Putative Antimicrobial Peptides Research Articles

Cloning and developmental expression of a family of pleurocidin-like antimicrobial peptides from winter flounder, Pleuronectes americanus (Walbaum)

Low molecular weight antimicrobial peptides are an important component of the innate immune system in animals, yet they have not been examined widely in fish. Of particular interest is their expression during development and in response to environmental conditions and disease. Here, we report the isolation of four genomic sequences encoding putative antimicrobial peptides from the winter flounder, Pleuronectes americanus (Walbaum), as well as reverse transcription-PCR products from two tissues that form the first defensive barrier to microbes — skin and intestine. Alignment of the predicted polypeptide sequences shows a conserved hydrophobic signal peptide of 22 amino acids followed by 25 amino acids that are identical (WF2) or homologous to the amino acid sequence of pleurocidin, followed by a conserved acidic portion. Southern hybridisation analysis indicates that related peptides are encoded in the genomes of other flatfish species. Northern and RT-PCR analyses of RNA from multiple tissues show that two of the pleurocidin genes are expressed predominantly in the skin whereas two other genes are expressed mainly in the intestine. RT-PCR assays of total RNA from larvae of different ages provide the first evidence of developmental expression of antimicrobial peptides in fish and indicate that the pleurocidin gene is first expressed at 13 days post-hatch in winter flounder.

Biological Characterization of Two Novel Cathelicidin-derived Peptides and Identification of Structural Requirements for Their Antimicrobial and Cell Lytic Activities

Cathelicidins are a family of myeloid antimicrobial peptide precursors that have been identified in several mammalian species (Zanetti, M., Gennaro, R., and Romeo, D. (1995) FEBS Lett. 374, 1-5). Two novel bovine congeners have been deduced from cDNA. Their C-terminal sequences of 27 and 28 residues correspond to putative antimicrobial peptides with a cationic N-terminal region predicted to assume an amphipathic alpha-helical conformation followed by a hydrophobic C-terminal tail. Peptides corresponding to these sequences have been chemically synthesized and shown to exert a potent antimicrobial activity against Gram-negative and Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus, and fungi. Both peptides are also cytotoxic to human erythrocytes and neutrophils, although at higher than microbicidal concentrations. The target selectivity has been improved by synthesizing truncated analogues, comprising only the 18 N-terminal residues, which show a great reduction in cytotoxic, but not in antimicrobial activity. The involvement of the C-terminal hydrophobic tail in the cytotoxic activity has been further demonstrated by inducing a major loss of activity in an analogue after replacing highly hydrophobic residues with more hydrophilic ones.