First vertebrate BRICHOS antimicrobial peptides: ß-hairpin host defense peptides in limbless amphibia lung resemble those of marine worms.

Innate immunity of invertebrates offers potent antimicrobial peptides (AMPs) against drug-resistant infections. To identify new worm ß-hairpin AMPs, we explored the sequence diversity of proteins with a BRICHOS domain, which comprises worm AMP precursors. Strikingly, we discovered new BRICHOS AMPs not in worms, but in caecilians, the least studied clade of vertebrates. Two precursor proteins from Microcaecilia unicolor and Rhinatrema bivittatum resemble SP-C lung surfactants and bear worm AMP-like peptides at C-termini. The analysis of M. unicolor tissue transcriptomes shows that the AMP precursor is highly expressed in the lung along with regular SP-C, suggesting a different, protective function. The peptides form right-twisted ß-hairpins, change conformation upon lipid binding, and rapidly disrupt bacterial membranes. Both peptides exhibit broad-spectrum activity against multidrug-resistant ESKAPE pathogens with 1-4 µM MICs and remarkably low toxicity, giving 40-70-fold selectivity towards bacteria. These BRICHOS AMPs, previously unseen in vertebrates, reveal a novel lung innate immunity mechanism and offer a promising antibiotics template.

L-Cysteine and N-acetyl-L-cysteine-mediated synthesis of nanosilver-based sols and hydrogels with antibacterial and antibiofilm properties.

The need of the synthesis of a new generation of medicines aimed at combating bacteria and biofilms that cause various infections is a great urgency. There has been a gradual decrease in the conventional techniques of treatment with the use of antibiotics. Consequently, much effort has focused on the search for new methods and approaches to obtain antibacterial drugs and determine their rational use such that microorganisms do not acquire resistance. Although silver nanoparticles (AgNPs) and silver nanoclusters (AgNCs) have exhibited certain levels of effectiveness against multidrug-resistant bacteria and biofilms, there are very few simple, cheap and easy-to-scale methods to obtain AgNPs and AgNCs with well-desired characteristics. In this study, we carried out the one-pot synthesis of sols and gels containing AgNPs and AgNCs using only L-cysteine (CYS) or N-acetyl-L-cysteine (NAC), as bioreducing/capping/gel-forming agents, and different silver salts - nitrate, nitrite and acetate. HRTEM, SAED, EDX mapping, AFM, SEM, EDX, ICP-MS and FTIR spectroscopy analysis confirmed the formation of spherical/elliptical CYS-AgNP and NAC-AgNC particles consisting of AgNPs or AgNCs "core" and CYS/Ag+ or NAC/Ag+ complexes "shell" with mean average diameters of 10 and 5 nm, respectively. UV-Vis spectroscopy fixed the localized surface plasmon resonance (LSPR) at 390-420 nm for the CYS-AgNPs systems and LSPR absence for the NAC-AgNCs ones. DLS and nanoparticle tracking analysis (NTA) data indicated that the mean average diameter of the particles is about 80 nm for the CYS-AgNPs systems and 20 nm for the NAC-AgNCs ones. The Zeta potential measurements showed that the particles possess positive and negative charge values for CYS-AgNPs and NAC-AgNCs systems, respectively. The prepared materials demonstrated the high antibacterial activity against the most common types of bacteria at the MIC range of 10-100 µM, wherein the effect of the NAC-AgNCs systems is 2 times stronger than that of the CYS-AgNPs ones. Both systems are non-toxic or have low-toxicity at 300 µM for normal human cells: erytrocytes, fibroblasts and macrophages. Sols and hydrogels in the concentration range of 20-40 µM showed the complete inhibition of the formation of biofilms from Acinetobacter baumannii and Pseudomonas aeruginosa, which belong to the ESKAPE pathogenes group and represent the most serious problem in practical medicine. NAC-AgNCs systems were the most active. The simple strategy of the preparation of AgNP/AgNC-based sols and gels, along with their pronounced antibacterial and antibiofilm activity, could open new perspectives for its applications in medicine.

