The first identified cathelicidin from tree frogs possesses anti-inflammatory and partial LPS neutralization activities

As of February 2017, approximately 7639 amphibian species have been described in the AmphibiaWeb database. However, only 20 cathelicidin-like antimicrobial peptides have been identified to date from 10 amphibian species. Half of these peptides were identified from genome sequences and have not yet been functionally characterized. In this study, a novel cathelicidin-like peptide designated cathelicidin-PP was purified from the skin of tree frog Polypedates puerensis. Cathelicidin-PP is a 32 residue peptide of sequence ASENGKCNLLCLVKKKLRAVGNVIKTVVGKIA. Circular dichroism spectroscopy indicated that cathelicidin-PP mainly adopts a beta-sheet structure in membrane-mimetic solutions. Cathelicidin-PP exhibits potent antimicrobial activity against bacteria and fungi, especially Gram-negative bacteria. Meanwhile, it shows low cytotoxicity toward mammalian cells. Scanning electron microscopy analysis indicated that cathelicidin-PP kills bacteria through the disruption of the bacterial cell membrane integrity. Furthermore, cathelicidin-PP exerts significant anti-inflammatory functions by inhibiting the lipopolysaccharide (LPS)-mediated generation of nitric oxide and pro-inflammatory cytokines, tumor necrosis factor-alpha, interleukin-1 beta, and interleukin-6. The MAPKs (ERK, JNK, and p38) and NF-kappa B signaling pathways are involved in the anti-inflammatory effect. Cathelicidin-PP caused partial neutralization of LPS in a dose-dependent manner. Quantitative PCR indicated that infection of tree frogs with bacteria causes increased expression of cathelicidin-PP in immune-related tissues. Taken together, cathelicidin-PP is the first identified cathelicidin-like peptide from tree frogs. Our findings demonstrate that in addition to direct bactericidal capacity, cathelicidin-PP also possesses immunomodulatory properties, including partial neutralization of LPS, and inhibiting the production of inflammatory cytokines.