RESUMEN
The inexorable decline in the armament of registered chemical insecticides has stimulated research into environmentally-friendly alternatives. Insecticidal spider-venom peptides are promising candidates for bioinsecticide development but it is challenging to find peptides that are specific for targeted pests. In the present study, we isolated an insecticidal peptide (Ae1a) from venom of the African spider Augacephalus ezendami (family Theraphosidae). Injection of Ae1a into sheep blowflies (Lucilia cuprina) induced rapid but reversible paralysis. In striking contrast, Ae1a was lethal to closely related fruit flies (Drosophila melanogaster) but induced no adverse effects in the recalcitrant lepidopteran pest Helicoverpa armigera. Electrophysiological experiments revealed that Ae1a potently inhibits the voltage-gated sodium channel BgNaV1 from the German cockroach Blattella germanica by shifting the threshold for channel activation to more depolarized potentials. In contrast, Ae1a failed to significantly affect sodium currents in dorsal unpaired median neurons from the American cockroach Periplaneta americana. We show that Ae1a interacts with the domain II voltage sensor and that sensitivity to the toxin is conferred by natural sequence variations in the S1-S2 loop of domain II. The phyletic specificity of Ae1a provides crucial information for development of sodium channel insecticides that target key insect pests without harming beneficial species.
Asunto(s)
Insecticidas/farmacología , Péptidos/farmacología , Venenos de Araña/química , Arañas/fisiología , Canales de Sodio Activados por Voltaje/química , Animales , Blattellidae/efectos de los fármacos , Dípteros/efectos de los fármacos , Drosophila melanogaster/efectos de los fármacos , Evaluación Preclínica de Medicamentos/métodos , Proteínas de Insectos/antagonistas & inhibidores , Proteínas de Insectos/metabolismo , Insecticidas/química , Lepidópteros/efectos de los fármacos , Canal de Sodio Activado por Voltaje NAV1.5/metabolismo , Péptidos/genética , Péptidos/aislamiento & purificación , Periplaneta/efectos de los fármacos , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacología , Arañas/química , Bloqueadores del Canal de Sodio Activado por Voltaje/farmacología , Canales de Sodio Activados por Voltaje/metabolismoRESUMEN
CsTx-1, the main neurotoxic acting peptide in the venom of the spider Cupiennius salei, is composed of 74 amino acid residues, exhibits an inhibitory cysteine knot motif, and is further characterized by its highly cationic charged C terminus. Venom gland cDNA library analysis predicted a prepropeptide structure for CsTx-1 precursor. In the presence of trifluoroethanol, CsTx-1 and the long C-terminal part alone (CT1-long; Gly-45-Lys-74) exhibit an α-helical structure, as determined by CD measurements. CsTx-1 and CT1-long are insecticidal toward Drosophila flies and destroys Escherichia coli SBS 363 cells. CsTx-1 causes a stable and irreversible depolarization of insect larvae muscle cells and frog neuromuscular preparations, which seem to be receptor-independent. Furthermore, this membranolytic activity could be measured for Xenopus oocytes, in which CsTx-1 and CT1-long increase ion permeability non-specifically. These results support our assumption that the membranolytic activities of CsTx-1 are caused by its C-terminal tail, CT1-long. Together, CsTx-1 exhibits two different functions; as a neurotoxin it inhibits L-type Ca(2+) channels, and as a membranolytic peptide it destroys a variety of prokaryotic and eukaryotic cell membranes. Such a dualism is discussed as an important new mechanism for the evolution of spider venomous peptides.
Asunto(s)
Evolución Molecular , Neurotoxinas/química , Venenos de Araña/química , Arañas/química , Animales , Canales de Calcio Tipo L/química , Canales de Calcio Tipo L/genética , Canales de Calcio Tipo L/metabolismo , Membrana Celular/química , Membrana Celular/genética , Membrana Celular/metabolismo , ADN Complementario/genética , Drosophila melanogaster , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Escherichia coli/metabolismo , Femenino , Músculo Esquelético/química , Músculo Esquelético/metabolismo , Neurotoxinas/genética , Estructura Terciaria de Proteína , Rana temporaria , Venenos de Araña/genética , Arañas/genética , Xenopus laevisRESUMEN
To investigate structural features modulating the biological activity of cupiennin 1 peptides from the spider Cupiennius salei, three truncated cupiennin 1d analogs were synthesized. The fact that their growth inhibiting effect on Gram-negative and Gram-positive bacteria, their lytic activity with human red blood cells and their insecticidal effect on Drosophila melanogaster correlates with structural properties shows that the hydrophobic N-terminal chain segment includes the major determinants of structure and activity. The polar C-terminus seems to modulate peptide accumulation at negatively charged cell surfaces via electrostatic interactions and has no important effect on the peptides' amphipathic secondary structure.