Rafael Herrmann

Positive cooperativity among insecticidal scorpion neurotoxins

The insecticidal activity of scorpion neurotoxic polypeptides increased 5-10-fold with no apparent increase in mammalian toxicity when a combination of two toxins was injected. Synergistic combinations could be predicted from binding studies and competitive displacement assays. Our results indicate that simultaneous expression in baculovirus or other transgenic organisms of the synergistic combinations of insecticidal toxins may result in more potent insect-selective biopesticides.

Isolation and characterization of a novel lepidopteran-selective toxin from the venom of South Indian red scorpion, Mesobuthus tamulus.

BackgroundScorpion venom contains insect and mammal selective toxins. We investigated the venom of the South Indian red scorpion, Mesobuthus tamulus for the purpose of identifying potent insecticidal peptide toxins.ResultsA lepidopteran-selective toxin (Buthus tamulus insect toxin; ButaIT) has been isolated from this venom. The primary structure analysis reveals that it is a single polypeptide composed of 37 amino acids cross-linked by four disulfide bridges with high sequence homology to other short toxins such as Peptide I, neurotoxin P2, Lqh-8/6, chlorotoxin, insectotoxin I5A, insect toxin 15 and insectotoxin I1. Three dimensional modeling using Swiss automated protein modeling server reveals that this toxin contains a short α-helix and three antiparallel β-strands, similar to other short scorpion toxins. This toxin is selectively active on Heliothis virescens causing flaccid paralysis but was non-toxic to blowfly larvae and mice.ConclusionThis is the first report of a Heliothine selective peptide toxin. Identification of diverse insect selective toxins offer advantages in employing these peptides selectively for pest control.

Effect of signal sequence and promoter on the speed of action of a genetically modified Autographa californica nucleopolyhedrovirus expressing the scorpion toxin LqhIT2

Abstract We report on the construction and optimization of recombinant Autographa californica nucleopolyhedrovirus engineered to express the insect-selective toxin IT2 from the scorpion Leiurus quinquestriatus hebraeus . We constructed a series of viruses expressing the synthetic Lqhit2 gene with different signal sequences or controlled by different promoters. The effect of the various viruses on speed of response was assayed in Heliothis virescens larvae. In addition, the performance of the optimum recombinant viral construct was compared with similar constructs carrying the Androctonus australis hector insect toxin controlled by two different promoters. There were no significant differences in speed of response of viruses with the early hr5/ie1 the early hr5/lef3 , or the early/late hr5/39K promoter driving toxin expression. However, the choice of signal sequence resulted in significant effects. The signal sequence from the bombyxin gene of the silkworm Bombyx mori , proved nominally the best. When the signal sequences were used from the following genes, the viruses acted significantly slower: AcMNPV gp67 , a lepidopteran adipokinetic hormone, a dipteran chymotrypsin, and the homologous Lqhit2 . Finally, the signal sequences of the genes for cuticle protein II of Drosophila melanogaster and of the insect toxins of the scorpions A. australis hector and Hottentota judaicus performed very poorly. The speed of action of AcMNPV, carrying the synthetic Lqhit2 gene with the bombyxin secretion signal and driven by the hr5/ie1 promoter [Ac.LqhIT2(hr5/ie1)], was compared to that of the same virus carrying the AaIT gene under the control of the p10 promoter, or the hr5/ie1 promoter in H. virescens , Trichoplusia ni , and Spodoptera exigua larvae. All recombinant viruses elicited the response significantly faster than the common progenitor wild-type virus in all tests. The response elicited by Ac.LqhIT2(hr5/ie1) was nominally faster than that of both viruses expressing AaIT in all insects tested.

Rapid purification and molecular modeling of AaIT peptides from venom of Androctonus australis

As recombinant viruses expressing scorpion toxins are moving closer toward the market, it is important to obtain large amounts of pure toxin for biochemical characterization and the evaluation of biological activity in nontarget organisms. In the past, we purified a large amount of Androctonus australis anti-insect toxin (AaIT) present in the venom of A. australis with an analytical reversed-phase column by repeated runs of crude sample. We now report 20 times improved efficiency and speed of the purification by employing a preparative reversed-phase column. In just two consecutive HPLC steps, almost 1 mg of AaIT was obtained from 70 mg crude venom. Furthermore, additional AaIT was obtained from side fractions in a second HPLC run. Recently discovered insect selective toxin, AaIT5, was isolated simultaneously from the same venom batch. It shows different biological toxicity symptoms than the known excitatory and depressant insect toxins. AaIT5 gave 100% mortality with a dose of less than 1.3 micrograms against fourth-instar tobacco budworms Heliothis virescens 24 h after injection. During the purification process, we implemented mass spectrometry in addition to bioassays to monitor the presence of AaIT and AaIT5 in the HPLC fractions. Mass spectrometric screening can unambiguously follow the purification process and can greatly facilitate and expedite the downstream purification of AaIT and AaIT5 eliminating the number of bioassays required. Further, electrospray ionization was compared with matrix-assisted desorption/ionization and evaluated as a method of choice for mass spectrometric characterization of fractions from the venom purification for it provided higher mass accuracy and relative quantitation capability. Molecular models were built for AaIT5, excitatory toxin AaIT4, and depressant toxin LqhIT2. Three-dimensional structure of AaIT5 was compared with structures of the other two toxins, suggesting that AaIT5 is similar to depressant toxins.