Identification and bioactivity assessment of a host-defence peptide, QUB-2654, from the skin secretion of the frog, Agalychnis callidryas

Abstract

Amphibian skin secretions contain a wide array of antimicrobial peptides with biological activities, providing an effective first-line of defence against microbial invasion. This thesis aims to characterise an antimicrobial peptide from the skin secretion of the Central American red-eyed tree frog, Agalychnis callidryas and to study its biological properties. Firstly, the cDNA sequence encoding an antimicrobial peptide (QUB-2654) precursor, was identified from the frog skin secretions extracted mRNAs through rapid-amplification of cDNA ends (RACE) PCR, molecular cloning and Sanger sequencing strategies. Next, the peptide was synthesised using a solid-phase peptide synthesiser, purified by reversed-phase high-performance liquid chromatography and corroborated by MALDI-TOF mass spectrometry. The purified peptide was tested for its antimicrobial activity, anti-lung cancer cell activity and haemolytic effect, by a series of bioassays. This study showed that QUB-2654 had significant bacteriostatic and bactericidal activities against a Gram-positive bacterium, Staphylococcus aureus (ATCC-CRM 6538) (MIC=3.125 µM, MBC=6.25 µM) and a Gram-negative bacterium, Escherichia coli (ATCC-CRM 8739) (MIC/MBC=6.25 µM), but the MIC value against Candida albicans (ATCC-CRM 10231) was not detected even at the highest peptide concentration. In addition, QUB-2654 statistically demonstrated anti-proliferative and killing effects against NCI-H838 human lung cancer cells at and above a concentration of 10 µM. The peptide concentration required to achieve half-maximal inhibition (IC50) of the NCI-H838 cells was 11.32 µM. This peptide was not haemolytic (<5% haemolysis) at the MBC values for S.aureus and E.coli, and at the IC50 value for NCI-H838 cells. These results suggest that peptide QUB-2654 has the potential to be developed as an antibacterial and anticancer drug.

Thesis is embargoed until 31 December 2027.

Date of Award	Dec 2022
Original language	English
Awarding Institution	Queen's University Belfast
Supervisor	Lei Wang (Supervisor), Tianbao Chen (Supervisor) & Xiaoling Chen (Supervisor)