Antimicrobial peptides (AMPs) are evolutionarily ancient factors of the innate immune system that serve as a crucial first line of defense for humans, animals, and plants against infection. This review focuses on the structural organization, biosynthesis, and biological functions of AMPs that possess a -hairpin spatial structure. Representatives of this class of AMPs are among the most active antibiotic molecules of animal origin. Due to their wide spectrum of activity and resistance to internal environmental factors, natural -hairpin AMPbased compounds might become the most promising drug candidates.

Being essential components of innate immune system, animal antimicrobial peptides (AMPs) also known as host-defense peptides came into sharp focus as possible alternatives to conventional antibiotics due to their high efficacy against a broad range of MDR pathogens and low rate of resistance development. Mammalian species can produce a set of co-localized AMPs with different structures and mechanisms of actions. Here we examined the combined antibacterial effects of cathelicidins, structurally diverse family of host-defense peptides found in vertebrate species. As a model we have used structurally distinct cathelicidins expressed in the leukocytes of goat Capra hircus. The recombinant analogs of natural peptides were obtained by heterologous expression in bacterial system and biological activities as well as the major mechanisms of antibacterial action of the peptides were investigated. As the result, the marked synergistic effect against wide panel of bacterial strains including extensively drug-resistant ones was observed for the pair of membranolytic α-helical amphipathic peptide ChMAP-28 and Pro-rich peptide mini-ChBac7.5Nα targeting a bacterial ribosome. ChMAP-28 was shown to damage the outer bacterial membrane at sub-inhibitory concentrations that could facilitate Pro-rich peptide translocation into the cell. Finally, resistance changes under a long-term continuous selective pressure of each individual peptide and the synergistic combination of both peptides were tested against Escherichia coli strains. The combination was shown to keep a high activity after the 26-days selection experiment in contrast to mini-ChBac7.5Nα used alone and the reference antibiotic polymyxin B. We identified the point mutation leading to amino acid substitution V102E in the membrane transport protein SbmA of the mini-ChBac7.5Nα-resistant strain obtained by selection. The experiments revealed that the presence of sub-inhibitory concentrations of ChMAP-28 restored the activity of mini-ChBac7.5Nα against this strain and clinical isolate with a weak sensitivity to mini-ChBac7.5Nα. The obtained results suggest a potential medical application of synergistic combinations of natural cathelicidins, which allows using a lower therapeutic dose and minimizes the risk of resistance development.

Arenicin-1, a β-sheet antimicrobial peptide isolated from the marine polychaeta Arenicola marina coelomocytes, has a potent, broad-spectrum microbicidal activity and also shows significant toxicity towards mammalian cells. Several variants were rationally designed to elucidate the role of structural features such as cyclization, a certain symmetry of the residue arrangement, or the presence of specific residues in the sequence, in its membranolytic activity and the consequent effect on microbicidal efficacy and toxicity. The effect of variations on the structure was probed using molecular dynamics simulations, which indicated a significant stability of the β-hairpin scaffold and showed that modifying residue symmetry and β-strand arrangement affected both the twist and the kink present in the native structure. In vitro assays against a panel of Gram-negative and Gram-positive bacteria, including drug-resistant clinical isolates, showed that inversion of the residue arrangement improved the activity against Gram-negative strains but decreased it towards Gram-positive ones. Variants with increased symmetry were somewhat less active, whereas both backbone-cyclized and linear versions of the peptides, as well as variants with R→K and W→F replacement, showed antimicrobial activity comparable with that of the native peptide. All these variants permeabilized both the outer and the inner membranes of Escherichia coli, suggesting that a membranolytic mechanism of action was maintained. Our results indicate that the arenicin scaffold can support a considerable degree of variation while maintaining useful biological properties and can thus serve as a template for the elaboration of novel anti-infective agents.

Endogenous antimicrobial peptides (AMPs) are among the earliest molecular factors in the evolution of animal innate immunity. In this study, novel AMPs named nicomicins were identified in the small marine polychaeta Nicomache minor in the Maldanidae family. Full-length mRNA sequences encoded 239-residue prepropeptides consisting of a putative signal sequence region, the BRICHOS domain within an acidic proregion, and 33-residue mature cationic peptides. Nicomicin-1 was expressed in the bacterial system, and its spatial structure was analyzed by circular dichroism and nuclear magnetic resonance spectroscopy. Nicomicins are unique among polychaeta AMPs scaffolds, combining an amphipathic N-terminal α-helix and C-terminal extended part with a six-residue loop stabilized by a disulfide bridge. This structural arrangement resembles the Rana-box motif observed in the α-helical host-defense peptides isolated from frog skin. Nicomicin-1 exhibited strong in vitro antimicrobial activity against Gram-positive bacteria at submicromolar concentrations. The main mechanism of nicomicin-1 action is based on membrane damage but not on the inhibition of bacterial translation. The peptide possessed cytotoxicity against cancer and normal adherent cells as well as toward human erythrocytes.