Studies on novel bioactive peptides and their precursors from the skin secretions of Australian Pelodryadinae frogs

The research presented in this thesis is centred on the transcriptomic, peptidomic and pharmacological analysis of bioactive peptides which have been isolated and characterised from Australian Pe/odryadinae Frogs. In this thesis, the lyophilised skin secretions from species of the Litoria genus of Australian Pe/odryadinae Frogs, L. caeru/ea, L. aurea and L. infrafrenata, have been studied using a rapid 3' - and 5' - RACE 'Shot-gun' cloning strategy for procurement of bioactive peptide precursor-encoding cDNAs, without causing any detrimental effects to the donor specimens. Two novel cDNAs encoding biosynthetic precursors of the neuropeptide analogue, caerulein, and several cDNAs encoding precursors of novel antimicrobial peptides, were identified for the first time. NCBI-BLAST searches revealed a unique pattern of strikingly-conserved (prepro-) regions within precursors in contrast to the sequences of obviously hypervariable mature peptide-encoding domains. This phenomenon does not only occur in species of the Australian hylid sub-family, Pe/odryadinae, but has also been.observed in Asian, European and North American ranids (sub-family Raninae) and South American hylids (sub family PhyIJomedusinae), indicating their probable origins from multiple duplications of an ancestral gene presumably before the fragmentation of Gondwana (ca. 150 million years ago). Surprisingly, there was only a limited degree of sequence identity between the caerulein precursors from two distantly-related frog lineages, Litoria and Xenopus,indicating a high degree of divergence of general gene product during natural selection but with a high degree of conservation of active mature peptide sequence-encoding domains. Synthetic replicates of bioactive peptides were accessed by pharmacological screening of several bioassays including antimicrobial, biofilm bactericidal, anticancer and erythrocyte cytotoxic potency analysis. The novel caerin peptide, which possessed two amino acid residue substitutions when compared with caerin 1.12, exhibited a high degree of efficacy in antimicrobial, biofilm eradication, time-kill kinetic and anticancer assays with a high degree of selectivity for prokaryotic over eukaryotic cells. Less effective, but still active aurem peptides would be restricted for use by their inherent cytotoxicity. Frenatin peptides, as predicted, displayed relatively poor functionality. Iris hoped that the novel data generated from studies described in this thesis will, in the future, provide a modicum of support for predictions of fundamental peptide function and the evolutionary relationships of peptides discovered in amphibian skins. Peptides from Australian Pelodryadinae frogs described here, may require additional modifications prior to consideration as therapeutic agents, primarily to help eliminate side-effects through reducing cytotoxicity.EThOS - Electronic Theses Online ServiceGBUnited Kingdo