Novel bioactive peptides from the defensive skin secretion of Phyllomedusinae frogs

Amphibian skin secretion contains a variety of bio-components, of which bioactive peptides are predominant. They produce such peptides to protect themselves from the threats in their environment like predatory attack and microorganism invasion. Antimicrobial peptides (AMPs) act as the first line of self-defence and plays an important role in preventing microbe infections via the skin contact. The skin secretion of phyllomedusinae tree frog contains massive quantities of AMPs, belonging to the dermaseptin superfamily, which exhibit remarkable antimicrobial activity against Grampositive bacteria, Gram-negative bacteria and fungi. In this thesis, we employed "shot-gun" molecular cloning and MS/MS sequencing techniques to identify novel AMPs from the skin secretion of selected Phyllomedusa species. Afterwards, we chemically synthesised these peptides to evaluate their biological activity as well as their cytotoxicity. In Chapter 3, a novel dermaspetin peptide was identified from the skin secretion of Phyllomedusa sauvagii, namely dermaseptin-PS3 (DPS3). This peptide exhibited moderate antimicrobial activity only against S. aureus, E. coli, and C. albicans (MICs are 512, 64, 128 mg/L, respectively). Additionally, DPS3 induced cell lysis on both cancer cells and red blood cells. Subsequently, one cationicity enhanced analogue and one hydrophobicity enhanced analogue were designed to determine the influence of both factors on the antimicrobial activity, anticancer activity and haemolytic activity. In Chapter 4, another novel dermaseptin peptide was identified by means of the same strategy, from the skin secretion of Phyllomedusa camba, namely dermaseptin-PC1 (DPC1). It is the first time a dermaseptin from has been identified in this species. DPC1 exhibited more potent antimicrobial activity against S. aureus, E. coli, and C. albicans (MICs are 64, 16, 64 mg/L, respectively) with a low degree of haemolytic activity. Furthermore, DPC1 showed anti-biofilm activity against both S. aureus and E. coli. In Chapter 5, we described the discovery of a prototype of a novel family of AMP from the skin secretion of Phyllomedusa hypochondrialis (now amended to Pithecopus hypochondrialis in RedList). This AMP showed a high degree of similarity to the chain B of distinctin, namely distinctin-like-peptide-PH (DLP-PH). DLP-PH showed antimicrobial activity against S. aureus, E. coli, and C. albicans at 256, 32, and 64 mg/L, and it also inhibited the growth of P. aeruginosa (MIC=64 mg/L). Subsequently, we investigated the biological function of both C- and N-terminal fragments of DLP-PH. The results of these chapters revealed that the genes of the dermaseptin superfamily are highly conserved, indicating the one-gene generated skin peptide library via gene duplication, focal hypermutation, and diversifying selection. Although these peptides display important antimicrobial and anticancer activities, the cytotoxicity and haemolysis cannot be nullified. Further investigation on reducing the membrane-lytic activity as well as improving biocompatibility should be taken into consideration. In conclusion, this thesis offered the chance to study novel dermaseptins and the prototype of novel antimicrobial templates.