Structural and Antimicrobial Features of Peptides Related to Myticin C

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Structural and antimicrobial features of peptides related to myticin C, a special defense molecule from the Mediterranean mussel Mytilus galloprovincialis Stefania Domeneghetti, Marco Franzoi, Nunzio Damiano, Rosa Norante, Nancy M. El Halfawy, Stefano Mammi, Oriano Marin, Massimo Bellanda, and Paola Venier J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b03491 • Publication Date (Web): 07 Oct 2015 Downloaded from http://pubs.acs.org on October 7, 2015

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Journal of Agricultural and Food Chemistry

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Structural and antimicrobial features of peptides related to myticin C, a special defense

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molecule from the Mediterranean mussel Mytilus galloprovincialis

3 4

Stefania Domeneghetti1§, Marco Franzoi1§, Nunzio Damiano2§, Rosa Norante3, Nancy M. El

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Halfawy4, Stefano Mammi5, Oriano Marin2,3, Massimo Bellanda5, Paola Venier1*

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1

Dept. of Biology, University of Padova, Via Ugo Bassi 58/B, 35131, Padova, Italy

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2

CRIBI Biotechnology Centre, Via Ugo Bassi 58/B, 35131 Padua, Italy

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3

Dept. of Biomedical Sciences, University of Padua, Via Ugo Bassi 58/B, 35131, Padova, Italy

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4

Dept. of Botany and Microbiology, Alexandria University, Moharam Bey 21511, 21526

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Alexandria, Egypt

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5

Dept. of Chemical Sciences, University of Padova, Via Marzolo 1, 35131, Padova, Italy

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§These authors equally contributed to the work

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*Corresponding author (e-mail: [email protected]; tel: 0039-0498276284)

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ABSTRACT

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Mussels (Mytilus spp.) have a large repertoire of cysteine-stabilized alphabeta peptides and myticin

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C (MytC) was identified in some hundreds of transcript variants after in vivo immunostimulation.

20

Using a sequence expressed in Italian mussels, we computed the MytC structure and synthesized

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the mature MytC and related peptide fragments (some of them also prepared in oxidized form) to

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accurately assess their antibacterial and antifungal activity. Only when tested at pH 5, the reduced

23

MytC as well as reduced and oxidized fragments including structural β-elements were able to

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inhibit Gram positive and negative bacteria (MIC range: 4-32 and 8-32 µM, respectively). Such

25

fragments caused selective E. coli killing (MBC: 8-32 µM) but scarcely inhibited two fungal

26

strains. In detail, the antimicrobial β-hairpin MytC[19-40]SOX, caused membrane-disrupting effects

27

in E. coli despite its partially ordered conformation in membrane-mimetic environments. In

28

perspective, MytC-derived peptides could be employed to protect acidic mucosal tissues, cosmetic

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and food products and, possibly, used as adjuvants in aquaculture.

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KEY WORDS

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Mytilus galloprovincialis, mussel, antimicrobial peptides, myticin C

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INTRODUCTION

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Cationic and cysteine-rich antimicrobial peptides (AMPs) play a central role in the innate immunity

34

of marine bivalves of the genus Mytilus. Arthropod-like defensins, mytilins, mytimycins and

35

myticins were originally isolated from acidified plasma fractions displaying antimicrobial activity

36

by high performance liquid chromatography (HPLC)1-3. The AA sequences of native myticin A

37

(MytA) and myticin B (MytB) were firstly obtained by mass spectrometry and Edman degradation

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and then confirmed by cDNA cloning and sequencing: their deduced molecular mass was consistent

39

with that of the purified peptides assuming the formation of four intramolecular disulfide bridges3.

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Though differing in 23 out of 96 amino acids, the MytA and MytB precursors are similarly

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organized in a highly conserved 20 AA signal peptide, a C-terminal extension of 36 AA, and a 2 ACS Paragon Plus Environment

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central 40 AA mature peptide which includes 8 invariant cysteines likely forming a canonical

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cysteine-stabilized alpha beta (CSαβ) motif, as in mussel mytilins and defensins. Actually, these

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AMP families diverge in primary sequence from one another and are unique to the Mytilus species4-

45

5

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Database of NCBI (pfam10690; http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml).

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The abundance of AMPs in hemocytes of adult mussels indicated these cells as the main production

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and storage site for both precursor and mature peptides3. While the anionic character of the C-

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terminal extension of mussel myticins may stabilize the positively charged central region within

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intracellular storage granules, the mature peptides are expected to exert their microbicidal role in

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pathogen-containing phagolysosomes, when released in the hemolymph and intercellular space or

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after massive cell degranulation4. The occurrence of MytA and MytB transcripts in tissues other

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than hemolymph reasonably depends on the circulating and infiltrating ability of hemocytes albeit

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other cells may play a role, such as mussel enterocytes in the case of mytilin B3,5.

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Myticin C (MytC) transcripts were later recognized in hemocytes of immunostimulated mussels as

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multiple sequence variants clustering apart from those of the already known MytA and MytB6. The

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deduced MytC pre-pro peptide showed similar organization, except for a slightly longer C-terminal

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extension of 40 AA (coding sequence: 100 AA). The virtual translation of 300 transcript variants

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denoting MytC in GenBank (e.g., AM498001.1, AM498018.1, AM498038.1, AM497990.1)

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suggested a molecular weight of 4.4 kDa, an isoelectric point at pH 8.8, and a 35% hydrophobicity

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ratio.

