Rapidly growing resistance of pathogenic bacteria to conventional antibiotics leads to inefficiency of traditional approaches of countering infections and determines the urgent need for a search of fundamentally new anti-infective drugs. Antimicrobial peptides (AMPs) of the innate immune system are promising candidates for a role of such novel antibiotics. However, some cytotoxicity of AMPs toward host cells limits their active implementation in medicine and forces attempts to design numerous structural analogs of the peptides with optimized properties. An alternative route for the successful AMPs introduction may be their usage in combination with conventional antibiotics. Synergistic antibacterial effects have been reported for a number of such combinations, however, the molecular mechanisms of the synergy remain poorly understood and little is known whether AMPs cytotoxicy for the host cells increases upon their application with antibiotics. Our study is directed to examination of a combined action of natural AMPs with different structure and mode of action (porcine protegrin 1, caprine bactenecin ChBac3.4, human alpha- and beta-defensins (HNP-1, HNP-4, hBD-2, hBD-3), human cathelicidin LL-37), and egg white lysozyme with varied antibiotic agents (gentamicin, ofloxacin, oxacillin, rifampicin, polymyxin B, silver nanoparticles) toward selected bacteria, including drug-sensitive and drug-resistant strains, as well as toward some mammalian cells (human erythrocytes, PBMC, neutrophils, murine peritoneal macrophages and Ehrlich ascites carcinoma cells). Using “checkerboard titrations” for fractional inhibitory concentration indexes evaluation, it was found that synergy in antibacterial action mainly occurs between highly membrane-active AMPs (e.g., protegrin 1, hBD-3) and antibiotics with intracellular targets (e.g., gentamicin, rifampcin), suggesting bioavailability increase as the main model of such interaction. In some combinations modulation of dynamics of AMP-bacterial membrane interaction in presence of the antibiotic was also shown. Cytotoxic effects of the same combinations toward normal eukaryotic cells were rarely synergistic. The obtained data approve that combined application of antimicrobial peptides with antibiotics or other antimicrobials is a promising strategy for further development of new approach for combating antibiotic-resistant bacteria by usage of AMP-based therapeutics. Revealing the conventional antibiotics that increase the activity of human endogenous AMPs against particular pathogens is also important for cure strategies elaboration.

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Rhesus monkey bone marrow expresses a cathelicidin whose C-terminal domain comprises a 37-residue alpha-helical peptide (RL-37) that resembles human LL-37. Like its human counterpart, RL-37 rapidly permeabilized the membranes of Escherichia coli ML-35p and lysed liposomes that simulated bacterial membranes. When tested in media whose NaCl concentrations approximated those of extracellular fluids, RL-37 was considerably more active than LL-37 against staphylococci. Whereas human LL-37 contains five acidic residues and has a net charge of ؉6, rhesus RL-37 has only two acidic residues and a net charge of ؉8. Speculating that the multiple acidic residues of human LL-37 reduced its efficacy against staphylococci, we made a peptide (LL-37 pentamide) in which each aspartic acid of LL-37 was replaced by an asparagine and each glutamic acid was replaced by a glutamine. LL-37 pentamide's antistaphylococcal activity was substantially greater than that of LL-37. Thus, although the precursor of LL-37 is induced in human skin keratinocytes by injury or inflammation, its insufficiently cationic antimicrobial domain may contribute to the success of staphylococci in colonizing and infecting human skin.Cathelin is a porcine leukocyte peptide with 96 amino acid residues. Its name is an acronym for cathepsin L inhibitor and reflects an early belief that it was a cysteine-protease inhibitor (28). Subsequently, the cathelin sequence was recognized as a conserved domain in the precursors of many mammalian antimicrobial peptides that are now collectively known as "cathelicidins" (42).Cathelin-associated antimicrobial peptides are structurally diverse (14). Many have an amphipathic alpha-helical structure, while others are ␤-sheet peptides with intramolecular cystine disulfide bonds and some have numerous proline or tryptophan residues. The bovine cathelicidin peptides include a cyclic dodecapeptide (29), a tryptophan-rich tridecapeptide called indolicidin (8, 31), two proline-and arginine-rich bactenecins (10, 30), and at least three alpha-helical bovine myeloid antimicrobial peptides (BMAPs) (13,33). Porcine cathelicidin peptides include three alpha-helical porcine myeloid antimicrobial peptides (PMAPs) (35,38,43), five ␤-sheet protegrins, (20,44), and several proline-rich molecules (2, 15, 28). Sheep and goats also possess multiple cathelicidins (3,17,24,32).In contrast to the above, only a single cathelicidin, human cationic antimicrobial peptide of 18 kDa (hCAP-18), is currently known to exist in humans. This propeptide, whose Cterminal domain constitutes the 37-residue antimicrobial peptide called LL-37, is produced constitutively by precursors of neutrophils in the bone marrow and is stored within the secondary (specific) granules of the neutrophil (33). hCAP-18 is also produced constitutively by epididymal epithelial cells, so that large concentrations of hCAP-18 are present in normal seminal plasma and the peptide coats the surfaces of normal spermatozoa (25). Recent evidence suggests that certain human lymphocyte populations a...

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