Arthropod antimicrobial peptides (AMPs) offer a promising source of new leads to address the declining number of novel antibiotics and the increasing prevalence of multidrug-resistant bacterial pathogens. AMPs with potent activity against Gram-negative bacteria and distinct modes of action have been identified in insects and scorpions, allowing the discovery of AMP combinations with additive and/or synergistic effects. Here, we tested the synergistic activity of two AMPs, from the dung beetle Copris tripartitus (CopA3) and the scorpion Heterometrus petersii (Hp1090), against two strains of Escherichia coli. We also tested the antibacterial activity of two hybrid peptides generated by joining CopA3 and Hp1090 with linkers comprising two (InSco2) or six (InSco6) glycine residues. We found that CopA3 and Hp1090 acted synergistically against both bacterial strains, and the hybrid peptide InSco2 showed more potent bactericidal activity than the parental AMPs or InSco6. Molecular dynamics simulations revealed that the short linker stabilizes an N-terminal 310-helix in the hybrid peptide InSco2. This secondary structure forms from a coil region that interacts with phosphatidylethanolamine in the membrane bilayer model. The highest concentration of the hybrid peptides used in this study was associated with stronger hemolytic activity than equivalent concentrations of the parental AMPs. As observed for CopA3, the increasing concentration of InSco2 was also cytotoxic to BHK-21 cells. We conclude that AMP hybrids linked by glycine spacers display potent antibacterial activity and that the cytotoxic activity can be modulated by adjusting the nature of the linker peptide, thus offering a strategy to produce hybrid peptides as safe replacements or adjuncts for conventional antibiotic therapy.

Anses INRAE Ecole Nationale Vétérinaire d'Alfort UMR BIPAR Laboratoire de Santé Animale F 94700 Maisons Alfort France

Department of Virology Veterinary Research Institute Hudcova 70 62100 Brno Czech Republic

Faculty of Science University of South Bohemia Branišovská 1160 31 37005 České Budějovice Czech Republic

Fraunhofer Institute for Molecular Biology and Applied Ecology Branch of Bioresources Ohlebersweg 12 35392 Giessen Germany

Institute for Insect Biotechnology Justus Liebig University of Giessen Heinrich Buff Ring 26 32 35392 Giessen Germany

Institute of Parasitology Biology Centre Czech Academy of Sciences Branišovská 1160 31 37005 České Budějovice Czech Republic

LOEWE Centre for Translational Biodiversity Genomics Senckenberganlage 25 60325 Frankfurt Germany

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