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

Abstract

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 β-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.

Design of Protegrin-1 Analogs with Improved Antibacterial Selectivity

“…Experiments were performed with male and female 8–10-week-old BALB/c mice (22–24 g, “Andreevka” laboratory animal nursery FMBA, Russia) according to the previously described method [ 27 ]. Briefly, a total of 3 groups of 5 mice per group were infected by intraperitoneal (i.p.)…”

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

“…At the same time, similar to abarenicins, capitellacin, and alvinellacin, HfBRI-25 is stabilized by two disulfide bonds. We used bacterial expression systems for the heterologous production of HfBRI-25 fused with the modified thioredoxin A-a highly soluble carrier protein that was successfully used for the recombinant production of other β-hairpin AMPs stabilized by disulfide bonds [5,8,19]. The fusion protein was expressed in E. coli BL21 (DE3) or ClearColi BL21(DE3) cells, and the obtained total cell lysates were fractionated by affinity chromatography.…”

“…4 Molecular masses were calculated by considering the presence of four Cys residues forming two disulfide bonds in the case of HfBRI-25. 5 Molecular masses were determined experimentally using MALDI-TOF mass spectrometry. (B) Spatial structures of HfBRI-25 and HfBRI-28 were modeled using the AlphaFold algorithm (ColabFold server [20]).…”

“…To date, BRICHOS-related AMPs have been identified in a number of different marine worms. Interestingly, a single structural family of such peptides have been described for each species: β-hairpin arenicins from A. marina [2,4], β-hairpin abarenicins from Abarenicola pacifica [5], β-hairpin alvinellacins from sister species Alvinella pompejana [6] and A. caudata [7], β-hairpin capitellacin from Capitella teleta [8], α-helical nicomicins bearing a Cterminal loop stabilized by a disulfide bridge from Nicomache minor [9], α-helical (or dimeric β-sheet, exact structure is unknown) polaricin from Amphitritides sp. [10].…”

“…Direct peptide isolation is a challenge due to the labor-consuming process of catching small solitary animals followed by the fine purification of peptides from immune cells and body fluids. Therefore, the in silico blasting of known sequences of various AMPs and their preprosequences in genome/transcriptome databases [5,6,8,10] or the RACE-based approach [9] were used to identify novel BRICHOSrelated AMPs in further studies.…”