Discovery, structural characterisation and targeted engineering of bioactive peptides from amphibian skin secretion

Amphibian skin secretions are remarkable sources of novel bioactive peptides. Among these, the antimicrobial peptides (AMPs) have demonstrated an outstanding efficacy in killing microorganisms via a general membranolytic mechanism, which may offer a prospect of solving specific target driven antibiotic-resistance. Here, the discovery of three novel defensive peptides is described from the skin secretion of the Indian cricket Frog, Fejervarya limnocharis and the Fujian large-headed frog, Limnonectes fujianensis, through the combination of ‘shot-gun’ cloning and MS/MS fragmentation sequencing. Subsequently, chemically-synthetic replicates were produced and subjected to bio-function evaluation.<br/><br/>In chapter 3, a novel brevinin-2 peptide was identified from the skin secretion of the Indian cricket Frog, Fejervarya limnocharis, namely QUB-3307. The results showed that QUB-3307 exhibited potent broad-spectrum antimicrobial activity, however, it induced significant cytotoxicity and haemolysis. To eliminate these side effects and reveal the function of the ‘Rana Box’ domain of QUB-3307, two truncated analogues, QUB-2531 and QUB-1376, were designed. QUB-2531 produced a low degree of cytotoxicity and haemolysis without decreasing the antimicrobial effect dramatically. However, QUB-1376 demonstrated lower antimicrobial potency. Interestingly, QUB-3307 and QUB-2531 exhibited an inhibitory effect on trypsin, which may be due to the presence of a typical Kunitz inhibitor motif, -KCK-, at their C-terminals. However, QUB-1376 failed to retain the inhibitory effect, which indicated that an intact Rana Box domain could contribute to the trypsin inhibition.<br/><br/>In chapter 4, a novel brevinin-1 peptide was identified from the skin secretion of the Indian cricket Frog, Fejervarya limnocharis, and was named QUB-2605. It demonstrated varying degrees of antimicrobial activities with high haemolysis. The modifications of the peptide were conducted to explain the structure-activity relationship from the N-terminus. QUB-2605 and its analogues shared the same family feature - the ‘Rana Box’- and the subfamily fragment feature motif, FLP-. Meanwhile, the introduction of Lys and Trp in the analogue peptide sequences revealed that antimicrobial activity of these designed analogues remained unchanged once the hydrophobicity and charge reached a threshold. Meanwhile, the correlation between hydrophobicity and haemolysis explained the structure-activity relationship. Hence, a new design idea that the hydrophobicity saturations in different situations related to antimicrobial activity could be the starting point for the generation of peptides with specific antimicrobial activity.<br/>In chapter 5, a novel temporin peptide was identified from the skin secretion of the Fujian large-headed frog (Limnonectes fujianensis), and was named QUB-1368. It demonstrated a broad-spectrum antimicrobial activity against several microorganisms except for Gram-negative bacteria. Target-modifications were carried out to further enhance the antimicrobial activity against Gram-negative bacteria via coupling QUB-1343 as the common functional domain of Onc112. Synergy checkerboard assay indicated that the hybrid peptide coupling of Onc112 and QUB-1368 contributed to functional improvement instead of cooperation of each fragment. Results indicated that hybrid peptide QUB-2696 exhibited its antimicrobial activity through multiple mechanisms, more than just membrane permeability.<br/><br/>In conclusion, the structure-activity relationships of AMPs were explained from modifications in both the N-terminus and C-terminus. Moreover, QUB-1952 and QUB-2696 represented excellent antimicrobial agents with high therapeutic indices.

Name dc:type.qualificationname

Doctor of Philosophy

Level dc:type.qualificationlevel

Doctoral Thesis

Grantor dc:publisher.institution

Queen's University Belfast

Year dc:date.issued

2020

Author dc:creator

• He, Haoyang

Advisors dc:contributor.advisor

• Zhou, Mei
• Wang, Lei
• Chen, Tianbao

• Amphibian skin secretion
• antimicrobial peptides (AMPs)
• structure-activity relationship
• molecular cloning
• hydrophobicity

Language dc:language

eng

Identifier

oai:pure.qub.ac.uk/portal:studenttheses/bac424ce-1521-4f2e-ab72-fdbaade5d141

OAI identifier oai:identifier

oai:pure.qub.ac.uk/portal:studenttheses/bac424ce-1521-4f2e-ab72-fdbaade5d141