Combined Use of Antimicrobial Peptides with Antiseptics against Multidrug-Resistant Bacteria: Pros and Cons.

Antimicrobial peptides (AMPs) are acknowledged as a promising template for designing new antimicrobials. At the same time, existing toxicity issues and limitations in their pharmacokinetics make topical application one of the less complicated routes to put AMPs-based therapeutics into actual medical practice. Antiseptics are one of the common components for topical treatment potent against antibiotic-resistant pathogens but often with toxicity limitations of their own. Thus, the interaction of AMPs and antiseptics is an interesting topic that is also less explored than combined action of AMPs and antibiotics. Herein, we analyzed antibacterial, antibiofilm, and cytotoxic activity of combinations of both membranolytic and non-membranolytic AMPs with a number of antiseptic agents. Fractional concentration indices were used as a measure of possible effective concentration reduction achievable due to combined application. Cases of both synergistic and antagonistic interaction with certain antiseptics and surfactants were identified, and trends in the occurrence of these types of interaction were discussed. The data may be of use for AMP-based drug development and suggest that the topic requires further attention for successfully integrating AMPs-based products in the context of complex treatment. AMP/antiseptic combinations show promise for creating topical formulations with improved activity, lowered toxicity, and, presumably, decreased chances of inducing bacterial resistance. However, careful assessment is required to avoid AMP neutralization by certain antiseptic classes in either complex drug design or AMP application alongside other therapeutics/care products.

Caprine Bactenecins as Promising Tools for Developing New Antimicrobial and Antitumor Drugs.

Proline-rich antimicrobial peptides (PR-AMPs) having a potent antimicrobial activity predominantly toward Gram-negative bacteria and negligible toxicity toward host cells, are attracting attention as new templates for developing antibiotic drugs. We have previously isolated and characterized several bactenecins that are promising in this respect, from the leukocytes of the domestic goat Capra hircus: ChBac5, miniChBac7.5N-α, and -ß, as well as ChBac3.4. Unlike the others, ChBac3.4 shows a somewhat unusual pattern of activities for a mammalian PR-AMP: it is more active against bacterial membranes as well as tumor and, to the lesser extent, normal cells. Here we describe a SAR study of ChBac3.4 (RFRLPFRRPPIRIHPPPFYPPFRRFL-NH2) which elucidates its peculiarities and evaluates its potential as a lead for antimicrobial or anticancer drugs based on this peptide. A set of designed structural analogues of ChBac3.4 was explored for antibacterial activity toward drug-resistant clinical isolates and antitumor properties. The N-terminal region was found to be important for the antimicrobial action, but not responsible for the toxicity toward mammalian cells. A shortened variant with the best selectivity index toward bacteria demonstrated a pronounced synergy in combination with antibiotics against Gram-negative strains, albeit with a somewhat reduced ability to inhibit biofilm formation compared to native peptide. C-terminal amidation was examined for some analogues, which did not affect antimicrobial activity, but somewhat altered the cytotoxicity toward host cells. Interestingly, non-amidated peptides showed a slight delay in their impact on bacterial membrane integrity. Peptides with enhanced hydrophobicity showed increased toxicity, but in most cases their selectivity toward tumor cells also improved. While most analogues lacked hemolytic properties, a ChBac3.4 variant with two additional tryptophan residues demonstrated an appreciable activity toward human erythrocytes. The variant demonstrating the best tumor/nontumor cell selectivity was found to more actively initiate apoptosis in target cells, though its action was slower than that of the native ChBac3.4. Its antitumor effectiveness was successfully verified in vivo in a murine Ehrlich ascites carcinoma model. The obtained results demonstrate the potential of structural modification to manage caprine bactenecins' selectivity and activity spectrum and confirm that they are promising prototypes for antimicrobial and anticancer drugs design.