Antimicrobial peptides (AMPs) are considered as promising antimicrobial agents due to their potent bioactivity. Palustrin-2 peptides were previously found to exhibit broad-spectrum antimicrobial activity with low haemolytic activity. Therefore, GL-29 was used as a template for further modification and study. Firstly, the truncated analogue, GL-22, was designed to examine the function of the ‘Rana box’, which was confirmed to have no impact on antimicrobial activity. The results of antimicrobial activity assessment against seven microorganisms demonstrated GL-22 to have a broad-spectrum antimicrobial activity, but weak potency against Candida albicans (C. albicans). These data were similar to those of GL-29, but GL-22 showed much lower haemolysis and lower cytotoxicity against HaCaT cells. Moreover, GL-22 exhibited potent in vivo activity at 4 × MIC against Staphylococcus aureus (S. aureus)-infected larvae. Several short analogues, from the C-terminus and N-terminus of GL-22, were modified to identify the shortest functional motif. However, the results demonstrated that the shorter peptides did not exhibit potent antimicrobial activity, and the factors that affect the bioactive potency of these short analogues need to be further studied.
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