Characterization of Antimicrobial Peptides from the Skin Secretions of Bornean Frogs belonging to the Family of Ranidae

Elizabeth Jega, Jenggut (2019) Characterization of Antimicrobial Peptides from the Skin Secretions of Bornean Frogs belonging to the Family of Ranidae. Masters thesis, Universiti Malaysia Sarawak (UNIMAS).

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Abstract

Antibiotic-resistant pathogens are now becoming a global problem in public health. Resistant microbes are difficult to treat and give impact to the nations, both economically and medically. Thus, this crisis necessitates the needs for alternative antimicrobial agents in which the bacteria has never been exposed to. Such alternative has been made possible by the discovery of antimicrobial peptides (AMPs) as the novel therapeutic agents which are found to confer a broad spectrum antibacterial activity. Recent discoveries of AMPs from frog skin secretions have become increasingly relevant to overcome the antibiotic resistance issue. The effectiveness of frog’s AMPs against microorganisms have made the frog peptides as a potential source for the development of novel antimicrobial agents. In this current research, the isolation, purification, identification and characterization of AMPs from the skin secretions of Bornean frog’s species were conducted. The species studied include Pulchrana glandulosa, Pulchrana signata, Pulchrana baramica, Odorrana hosii, Meristogenys jerboa and Chalcorana raniceps that inhabit the south-eastern region of Sarawak. Results of the preliminary study using disk diffusion test revealed encouraging antimicrobial activity in all of the tested species. Secretions were then partially purified and fractionated to near homogeneity using the high performance liquid chromatography (HPLC) technique. Microdilution steps were used to investigate the AMPs’ potency against Escherichia coli, Staphylococcus aureus and Candida albicans. Further analysis by CD spectrum and protein sequencing were then undertaken to elucidate the primary and secondary structures of the purified peptides. The results showed that the tested species possessed encouraging antimicrobial and antifungal activities. The activity of skin secretions’ peptides from M. jerboa are most potent with MIC= 12.5 µg/mL against E.coli isolates and MIC = 25 µg/mL against S. aureus isolates. Nonetheless, Pulchrana glandulosa, Pulchrana signata, Odorrnana hosii, Pulchrana baramica and Chalcorana iv raniceps shown promising results with MIC = 25, 50, 25, 125 and 100 µg/mL respectively against E.coli isolates. P. signata, C. raniceps, O. hosii and P. baramica inhibit S. aureus isolates at a minimum concentration of within 50 to 1000 µg/mL. All peptides showed moderate haemolytic activity at the concentration between 1.56 µg/mL to 12.5 µg/mL. Incubation of frog skin secretions in human red blood cell (RBC) for 1 hour at a concentration of more than 25 µg/mL demonstrated 50 percent lysis of the RBC, thus suggesting that AMPs, although not all, has moderate toxicity at the concentrations effective to kill bacteria. The CD spectrum of the peptides from all frog species showed that the dominant secondary structures are in the forms of β-pleated sheets and random coils with low composition of -helices. Mass spectrometry analysis revealed the peptides assigned for P. signata, C. raniceps and M. jerboa as Ranateurin-2 (GLFVQLVDDDPAYFDAK, Mr = 1911 Da), Distinctin (NLVTLATATTG, Mr = 1060 Da) and Magainin-2 (GIGMHLIAGIAK, Mr = 1195 Da) respectively. Two AMPs were isolated from O. hosii which are Odorannain-1 (VYSEVNSLSR, Mr = 1152 Da) and Odorannain-B (AAGVAAWLPLAR, Mr = 1194 Da). The peptide isolated from P. baramica is similar to a hypothetical protein (FPNDTAAR) found in Xenopous tropicalis whereas a peptide related to the cationic trypsin was isolated from P. glandulosa skin secretion

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