Isolation of dermatoxin from frog skin, an antibacterial peptide encoded by a novel member of the dermaseptin genes family

A 32-residue peptide, named dermatoxin, has been extracted from the skin of a single specimen of the tree frog Phyllomedusa bicolor, and purified to h omogeneity using a four-step protocol. Mass spectral analysis and sequencin g of the purified peptide, as well as chemical synthesis and cDNA analysis were consistent with the structure: SLGSFLKGVGTTLASVGKVVSDQF GKLLQAGQ. This peptide proved to be bactericidal towards mollicutes (wall-less eubacteria ) and Gram-positive eubacteria, and also, though to a lesser extent, toward s Gram-negative eubacteria. Measurement of the bacterial membrane potential revealed that the plasma membrane is the primary target of dermatoxin. Obs ervation of bacterial cells using reflected light fluorescence microscopy a fter DNA-staining was consistent with a mechanism of cell killing based upo n the alteration of membrane permeability rather than membrane solubilizati on, very likely by forming ion-conducting channels through the plasma membr ane. CD spectroscopy and secondary structure predictions indicated that der matoxin assumes an amphipathic alpha-helical conformation in low polarity m edia which mimic the lipophilicity of the membrane of target microorganisms . PCR analysis coupled with cDNA cloning and sequencing revealed that derma toxin is expressed in the skin, the intestine and the brain. Preprodermatox in from the brain and the intestine have the same sequence as the skin prep roform except for two amino-acid substitutions in the preproregion of the b rain precursor. The dermatoxin precursor displayed the characteristic featu res of preprodermaseptins, a family of peptide precursors found in the skin of Phyllomedusa ssp. Precursors of this family have a common N-terminal pr eproregion followed by markedly different C-terminal domains that give rise to 19-34-residue peptide antibiotics named dermaseptins B and phylloxin, a nd to the D-amino-acid-containing opioid heptapeptides dermorphins and delt orphins. Because the structures and cidal mechanisms of dermatoxin, dermase ptins B and phylloxin are very different, dermatoxin extends the repertoire of structurally and functionally diverse peptides derived from the rapidly evolving C-terminal domains of precursors of the dermaseptins family.