Molecular cloning and bioactivity assessment of a bioactive peptide QUB-1509 from the skin secretion of the Asian frog Hylarana guentheri

Abstract

The abuse of antibiotics has resulted in the increase of drug-resistant bacteria and as numerous people have now died from super-bacterial infections worldwide, the quest for and development of novel antibiotics is urgent and significant. Temporins are among the smallest peptides that have been discovered to disrupt the bacterial membrane directly which makes it difficult for bacteria to develop resistance.

In this study, the antimicrobial peptide QUB-1509, which was isolated from the skin secretion of the Asian frog, Hylarana guentheri, and belongs to the temporin family, was focused upon. A cDNA library was constructed by using the isolated mRNA from the skin secretion and ‘shotgun’ cloning was employed as an interrogation technique. After the mature sequence of the peptide was deduced from cloned precursor-encoding cDNA, solid phase peptide synthesis (SPPS) was employed to obtain the crude peptide. Reversed-Phase High Performance Liquid Chromatography (RP-HPLC) and Matrix-Assisted Laser Desorption Ionization, Time-of-Flight (MALDI-TOF) mass spectrometry were used to purify and identify QUB-1509. Bioassay of QUB-1509 showed that it possessed a broad-spectrum antibacterial activity against S. aureus (MIC=32 μM, MBC=64 μM), E. coli (MIC=128 μM, MBC=256 μM), C. albicans (MIC=128 μM, MBC=256 μM). QUB-1509 also showed cytotoxicity towards both HCT116 and U251MG human cancer cell lines. In the haemolysis assay, QUB-1509 showed low cytotoxicity towards horse red blood cells with no more than 15% haemolysis at 512 μM. These results have laid the foundation for the development of QUB-1509 as a potential antibacterial therapeutic.

Date of Award	Dec 2020
Original language	English
Awarding Institution	Queen's University Belfast
Supervisor	Lei Wang (Supervisor) & Mei Zhou (Supervisor)