Purification, Structural Characterization And CDNA Cloning Of Skin Secretion Of Hylarana Guentheri

Linear, amphipathic and cationic antimicrobial peptides have been previously reported from a wide range of amphibian species especially frogs of the genus Rana. Such antimicrobial peptides are attracting increasing attention in pharmacological applications because they mainly act by permeabilizing and disrupting the target cell membrane with low degree of bacterial resistance. The guenther's frog, Hylarana guentheri, is widely distributed in Southern China. It is commonly the dominating amphibian species even where the amphibian population is declining.The present study describes the purification and the structural and biological characterization of five novel peptides(AMP1, AMP2, AMP3, AMP4 and AMP5) with antimicrobial activity from electrically stimulated skin secretions of H. guentheri. Respectively, the primary structure of AMP1 ,AMP2, AMP3, AMP4 and AMP5 are GFSSLFKAGAKYLLKSVGKAGAQQLACKAANNCA, GVITDALK GAAKTVAAELLRKAHCKLTNSC, VIDDLKKVAKKVRRELLCKKHHKKLN, SIWEGIKNAGKGFLVSILDKVRCKVAGGCNP and FLPLLFGAISHLL-NH2. The minimum inhibitory concentrations (MICs) of the five antimicrobial peptides (AMPs) against strains of Staphylococcus aureus FDA209P range from 9.8μg/mL to 44.3μg/mL. The MIC value of AMP2 and AMP4 against Escherichia coli O111, Escherichia coli ATCC 25922 are 8.2μg/mL, 8.2μg/mL and 19.6μg/mL, 9.8μg/mL, respectively. The activity of all AMPs towards Bacillus subtilis ATCC 6633 are weak, the MIC values above 64μg/mL. Furthermore, the cDNAs of two novel members of brevinin-2 family, were subsequently cloned by using a 3'- and 5'- RACE strategy. This is the first report of antimicrobial peptides from Hylarana guentheri.