In our previous study, temporin-GHaR (GHaR) showed potent antimicrobial activity with strong hemolytic toxicity. To overcome its weakness, we designed GHaR6R, GHaR7R, GHaR8R, GHaR9R, and GHaR9W by changing the number of positive charges and the hydrophobic surface of GHaR. With the exception of GHaR7R, the hemolytic toxicity of the derived peptides had been reduced, and the antimicrobial activities remained close to the parent peptide (except for GHaR9R). GHaR6R, GHaR7R, GHaR8R, and GHaR9W exhibited a great bactericidal effect on Streptococcus mutans (S. mutans), which is one of the main pathogens causing dental caries. According to the membrane permeation and scanning electron microscope (SEM) analysis, these derived peptides targeted to the cell membranes of planktonic bacteria, contributing to the disruption of the membrane integrity and leakage of the intracellular contents. Moreover, they inhibited the formation of biofilms and eradicated the mature biofilms of S. mutans. Compared with GHaR7R, the derived peptides showed less cytotoxicity to human oral epithelial cells (HOECs). The derived peptides are expected to be the molecular templates for designing antibacterial agents to prevent dental caries.

The abuse of antibiotics has led to the emergence of multidrug-resistant bacteria, which is becoming a serious worldwide problem people have to face. In our previous study, temporin-GHa (GHa) cloned from Hylarana guentheri showed antimicrobial activity against Gram-positive bacteria. In order to improve its therapeutic potential, we used a template-based and a database-assisted design to obtain three derived peptides by replacing the histidine at both ends of GHa with lysine, which exhibited faster and stronger bactericidal activity and a broader spectrum than the parent peptide. GHaK and GHa4K targeted to the bacterial membrane to exert their antibacterial activities at a faster membrane damage rate. The derived peptides inhibited the initial adhesion and the formation of Staphylococcus aureus biofilms, and eradicated the mature biofilms, which indicated that the derived peptides effectively penetrated the biofilm and killed bacteria. The therapeutic index (TI) and cell selectivity index (CSI) of the derived peptides increased significantly, which means a broader therapeutic window of the derived peptides. The derived peptides with improved activity and cell selectivity have the potential to be the promising candidates for the treatment of S. aureus infections. Our research also provides new insights into the design and development of antimicrobial peptides.