The Relationship Between Antimicrobial Activity And Structure Of Designed Analogues Based On Temporin-1CEb, An AMP From Chinese Brown Frog, Rana Chensinensis

Antimicrobial peptides (AMPs),small molecule peptides encoded by the specific genes,have broad-spectrum antimicrobial activity. For the multiple-antibiotic resistant strains, AMPs play an important role in the development of novel antibiotics because of their broad-spectrum antimicrobial activities and specific antimicrobial mechanism.Temporin-1CEb, a natural antimicrobial peptide purified from the skin secretions of Chinese brown frog, Rana chensinensis., contains 12 amino acid residues and its primary structure is ILPILSLIGGLL-NH2. Temporin-1CEb is identified as the member of temporin peptide family, consists of these antimicrobial peptides with small molecular weight. Temporin-1CEb displays low antimicrobial activity against Gram-positive bacteria, has no inhibitory effects on the growth of Gram-negative bacteria. Temporin-1CEb also exhibits a high hemolytic activity.In this study, we chose temporin-1CEb as a template to design a series of temporin-1CEb analogues for increasing the antimicrobial activity and decreasing the hemolytic activity by modifying cationic and hydrophobic characterizes, and the degree ofα-helix of peptide. Compare to temporin-1CEb, Anal-1-Anal-8 exhibited higher antimicrobial activity and lower hemolytic activity. Anal-1,Anal-2,Anal-3,Anal-4,Anal-6 and Anal-7 exhibited five up ten times antimicrobial activity in eight of designed temporin-1CEb analogues. Especially, Anal-2 and Anal-6 showed the potential to become a novel antimicrobial agent because of its high antimicrobial activity against both Gram-positive and Gram-negative bacteria with low cytotoxicity.The results of activity and structure of peptide indicate that antimicrobial activity of peptides can be increased by improving the cationicity and amphiphilicity by an appropriate substitution of L-lysine. High hydrophobicity and the degree ofα-helix is correlated with higher hemolytic activity.To clarify the mechanism of action,we examined the mechanism of Anal-2, one analogue of temporin-1CEb on cytoplasmic membrane of bacterium. The results of scanning electron microscopy (SEM) and cytoplasmic membrane depolarization indicated that the membrane were disrupted by of the antimicrobial peptide. Finally, bacteria were disintegrated into small fragments and led to the death.