Novel BRICHOS-related defensin-like antimicrobial peptide from the marine polychaeta Arenicola marina

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Abstract

To date, polychaetes remain a poorly studied class of invertebrate animals in the context of clarification of their immune system functioning and, in particular, of antimicrobial peptides (AMPs) biodiversity. AMPs, also known as host defense peptides, play a key role in host protection from various pathogens and regulation of the species composition of symbiotic microbes. The study of biosynthesis of AMPs in polychaetes has revealed an interesting pattern, namely so-called BRICHOS domain in the precursor proteins of a number of such peptides. The conserved structure of this domain allows to perform a bioinformatic search for AMP precursors in polychaete transcriptomes. In this work, we found and studied a new BRICHOS-associated AMP from the lugworm Arenicola marina, which represents a structural family of defensin-like peptides stabilized by four disulfide bonds, not previously identified in marine worms. The peptide, designated as AmBRI-44a, contained 44 amino acid residues and was obtained by heterologous expression in Escherichia coli. AmBRI-44a was shown to have a specific activity against a narrow spectrum of Gram-positive bacteria and did not exhibit pronounced cytotoxic effects on eukaryotic cell line HEK293T. A potential mechanism of the antibacterial action of this peptide may be associated with inhibition of bacterial cell wall biosynthesis, as indicated by genetic and phenotypic analysis of selected AmBRI-44a-resistant bacteria Bacillus licheniformis B-511. The results obtained allow us to consider the novel peptide AmBRI-44a as a candidate compound for the development of an antibiotic agent that could potentially be effective in the treatment of infectious diseases mediated by multidrug-resistant Gram-positive bacteria.

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V. N. Safronova

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

Email: ovch@ibch.ru
Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997

P. V. Panteleev

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

Email: ovch@ibch.ru
Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997

R. N. Kruglikov

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

Email: ovch@ibch.ru
Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997

I. A. Bolosov

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

Email: ovch@ibch.ru
Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997

E. I. Finkina

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

Email: ovch@ibch.ru
Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997

T. V. Ovchinnikova

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

Author for correspondence.
Email: ovch@ibch.ru
Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997

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Supplementary files

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2. Fig. 1. Primary structure of novel BRICHOS-associated peptides from A. marina. (a) Structural organization of precursor proteins of novel cysteine-rich peptides. TM – transmembrane (anchor) region; KR – site of potential processing by furin-like proteases (marked with “scissors”); AMP – region corresponding to mature peptide; (b) alignment of amino acid sequences of peptide AmIMP2 [18], novel peptides AmBRI-44a and AmBRI-44b from A. marina and hevein from Hevea brasiliensis (UniProt: P02877). Cysteine ​​residues are shown in black, arginine and lysine residues in gray. Aspartic and glutamic acid residues are underlined. The square brackets show the order of closure of the four disulfide bonds.

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3. Fig. 2. Preparation of the recombinant peptide AmBRI-44a and its structural characterization. (a) – Chromatogram of the purification of the AmBRI-44a peptide by RP-HPLC. The yield of the peptide was detected by the change in optical absorption at wavelengths of 214 nm (gray line) and 280 nm (black line). The fraction containing the target peptide is marked with an asterisk in the chromatogram; (b) – MALDI mass spectrometric analysis of the RP-HPLC fraction corresponding to the target peptide; (c) – circular dichroism (CD) spectra of the purified recombinant peptide AmBRI-44a in water and in dodecylphosphocholine (DPC) micelles. (d) – analysis of the secondary structure of the AmBRI-44a peptide using CD spectroscopy data and the CONTINLL program [35]; (d) – modeling of the spatial structure of the AmBRI-44a peptide was carried out using the AlphaFold2 algorithm (the ColabFold program [36]), the visualization of the model was obtained in the PyMol program. Disulfide bonds are highlighted in black; (e) – amino acid sequence of the AmBRI-44a peptide. Basic amino acid residues are highlighted in gray, acidic ones are underlined. Square brackets indicate the possible nature of the closure of disulfide bonds (in accordance with the predicted model of the spatial structure of the peptide).

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4. Fig. 3. Biological activity of the recombinant peptide AmBRI-44a. (a) – Antimicrobial activity against gram-positive bacteria and yeast-like fungi; (b) – cytotoxicity of the peptide against human cells.

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5. Fig. 4. The putative mechanism of antibacterial action of the AmBRI-44a peptide. (a) – Induction of resistance of Bacillus licheniformis B-511 bacteria to the AmBRI-44a peptide (initial MIC value 0.25 μM). After 27 days of successive passages in the presence of the AmBRI-44a peptide, the bacterial culture capable of growing at the maximum concentration of AMP was subcultured on MHB agar medium for 3 days, after which the final MIC value was determined; (b) – structural organization of the WalK sensor histidine kinase of B. licheniformis bacteria, which consists of five domains: PAS* (PER-ARNT-SIM sensor) – extracellular sensor domain; HAMP domain of the WalK protein (the abbreviation reflects the presence of the domain in the following proteins: Histidine kinases, Adenylyl cyclases, Methyl-accepting chemotaxis proteins, Phosphatases) is a cytoplasmic domain responsible for phosphorylation during signal transduction; PAS is a cytoplasmic sensor domain; HisKA is a dimerizing domain responsible for histidine autophosphorylation; HATPase is an ATPase domain of histidine kinase; (c) alignment of amino acid sequences of the HAMP domain of WalK histidine kinase for different bacteria. Previously discovered residues for which polymorphism and the corresponding mutant phenotype of vancomycin resistance in S. aureus bacteria were shown (according to data in [32, 37]) are highlighted in black. The WalK[R217S] mutation found in this study in B. licheniformis is highlighted in gray.

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