Membrane mechanism of temporin-1CEc, an antimicrobial peptide isolated from the skin secretions of Rana chensinensis, and its systemic analogs

Antimicrobial peptides (AMPs) are new and powerful target molecules in the development of new antibacterial agents. Temporin-1CEc, a natural peptide isolated and purified from the skin secretions of the Chinese brown frog Rana chensinensis, exhibits low or no antibacterial activity against gram-negative and gram-positive bacteria, which limits its potential therapeutic use; however, it displays low hemolysis to human erythrocytes. Here, a series of temporin-1CEc analogs was designed and synthesized by amino acid residue substitutions based on cationicity, hydrophobicity, amphipathicity and secondary structure to understand the structure-activity relationships of this peptide in depth. The results showed that all of the analogs, except for 2K and 4K, had significantly improved antibacterial activity against the tested standard bacterial strains and multidrug-resistant bacterial strains compared to temporin-1CEc. 2K2L and 2K4L, but not 4K2L and 4K4L, showed the strongest antibacterial activity compared with their parent peptides 2K and 4K, suggesting that peptide hydrophobicity plays a more important role in antibacterial activity than cationicity for this series of AMPs. However, the antibacterial activity of the 6 Trp-containing analogs of 2K4L decreased with a further increase in hydrophobicity based on the results of 2K4L, indicating that it is more important to balance cationicity and hydrophobicity. Moreover, an increase in AMP hydrophobicity led to hemolysis. Notably, all of the peptides adopted α-helical structures in 50% trifluoroethanol/water and 30 mM SDS solutions. 2K2L and 2K4L displayed broad-spectrum antibacterial activity against sensitive and multidrug-resistant bacteria, effectively killing the tested multidrug resistant strain Staphylococcus epidermidis (MRSE1208). 2K2L and 2K4L were able to increase the permeability of the outer and inner membranes by depolarization and disturb the integration of the cytoplasmic membrane of MRSE1208 cells, leading to leakage of its cellular contents. In addition, 2K2L and 2K4L at low concentrations inhibited biofilm formation and degraded mature 1-day-old MRSE1208 biofilms. Notably, 2K2L and 2K4L inhibited the formation of MRSE1208 biofilms at concentrations below its MIC value, suggesting that the peptide may exert an inhibitory effect through not only direct antimicrobial activity but also a biofilm-specific mechanism. Collectively, these results suggest that 2K2L and 2K4L could be effective antibiotics against multidrug-resistant bacterial strains.