Novel BRICHOS-Related Antimicrobial Peptides from the Marine Worm Heteromastus filiformis: Transcriptome Mining, Synthesis, Biological Activities, and Therapeutic Potential.

Marine polychaetes represent an extremely rich and underexplored source of novel families of antimicrobial peptides (AMPs). The rapid development of next generation sequencing technologies and modern bioinformatics approaches allows us to apply them for characterization of AMP-derived genes and the identification of encoded immune-related peptides with the aid of genome and transcriptome mining. Here, we describe a universal bioinformatic approach based on the conserved BRICHOS domain as a search query for the identification of novel structurally unique AMP families in annelids. In this paper, we report the discovery of 13 novel BRICHOS-related peptides, ranging from 18 to 91 amino acid residues in length, in the cosmopolitan marine worm Heteromastus filiformis with the assistance of transcriptome mining. Two characteristic peptides with a low homology in relation to known AMPs-the α-helical amphiphilic linear peptide, consisting of 28 amino acid residues and designated as HfBRI-28, and the 25-mer ß-hairpin peptide, specified as HfBRI-25 and having a unique structure stabilized by two disulfide bonds-were obtained and analyzed as potential antimicrobials. Interestingly, both peptides showed the ability to kill bacteria via membrane damage, but mechanisms of their action and spectra of their activity differed significantly. Being non-cytotoxic towards mammalian cells and stable to proteolysis in the blood serum, HfBRI-25 was selected for further in vivo studies in a lethal murine model of the Escherichia coli infection, where the peptide contributed to the 100% survival rate in animals. A high activity against uropathogenic strains of E. coli (UPEC) as well as a strong ability to kill bacteria within biofilms allow us to consider the novel peptide HfBRI-25 as a promising candidate for the clinical therapy of urinary tract infections (UTI) associated with UPEC.

Design of Protegrin-1 Analogs with Improved Antibacterial Selectivity.

Protegrin-1 (PG-1) is a cationic ß-hairpin pore-forming antimicrobial peptide having a membranolytic mechanism of action. It possesses in vitro a potent antimicrobial activity against a panel of clinically relevant MDR ESKAPE pathogens. However, its extremely high hemolytic activity and cytotoxicity toward mammalian cells prevent the further development of the protegrin-based antibiotic for systemic administration. In this study, we rationally modulated the PG-1 charge and hydrophobicity by substituting selected residues in the central ß-sheet region of PG-1 to design its analogs, which retain a high antimicrobial activity but have a reduced toxicity toward mammalian cells. In this work, eight PG-1 analogs with single amino acid substitutions and five analogs with double substitutions were obtained. These analogs were produced as thioredoxin fusions in Escherichia coli. It was shown that a significant reduction in hemolytic activity without any loss of antimicrobial activity could be achieved by a single amino acid substitution, V16R in the C-terminal ß-strand, which is responsible for the PG-1 oligomerization. As the result, a selective analog with a ≥30-fold improved therapeutic index was obtained. FTIR spectroscopy analysis of analog, [V16R], revealed that the peptide is unable to form oligomeric structures in a membrane-mimicking environment, in contrast to wild-type PG-1. Analog [V16R] showed a reasonable efficacy in septicemia infection mice model as a systemic antibiotic and could be considered as a promising lead for further drug design.

Novel ß-Hairpin Peptide from Marine Polychaeta with a High Efficacy against Gram-Negative Pathogens.

In recent years, new antibiotics targeting multidrug resistant Gram-negative bacteria have become urgently needed. Therefore, antimicrobial peptides are considered to be a novel perspective class of antibacterial agents. In this study, a panel of novel BRICHOS-related ß-hairpin antimicrobial peptides were identified in transcriptomes of marine polychaeta species. Two of them-abarenicin from Abarenicola pacifica and UuBRI-21 from Urechis unicinctus-possess strong antibacterial potential in vitro against a wide panel of Gram-negative bacteria including drug-resistant strains. Mechanism of action assays demonstrate that peptides disrupt bacterial and mammalian membrane integrity. Considering the stronger antibacterial potential and a low ability of abarenicin to be bound by components of serum, this peptide was selected for further modification. We conducted an alanine and arginine scanning of abarenicin by replacing individual amino acids and modulating hydrophobicity so as to improve its antibacterial potency and membrane selectivity. This design approach allowed us to obtain the Ap9 analog displaying a high efficacy in vivo in the mice septicemia and neutropenic mice peritonitis models. We demonstrated that abarenicin analogs did not significantly induce bacterial resistance after a four-week selection experiment and acted on different steps of the biofilm formation: (a) killing bacteria at their planktonic stage and preventing biofilm formation and (b) degrading pre-formed biofilm and killing embedded bacteria. The potent antibacterial and antibiofilm activity of the abarenicin analog Ap9 with its high efficacy in vivo against Gram-negative infection in mice models makes this peptide an attractive candidate for further preclinical investigation.