The dermal glands of frogs produce antimicrobial peptides that protect the skin against noxious microorganisms and assist in wound repair. The sequences of these peptides are very dissimilar, both within and between species, so that the 5000 living anuran frogs may produce ≈ 100 000 different antimicrobial peptides. The antimicrobial peptides of South American hylid frogs are derived from precursors, the preprodermaseptins, whose signal peptides and intervening sequences are remarkably conserved, but their C‐terminal domains are markedly diverse, resulting in mature peptides with different lengths, sequences and antimicrobial spectra. We have used the extreme conservation in the preproregion of preprodermaseptin transcripts to identify new members of this family in Australian and South American hylids. All these peptides are cationic, amphipathic and α‐helical. They killed a broad spectrum of microorganisms and acted in synergy. 42 preprodermaseptin gene sequences from 10 species of hylid and ranin frogs were analyzed in the context of their phylogeny and biogeography and of geophysical models for the fragmentation of Gondwana to examine the strategy that these frogs have evolved to generate an enormous array of peptide antibiotics. The hyperdivergence of modern antimicrobial peptides and the number of peptides per species result from repeated duplications of a ≈ 150‐million‐year‐old ancestral gene and accelerated mutations of the mature peptide domain, probably involving a mutagenic, error‐prone, DNA polymerase similar to Escherichia coli Pol V. The presence of antimicrobial peptides with such different structures and spectra of action represents the successful evolution of multidrug defense by providing frogs with maximum protection against infectious microbes and minimizing the chance of microorganisms developing resistance to individual peptides. The hypermutation of the antimicrobial domain by a targeted mutagenic polymerase that can generate many sequence changes in a few steps may have a selective survival value when frogs colonizing a new ecological niche encounter different microbial predators.

Antimicrobial peptides that play a role in host defence against competing or pathogenic microorganisms are small proteins, typically 10-50 residues long, interact with lipid bilayers to alter cell-membrane permeability, which often leads to cell death. Structural studies have revealed that the peptide secondary structures include a-helices, b-sheet structures stabilized by two or three disulfide bonds, and extended structures with overrepresentation of one or two amino acids (W, P, H or G) [1]. However, all these peptides, regardless of the secondary structure or length, are cationic and exhibit amphipathic properties upon interaction with lipid bilayers, with apolar amino acid residues segregating from the hydrophilic residues on opposite sides of the 3D structure. These structural elements are believed to play a crucial role in the binding of cationic host [468][469][470][471][472][473][474][475][476][477][478][479][480] demonstrated that this peptide adopted a non-amphipathic a-helical conformation in trifluoroethanol ⁄ water mixtures, but was highly aggregated in aqueous solutions and in the presence of sodium dodecyl sulfate micelles. Circular dichroism, FTIR and attenuated total reflectance FTIR spectroscopies, combined with a surface plasmon resonance study, show that Drs S9 forms stable and ordered b-sheet aggregates in aqueous buffers or when bound to anionic or zwitterionic phospholipid vesicles. These structures slowly assembled into amyloid-like fibrils in aqueous environments via spherical intermediates, as revealed by electron microscopy and Congo red staining. Drs S9 induced the directional migration of neutrophils, T lymphocytes and monocytes. Interestingly, the antimicrobial and chemotactic activities of Drs S9 are modulated by its amyloid-like properties. Whereas spherical oligomers of Drs S9 exhibit antimicrobial activity, the soluble, weakly selfassociated forms of Drs S9 act on human leukocytes to promote chemotaxis and ⁄ or immunological response activation in the same range of concentration as amyloidogenic peptides Ab(1-42), the most fibrillogenic isoform of amyloid beta peptides, and the prion peptide PrP(106-126).

The dermaseptins S are closely related peptides with broad-spectrum antibacterial activity that are produced by the skin of the South American hylid frog, Phyllomedusa sauvagei. These peptides are polycationic (Lys-rich), alpha-helical, and amphipathic, with their polar/charged and apolar amino acids on opposing faces along the long axis of the helix cylinder. The amphipathic alpha-helical structure is believed to enable the peptides to interact with membrane bilayers, leading to permeation and disruption of the target cell. We have identified new members of the dermaseptin S family that do not resemble any of the naturally occurring antimicrobial peptides characterized to date. One of these peptides, designated dermaseptin S9, GLRSKIWLWVLLMIWQESNKFKKM, has a tripartite structure that includes a hydrophobic core sequence encompassing residues 6-15 (mean hydrophobicity, +4.40, determined by the Liu-Deber scale) flanked at both termini by cationic and polar residues. This structure is reminiscent of that of synthetic peptides originally designed as transmembrane mimetic models and that spontaneously become inserted into membranes [Liu, L., and Deber, C. M. (1998) Biopolymers 47, 41-62]. Dermaseptin S9 is a potent antibacterial, acting on gram-positive and gram-negative bacteria. The structure of dermaseptin S9 in aqueous solution and in TFE/water mixtures was analyzed by circular dichroism and two-dimensional NMR spectroscopy combined with molecular dynamics calculations. Dermaseptin S9 is aggregated in water, but a monomeric nonamphipathic alpha-helical conformation, mostly in residues 6-21, is stabilized by the addition of TFE. These results, combined with membrane permeabilization assays and surface plasmon resonance analysis of the peptide binding to zwitterionic and anionic phospholipid bilayers, demonstrate that spatial segregation of hydrophobic and hydrophilic/charged residues on opposing faces along the long axis of a helix is not essential for the antimicrobial activity of cationic alpha-helical peptides.