ABSTRACT
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.
Subject(s)
Evolution, Molecular , Neurotoxins/chemistry , Spider Venoms/chemistry , Spiders/chemistry , Animals , Calcium Channels, L-Type/chemistry , Calcium Channels, L-Type/genetics , Calcium Channels, L-Type/metabolism , Cell Membrane/chemistry , Cell Membrane/genetics , Cell Membrane/metabolism , DNA, Complementary/genetics , Drosophila melanogaster , Escherichia coli/genetics , Escherichia coli/growth & development , Escherichia coli/metabolism , Female , Muscle, Skeletal/chemistry , Muscle, Skeletal/metabolism , Neurotoxins/genetics , Protein Structure, Tertiary , Rana temporaria , Spider Venoms/genetics , Spiders/genetics , Xenopus laevisABSTRACT
The urgent need for new drug development for African trypanosomiasis is widely recognized. This requires reliable and informative high-throughput assays. Currently, drug action is determined with a fluorimetric/colorimetric assay based on the metabolism of the dye Alamar Blue (resazurin) by live cells. However, this assay does not easily distinguish between cell death and growth arrest, or supply information about the rate at which test compounds affect these parameters. We report here an alternative fluorimetric assay, based on the interaction of propidium iodide with DNA, that allows either real-time monitoring of cell viability or the generation of EC(50) values at a predetermined time-point. The assay is highly sensitive and fluorescence readings easily correlate to numbers of parasites or DNA content. The EC(50) values were highly similar to those obtained with the standard Alamar Blue assay. The procedure lends itself readily to applications in drug development or resistance monitoring.