Mechanistic insights on the antibacterial action of the kyotorphin peptide derivatives revealed by in vitro studies and Galleria mellonella proteomic analysis

Objectives: The selected kyotorphin derivatives were tested to improve their antimicrobial and antibiofilm activity. The antimicrobial screening of the KTP derivatives were ascertained in the representative strains of bacteria, including Streptococcus pneumoniae, Streptococcus pyogenes, Escherichia coli and Pseudomonas aeruginosa. Methods: Kyotorphin derivatives, KTP-NH₂, KTP–NH₂–DL, IbKTP, IbKTP-NH₂, MetKTP-DL, MetKTP-LD, were designed and synthesized to improve lipophilicity and resistance to enzymatic degradation. Peptides were synthesized by standard solution or solid-phase peptide synthesis and purified using RP-HPLC, which resulted in >95 % purity, and were fully characterized by mass spectrometry and ¹H NMR. The minimum inhibitory concentrations (MIC) determined for bacterial strains were between 20 and 419 μM. The direct effect of IbKTP-NH₂ on bacterial cells was imaged using scanning electron microscopy. The absence of toxicity, high survival after infection and an increase in the hemocytes count was evaluated by injections of derivatives in Galleria mellonella larvae. Proteomics analyses of G. mellonella hemolymph were performed to investigate the underlying mechanism of antibacterial activity of IbKTP-NH₂ at MIC. Results: IbKTP-NH₂ induces morphological changes in bacterial cell, many differentially expressed proteins involved in DNA replication, synthesis of cell wall, and virulence were up-regulated after the treatment of G. mellonella with IbKTP-NH₂. Conclusion: We suggest that this derivative, in addition to its physical activity on the bacterial membranes, can elicit a cellular and humoral immune response, therefore, it could be considered for biomedical applications.