Abstract
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.
Highlights
Over recent years, a growing number of bacterial species became resistant to clinically significant antibiotics
Natural goat cathelicidins do not undergo significant post-translational modifications, heterologous expression in E. coli of the peptides fused with a carrier protein seems to be a reasonable approach for their production
Being the C-terminal part of a large carrier protein, apidaecins were proved to retain the ability to effectively inhibit the growth of bacterial cells during heterologous expression in E. coli (Taguchi et al, 1994)
Summary
A growing number of bacterial species became resistant to clinically significant antibiotics. Cathelicidins, one of the major groups of animal AMPs, are known to be the key molecular factors of innate immunity of most vertebrate species, from hagfish to human (Kosciuczuk et al, 2012). The precursors of cathelicidins are produced in immune and epithelia cells and contain the N-terminal part of 99–114 amino acid residues which is known as the cathelin domain. This structure is highly conserved among vertebrates, whereas the C-terminal domain, encoding the mature peptide, shows substantial heterogeneity. Neutrophils of some artiodactyls, including goats, do not contain defensin-like AMPs (Zhao et al, 1999), suggesting a key role of cathelicidins in the protection of these animals against pathogens. Study of artiodactyl cathelicidins can provide new molecular insight into their role in the host defense
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