Peptidomic approach identifies cruzioseptins, a new family of potent antimicrobial peptides in the splendid leaf frog, Cruziohyla calcarifer.

UNLABELLED Phyllomedusine frogs are an extraordinary source of biologically active peptides. At least 8 families of antimicrobial peptides have been reported in this frog clade, the dermaseptins being the most diverse. By a peptidomic approach, integrating molecular cloning, Edman degradation sequencing and tandem mass spectrometry, a new family of antimicrobial peptides has been identified in Cruziohyla calcarifer. These 15 novel antimicrobial peptides of 20-32 residues in length are named cruzioseptins. They are characterized by having a unique shared N-terminal sequence GFLD- and the sequence motifs -VALGAVSK- or -GKAAL(N/G/S) (V/A)V- in the middle of the peptide. Cruzioseptins have a broad spectrum of antimicrobial activity and low haemolytic effect. The most potent cruzioseptin was CZS-1 that had a MIC of 3.77μM against the Gram positive bacterium, Staphylococcus aureus and the yeast Candida albicans. In contrast, CZS-1 was 3-fold less potent against the Gram negative bacterium, Escherichia coli (MIC 15.11μM). CZS-1 reached 100% haemolysis at 120.87μM. Skin secretions from unexplored species such as C. calcarifer continue to demonstrate the enormous molecular diversity hidden in the amphibian skin. Some of these novel peptides may provide lead structures for the development of a new class of antibiotics and antifungals of therapeutic use. BIOLOGICAL SIGNIFICANCE Through the combination of molecular cloning, Edman degradation sequencing, tandem mass spectrometry and MALDI-TOF MS we have identified a new family of 15 antimicrobial peptides in the skin secretion of Cruziohyla calcarifer. The novel family is named "Cruzioseptins" and contains cationic amphipathic peptides of 20-32 residues. They have a broad range of antimicrobial activity that also includes effective antifungals with low haemolytic activity. Therefore, C. calcarifer has proven to be a rich source of novel peptides, which could become leading structures for the development of novel antibiotics and antifungals of clinical application.