The cDNAs encoding antimicrobial peptides (AMPs) in the skin of Hylarana guentheri were identified, namely temporin (five peptides, termed temporin-GHa-GHd and temporin-GUa), brevinin-1 (one peptide, brevinin-1GUb), and brevinin-2 (eight peptides, brevinin-2GHd-2GHj, and brevinin-2GHb). Eleven of the 14 peptides have novel primary structures. Synthesized temporin GHa-GHd have broad-spectrum antimicrobial activities against Gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis), Gram-negative bacteria (Escherichia coli, Vibrio alginolyticus, and Pseudomonas aeruginosa), as well as fungus (Candida albicans). Among these tested strains, S. aureus was the most sensitive to temporin-GHa-GHd with minimum inhibitory concentration (MIC) values between 6.8 and 12.9 μM. They also exhibited antimicrobial activities against Methicillin-resistant S. aureus with the MIC ranging from 12.7 to 51.7 μM. Interestingly, secondary structure prediction shows that there is no α-helix in temporin-GHb, which illustrates that α-helix is not required for the antimicrobial activity of temporin-GHb. NaCl (at final concentrations of 0.15-2 M) decreased the antimicrobial activity of temporin-GHa-GHd slightly, while human serum and S. aureus V8 proteinase had no effect on the antimicrobial activity. Scanning electron microscopy images of E. coli and S. aureus showed that the surface of microbial cells was considerably rough and shrived after 1 h of treatment with temporin-GHa-GHd at 37°C. The stabilities of temporin-GHa-GHd in human serum or in S. aureus V8 proteinase make them to be promising candidates of novel antimicrobial agents or models for the development of novel AMPs.

Temporin-GHc (GHc) and temporin-GHd (GHd) produced by the frog Hylarana guentheri had shown broad-spectrum antibacterial activities against bacteria and fungi. In this study, we investigated whether they exert antibacterial and antibiofilm activities against cariogenic bacteria, Streptococcus mutans. GHc and GHd adopt the random coil conformation in aqueous solution and convert to α-helix in membrane mimetic environments by using circular dichroism spectroscope. They are positively charged by histidine, with the polar and nonpolar amino acids on opposing faces along the helix. The amphipathicity enabled the peptides to target at bacterial membrane, leading to an increase in membrane permeation and disruption of S. mutans, which allowed the peptides to bind with genomic DNA. GHc and GHd completely impeded the initial attachment of biofilm formation and disrupted preformed S. mutans biofilms. The expression of exopolysaccharide (EPS) biosynthesis genes which synthesize glucosyltransferases in S. mutans was downregulated by exposing to GHc or GHd, contributing to the decrease of soluble and insoluble EPS. GHc and GHd exhibited selectivity toward S. mutans in the presence of human erythrocytes, and no cytotoxicity toward human oral epithelial cells was observed at a concentration of 200 μM. These results laid the foundation for the development of GHc and GHd as potential anti-caries agents.

Brevinin-GR23 (B-GR23) was a brevinin-2 like antimicrobial peptide, which had antimicrobial activity against Staphylococcus aureus with minimum inhibitory concentration (MIC) of 16 μM. B-GR23 increased the bacterial membrane permeation, leading to the damage of membrane integrity and the leakage of genomic DNA, then causing the cell death. The peptide nearly inhibited all plantonic bacteria to start the initial attachment of biofilm at the concentration of 1 × MIC. Whereas the disruption rates on immature and mature biofilm decreased from 60% to 20%. B-GR23 reduced the production of extracellular polysaccharides (EPS) in the planktonic growth of S. aureus, which is a crucial structure of biofilm formation. B-GR23 with the concentration of ½ × MIC inhibited 50% water-soluble EPS, and 48% water-insoluble EPS, which contributed to the antibiofilm activity. B-GR23 had no significant toxicity to human blood cells under-tested concentration (200 μM), making it a potential template for designing antimicrobial peptides.

No abstract