologists to isolate and characterise just some of the toxins produced by such a unique group of arachnids and examines the potential applications of these novel peptides.

Isolation of delta-missulenatoxin-Mb1a, the major vertebrate-active spider delta-toxin from the venom of Missulena bradleyi (Actinopodidae).

The present study describes the isolation and pharmacological characterisation of the neurotoxin delta-missulenatoxin-Mb1a (delta-MSTX-Mb1a) from the venom of the male Australian eastern mouse spider, Missulena bradleyi. This toxin was isolated using reverse-phase high-performance liquid chromatography and was subsequently shown to cause an increase in resting tension, muscle fasciculation and a decrease in indirect twitch tension in a chick biventer cervicis nerve-muscle bioassay. Interestingly, these effects were neutralised by antivenom raised against the venom of the Sydney funnel-web spider Atrax robustus. Subsequent whole-cell patch-clamp electrophysiology on rat dorsal root ganglion neurones revealed that delta-MSTX-Mb1a caused a reduction in peak tetrodotoxin (TTX)-sensitive sodium current, a slowing of sodium current inactivation and a hyperpolarising shift in the voltage at half-maximal activation. In addition, delta-MSTX-Mb1a failed to affect TTX-resistant sodium currents. Subsequent Edman degradation revealed a 42-residue peptide with unusual N- and C-terminal cysteines and a cysteine triplet (Cys(14-16)). This toxin was highly homologous to a family of delta-atracotoxins (delta-ACTX) from Australian funnel-web spiders including conservation of all eight cysteine residues. In addition to actions on sodium channel gating and kinetics to delta-ACTX, delta-MSTX-Mb1a caused significant insect toxicity at doses up to 2000 pmol/g. Delta-MSTX-Mb1a therefore provides evidence of a highly conserved spider delta-toxin from a phylogenetically distinct spider family that has not undergone significant modification.

Clinical and in vitro evidence for the efficacy of Australian red-back spider (Latrodectus hasselti) antivenom in the treatment of envenomation by a Cupboard spider (Steatoda grossa).

We report the case of a 22-year-old female who was bitten on the shoulder by a spider subsequently identified as a female Cupboard spider (Steatoda grossa). She developed nausea, vomiting, and severe local and regional pain, similar to that seen in latrodectism. Symptoms were treated successfully with red-back spider antivenom (RBSAV). We also present in vitro data, which supports this clinical observation, and suggests that S. grossa venom is immunogenically reactive with both RBSAV and latrotoxin (LTx)-specific antibodies by Western blotting. Moreover, the effects of S. grossa venom on the isolated chick biventer cervicis nerve-muscle preparation are dose-dependent and similar to those seen with Latrodectus spp. venoms. S. grossa venom produced a sustained muscle contracture which could be prevented by pre-incubation of venom with RBSAV. Venom effects could also be reversed by the addition of antivenom after application of venom to the preparation. Although severe envenomation is uncommon following the bite of Steatoda spp. it may resemble latrodectism. These results indicate that RBSAV is likely to be effective in reversing symptoms of envenomation and should be considered in the treatment of patients with distressing or persisting symptoms.

Cloning and activity of a novel α-latrotoxin from red-back spider venom.

The venom of the European black widow spider Latrodectus tredecimguttatus (Theridiidae) contains several high molecular mass (110-140 kDa) neurotoxins that induce neurotransmitter exocytosis. These include a vertebrate-specific α-latrotoxin (α-LTX-Lt1a) responsible for the clinical symptoms of latrodectism and numerous insect-specific latroinsectoxins (LITs). In contrast, little is known about the expression of these toxins in other Latrodectus species despite the fact that envenomation by these spiders induces a similar clinical syndrome. Here we report highly conserved α-LTX, α-LIT and δ-LIT sequence tags in Latrodectus mactans, Latrodectus hesperus and Latrodectus hasselti venoms using tandem mass spectrometry, following bioassay-guided separation of venoms by liquid chromatography. Despite this sequence similarity, we show that the anti-α-LTX monoclonal antibody 4C4.1, raised against α-LTX-Lt1a, fails to neutralize the neurotoxicity of all other Latrodectus venoms tested in an isolated chick biventer cervicis nerve-muscle bioassay. This suggests that there are important structural differences between α-LTXs in theridiid spider venoms. We therefore cloned and sequenced the α-LTX from the Australian red-back spider L. hasselti (α-LTX-Lh1a). The deduced amino acid sequence of the mature α-LTX-Lh1a comprises 1180 residues (∼132kDa) with ∼93% sequence identity with α-LTX-Lt1a. α-LTX-Lh1a is composed of an N-terminal domain and a central region containing 22 ankyrin-like repeats. The presence of two furin cleavage sites, conserved with α-LTX-Lt1a, indicates that α-LTX-Lh1a is derived from the proteolytic cleavage of an N-terminal signal peptide and C-terminal propeptide region. However, we show that α-LTX-Lh1a has key substitutions in the 4C4.1 epitope that explains the lack of binding of the monoclonal antibody.

Soluble expression of a functional recombinant cytolytic immunotoxin in insect cells.

We have previously described the production of a recombinant melittin-based cytolytic immunotoxin (IT), scFv-mel-FLAG, in bacterial cells. While the IT exhibited specific cytotoxicity for a human lymphoblastoid cell line, HMy2, yields from expression were low. Here, we describe a baculovirus expression system for the overexpression and secretion of scFv-mel-FLAG. A novel snake phospholipase A2 inhibitor signal peptide was used to aid in the secretion of the immunotoxin. Sf21 insect cells infected with the recombinant virus secreted soluble scFv-mel-FLAG into the culture medium from which it was purified directly on an affinity column. The final yield of scFv-mel-FLAG was estimated at 3-5 mg/L, which was an improvement of 30-fold compared to expression in the prokaryotic system. The cell binding characteristics of the recombinant IT were assessed by flow cytometry using the antigen expressing cell line HMy2. ScFv-mel-FLAG bound specifically to HMy2 cells in direct binding assays and this binding was completely inhibited in the presence of an excess of soluble antigen. Significant cytotoxicity for HMy2 cells, measured by leakage of cytosolic LDH, was also observed for the IT at a concentration of 60 pmol/10(4) cells. Cytotoxicity was concentration dependent and was specific for antigen-positive cells. Thus the baculovirus expression system, under the control of a novel secretion signal, can be used for the production of soluble and functional recombinant cytolytic immunotoxins. To our knowledge, this is the first report of expression of a recombinant immunotoxin in the baculovirus expression vector system.