Molecular cloning and characterization of antimicrobial peptides from skin of Hylarana guentheri

<span class="paragraphSection"><div class="boxTitle">Abstract</div>The cDNAs encoding antimicrobial peptides (AMPs) in the skin of <span style="font-style:italic;">Hylarana guentheri</span> 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 (<span style="font-style:italic;">Staphylococcus aureus</span> and <span style="font-style:italic;">Bacillus subtilis</span>), Gram-negative bacteria (<span style="font-style:italic;">Escherichia coli</span>, <span style="font-style:italic;">Vibrio alginolyticus</span>, and <span style="font-style:italic;">Pseudomonas aeruginosa</span>), as well as fungus (<span style="font-style:italic;">Candida albicans</span>). Among these tested strains, <span style="font-style:italic;">S. aureus</span> 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 <span style="font-style:italic;">S. aureus</span> 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 <span style="font-style:italic;">S. aureus</span> V8 proteinase had no effect on the antimicrobial activity. Scanning electron microscopy images of <span style="font-style:italic;">E. coli</span> and <span style="font-style:italic;">S. aureus</span> 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 <span style="font-style:italic;">S. aureus</span> V8 proteinase make them to be promising candidates of novel antimicrobial agents or models for the development of novel AMPs.</span>

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