Antibacterial activity of six novel peptides from Tityus discrepans scorpion venom. A fluorescent probe study of microbial membrane Na+ permeability changes.

ABSTRACT

Six novel peptides (named bactridines) were isolated from Tityus discrepans scorpion venom. From mass spectrometry molecular masses were 6916, 7362, 7226, 7011, 7101 and 7173 Da (bactridines 1-6). Bactridines 1 and 2 were sequenced by Edman degradation. The sequences and in silico analysis, indicated that they are positively charged polypeptides comprised of 61 and 64 amino acids (AA), respectively, bactridine 1 and bactridine 2 containing 4 disulfide bridges. Bactridine 1 was only toxic to cockroaches and crabs, and bactridine 2-6 were only toxic to mice. Bactridine 1 has a 78% sequence identity with ardiscretin. Ardisctretin is an insect specific sodium toxin which also produces a small depolarization and induces repetitive firing in squid axons resembling those of DDT [1,10(pchlorobenzyl) 2-trichloretane] in its ability to slow down action potential, to induce repetitive firing. Measured as the minimal inhibitory concentration, bactridines had high antibacterial activity against a wide range of gram positive and gram negative bacteria. Complete bacterial growth inhibition occurred at concentrations from 20 to 80 microM depending on the bacteria and peptide tested. Effects on membrane Na(+) permeability induced by bactridines were observed on Yersinia enterocolitica loaded with 1 microM CoroNa Red. CoroNa Red fluorescence leakage from bacteria was observed after exposure to 0.3 microM of any bactridine tested, indicating that they modified Na(+) membrane permeability. This effect was blocked by 10 microM amiloride and by 25 microM mibefradil drugs that affect Na(+) and Ca(2+) channels respectively. We found no evidence of changes of K(+) or Ca(2+) concentrations neither inside nor outside the bacteria in experiments using the fluorescent dyes Fluo 4AM (10 microM) and PBFI (20 microM).