Porcine leukocytes contained three homologous peptides, PG‐1, 2 and 3, that manifested potent microbicidal activity against Escherichia coli, Listeria monocytogenes and Candida albicans in vitro. The peptides (‘protegrins’) were composed of 16 (PG‐2) or 18 amino acid residues, and, like tachyplesins (broad‐spectrum antibiotic peptides of horseshoe crab hemocytes), they contained two intramolecular cystine disulfide bonds. Considerably smaller than defensins, protegrins nevertheless showed substantial homology to them, especially to the ‘corticostatic’ rabbit defensin, NP‐3a. The relatively simple structure of protegrins should provide useful prototypes for constructing congeners with selectively enhanced host defense activities.

No abstract

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.

We purified three homologous antimicrobial peptides ('gallinacins') from chicken leukocytes, examined their antimicrobial activity in vitro, and established their primary sequences by a combination of gas phase microsequencing and on-line IX-ESI-MS analysis of endo-and exoprotease peptide digests. The peptides contained 3&39 amino acid residues, were relatively cationic due to their numerous lysine and arginine residues, and each contained 3 intramolecular cystine disulfide bonds. Gallinacins showed primary sequence homology to the recently delineated p-defensin family, heretofore found only in the respiratory epithelial cells and neutrophils of cattle, suggesting that /3-defensins originated at least 250 million years ago, before avian and mammalian lineages diverged. The 9 invariant residues (6 cysteines, 2 glycines and 1 proline) common to avian gallinacins and bovine p-defensins are likely to constitute the essential primary structural motif of this ancient family of host-defense peptides.

The broad spectrum activity of amphiphilic helical AMPs is based on multiple stresses resulting from interactions with microbial membranes; however, rather than killing through formation of pores, the AMPs appear to interfere with the coordinated and highly dynamic functioning of membrane bound multienzyme complexes such as electron transport chains and cell wall or lipid biosynthesis machineries.