β-Hairpin antimicrobial peptides are among the most potent peptide antibiotics of animal origin. Arenicins, isolated earlier from marine polychaeta lugworm Arenicola marina, belong to a family of β-hairpin antimicrobial peptides and display a broad spectrum of biological activities. However, despite being potent antimicrobials, arenicins are partially unapplicable as therapeutics as a result of their relatively high cytotoxicity against mammalian cells. In this study, a template-based approach was used to create therapeutically valuable analogs of arenicin-1 and identify amino acid residues important for antibacterial and cytotoxic activities of the peptide. The plasmids encoding recombinant analogs were constructed by mutagenesis technique based on inverse PCR amplification of the whole arenicin-1 expression plasmid. The analogs were produced as a part of the fusion proteins in Escherichia coli. It was shown that an obvious reduction in hemolytic activity without lose of antimicrobial activity can be achieved by a single amino acid substitution in the non-polar face of the molecule with hydrophilic residues such as serine and arginine. As the result, the selective analog with 50-fold improved therapeutic index was developed. The circular dichroism spectra demonstrated that the secondary structure of the analog was similar to the natural arenicin-1 in water solution and sodium dodecyl sulfate micelles but significantly differed in the presence of dodecylphosphocholine micelles mimicking mammalian membranes. Similarly to arenicin-1, the designed analog killed bacteria via induction of the membrane damage, assessed using the fluorescent dye SYTOX Green uptake. Our results afford molecular insight into mechanism of antimicrobial action of the designed arenicin analogs and their possible clinical application.

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.

Endogenous antimicrobial peptides (AMPs) are evolutionary ancient molecular factors of innate immunity that play a key role in host defense. Among the most active and stable under physiological conditions AMPs are the peptides of animal origin that adopt a β-hairpin conformation stabilized by disulfide bridges. In this study, a novel BRICHOS-domain related AMP from the marine polychaeta Capitella teleta, named capitellacin, was produced as the recombinant analogue and investigated. The mature capitellacin exhibits high homology with the known β-hairpin AMP family—tachyplesins and polyphemusins from the horseshoe crabs. The β-hairpin structure of the recombinant capitellacin was proved by CD and NMR spectroscopy. In aqueous solution the peptide exists as monomeric right-handed twisted β-hairpin and its structure does not reveal significant amphipathicity. Moreover, the peptide retains this conformation in membrane environment and incorporates into lipid bilayer. Capitellacin exhibits a strong antimicrobial activity in vitro against a wide panel of bacteria including extensively drug-resistant strains. In contrast to other known β-hairpin AMPs, this peptide acts apparently via non-lytic mechanism at concentrations inhibiting bacterial growth. The molecular mechanism of the peptide antimicrobial action does not seem to be related to the inhibition of bacterial translation therefore other molecular targets may be assumed. The reduced cytotoxicity against human cells and high antibacterial cell selectivity as compared to tachyplesin-1 make it an attractive candidate compound for an anti-infective drug design.

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 β-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 β-hairpin AMPbased compounds might become the most promising drug candidates.

New bioengineering approaches are required for development of more active and less toxic antimicrobial peptides. In this study we used β-hairpin antimicrobial peptide arenicin-1 as a template for design of more potent antimicrobials. In particular, six shortened 17-residue analogs were obtained by recombinant expression in Escherichia coli. Besides, we have introduced the second disulfide bridge by analogy with the structure of tachyplesins. As a result, a number of analogs with enhanced activity and cell selectivity were developed. In comparison with arenicin-1, which acts on cell membranes with low selectivity, the most potent and promising its analog termed ALP1 possessed two-fold higher antibacterial activity and did not affect viability of mammalian cells at concentration up to 50 μM. The therapeutic index of ALP1 against both Gram-positive and Gram-negative bacteria was significantly increased compared with that of arenicin-1 while the mechanism of action remained the same. Like arenicin-1, the analog rapidly disrupt membranes of both stationary and exponential phase bacterial cells and effectively kills multidrug-resistant Gram-negative bacteria. Furthermore, ALP1 was shown to bind DNA in vitro at a ratio of 1:1 (w/w). The circular dichroism spectra demonstrated that secondary structures of the shortened analogs were similar to that of arenicin-1 in water solution, but significantly differed in membrane-mimicking environments. This work shows that a strand length is one of the key parameters affecting cell selectivity of β-hairpin antimicrobial peptides.