Molecular cloning, structural characterisation and functional study of peptides from amphibian skin secretion
The skin secretions of amphibians contain large amounts of biologically-active compounds, such as antimicrobial peptides (AMPs), anticancer peptides and trypsin inhibitors. Several kinds of AMPs, isolated and characterised from the skin of amphibians, have been studied by researchers to provide novel kinds of drugs and pharmacological agents for humans to treat diseases. In this study, the skin secretions of the Chinese Piebald Odorous Frog (Odorrana schmackeri) and the European Edible Frog (Pelophylax kl. esculentus) were collected using an electrical stimulation method. Afterwards, three novel peptide (bombesin-OS, bombesin-PE and QUB2455) sequences were deduced from cDNAs and then confirmed by high performance liquid chromatography (HPLC) and tandem mass spectrometry (MS/MS). The pharmacological effects of bombesin-OS and bombesin-PE were tested. In addition, the antimicrobial activity and antibiofilm activity of AMPs were assessed and their haemolysis activity was also tested. Apart from these, the relationship between structure and function of AMPs was determined by means of CD experiments and secondary structure predictions. Moreover, the antimicrobial activity mechanism of AMPs was evaluated. In Chapter 3, the effects of bombesin-OS and bombesin-PE on smooth muscle were determined in bladder, uterus, and ileum. Both bombesin-OS and bombesin-PE possessed significant contractile activity on bladder, uterus and ileum. In Chapter 4, QUB2455 was found to exhibit strong bactericidal activity especially against Gram-positive organisms. Moreover, our results revealed that QUB2455 demonstrated strong antimicrobial activity against MRSA and E. faecalis. Additionally, QUB2455 showed low cytotoxicity against horse blood erythrocytes and cancer cells. It also possessed a higher TI value than ampicillin. The time-killing against MRSA of QUB2455 illustrated that QUB2455 could kill MRSA rapidly. In addition, QUB2455 possessed strong antibiofilm ability against S. aureus, MRSA, E. faecalis and C. albicans. Our results also revealed that QUB2455 caused increased membrane permeability in MRSA. The stability of QUB2455 revealed that QUB2455 retained good antimicrobial activity in the presence of high concentrations of salts, different pH values, high temperatures and even in serum. Moreover, three peptides, QUB1705, QUB1155, QUB1227, were designed. Our data revealed that all of the three designed peptides showed negligible antimicrobial activity. In Chapter 5, three short peptides, QUB1361, QUB1274 and QUB1457, were designed based on natural antimicrobial peptides, and it was confirmed that QUB1361, QUB1274 and QUB1457 possessed strong broad-spectrum bactericidal activity. Moreover, QUB1361, QUB1457 and QUB1274 possessed antibiofilm activity especially against S. aureus and MRSA. QUB1457 demonstrated the highest TI values for tested organisms among these designed short peptides. Additionally, QUB1457 retained good antimicrobial activity in different environments. The three short peptides showed low cytotoxity against horse blood erythrocytes and cancer cells. Moreover, QUB1457 also showed a rapid time-killing ability against MRSA. The membrane permeability results revealed that QUB1361, QUB1274 and QUB1457 demonstrated outer membrane and inner membrane permeability increases in E. coli. In conclusion, our results revealed that QUB2455 and QUB1457 are good candidates for treatment of bacterial infections such as those of S. aureus and MRSA. Thesis embargoed until 31st May 2024
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