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High throughput amplicon sequencing revealed synonymous and non-synonymous single

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nucleotide changes along the whole MytC precursor transcript of farmed mussels from three

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European countries7. Such molecular diversity is consistent with the rapid and adaptive evolution of

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peptides actively recruited to target infectious agents evolving more quickly8-10. Actually, myticin

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precursor transcripts were the most abundant (20.2%) in hemocytes of adult mussels injected with

. The molecular hallmarks of myticin pre-pro peptides are described in the Conserved Domain



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heat-killed bacteria or poly I:C compared with mytilins (14.3%), defensins (1.2%) and very rare

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mytimycins6,11.

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Other cysteine-rich AMPs were subsequently identified in mussel: big defensins and mytimacins12,

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and an unusual myticusin-1 identified in M. coruscus was traced in M. galloprovincialis13. Seven

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sequences expressed in Mytilus coruscus and highly similar to the M. galloprovincialis myticins are

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present in GenBank (GU324724.1-GU324731.1, unpublished). The variety of mussel cysteine-rich

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peptides is currently under study and their functional roles have still to be disentangled14.

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Consistent with the onset of immune competence during development, MytC transcripts were

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detected in mussel oocytes, possibly a maternal effect, at levels raising beyond the settling transition

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at 15-18 days post-fertilization and peaking in the adult hemocytes6,15. Using specific riboprobes

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and antisera, granular hemocytes were confirmed as source of MytC transcripts and peptides15.

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The functional roles suggested for the mussel myticins span from antibacterial and antiviral effects

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to chemotactic and immunomodulatory behaviour. Native MytA and MytB preferentially killed

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Gram positive (G+; 2.25-4.5 µM MBC on Micrococcus luteus) than Gram negative (G-) bacteria

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and MytB also inhibited the fungus Fusarium oxysporum (MIC: 5-10 µM)3. The infectivity of

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VHSV, an enveloped virus causing hemorrhagic septicemia in fish, was significantly inhibited in

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Chinook salmon embryo cells transiently transfected with different GFP-MytC plasmids16. In the

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same study, raw lysates of the MytC-transfected cells induced hemocyte chemotaxis at variable

85

degree and some immune-related hemocyte genes appeared up-regulated at 48-72 h after in vivo

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injection of one MytC construct. As proof-of-concept, one chemically synthesized variant of the

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mature MytC partially decreased the VHSV infectivity in cultured fish cells at 62.5-125 µM range

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and significantly induced mussel hemocyte chemotaxis in filtered sea water at 0.025-0.25 µM

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range17. The same peptide inhibited the E. coli growth in the range 25-250 µM (70% reduction at

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250 µM) only when prepared at pH 3, a condition suggested to increase the α-helix component in


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the peptide structure, to cause aggregation of anionic phospholipids and destabilization of

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negatively charged membranes17.

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MATERIALS AND METHODS

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Modelling of the mature MytC. Precursor transcript variants of MytC were downloaded as

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nucleotide sequences from GenBank and Sequence Read Archive (http://www.ncbi.nlm.nih.gov).

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The AA conservation profile of the mature peptide only was achieved with the freely available tool

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WebLogo v3.4 using the sample SRR286640 (hemocytes sequence reads from mussels farmed in

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the Venice lagoon area)7. The 3D model of the mature peptide was computed with I-tasser18 and

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then refined with GROMACS v. 4.6.519 (www.gromacs.org).

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Based on Cα distances, disulfide bonds fully compatible with the cysteine positions (Cys 5-24, 10-

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33, 14-35, 19-38) were imposed. For all simulations, the Charmm forcefield was used in a

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5.188×5.188×3.630 nm cell filled with SPC/E water model, in isothermal-isobaric ensemble

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(NPT)20.

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A 1.2 nm distance cut-off was imposed both for the Lennard-Jones vdW potential and for the

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electrostatic interactions, using the particle mesh Ewald (PME) method with Fourier spacing 0.12

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and PME order 4. Berendsen and Parrinello-Rahman’s temperature-pressure coupling was chosen

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for solvent equilibration and simulated annealing, respectively, with isotropic pressure coupling.

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After a minimization of 2000 steps, the Linear Constraint Solver (LINCS) method for bond

109

constraints was chosen. For solvent equilibration, a 2 ps molecular dynamics simulation was

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performed at 300 K, with constrained protein atom positions. Finally, simulated annealing of 10 ns

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was performed with annealing time of 0, 2 and 8 ns, at temperatures of 300, 365 and 280 K for

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protein and 300, 320 and 280 K for water, respectively. For the 100 ns simulation, the same

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experimental protocol was used, except that the simulated annealing was replaced with free

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dynamics at 300 K.



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The superpose function of PyMOL (www.pymol.org) was used to overlay ribbon peptide structures;

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the homolog search in the PDB was performed via DALI server21.

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Peptide Synthesis. Synthetic peptides were prepared by the solid phase peptide synthesis method

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using a multiple peptide synthesizer (Syro II, MultiSynTech GmbH) on an LL Rink Amide resin

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(Novabiochem, Bad Soden, Germany) (Table 1). The fluoren-9-ylmethoxycarbonyl (Fmoc)

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strategy22 was used, utilizing 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium

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hexafluorophosphate (HATU) as coupling reagent23. The side-chain protected amino acid building

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blocks used were: N-α-Fmoc-Nω-(2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl)-l-arginine,

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N-α-Fmoc-O-tert-butyl-l-tyrosine, N-α-Fmoc-O-tert-butyl-l-serine, N-α-Fmoc-Nε-(tert-

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butyloxycarbonyl)-l-lysine, N-α-Fmoc-N(im)-trityl-l-histidine, N-α-Fmoc-S-trityl-cystine, Fmoc-

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Gly-Ser(Psi(Me, Me)pro)-OH, Fmoc-Thr(tBu)-Ser(Psi(Me, Me)pro)-OH. Cleavage of the peptides

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was performed by reacting the peptidyl-resins with a mixture containing TFA/ethanedithiol

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95%/5% for 2.5 h, and then precipitated by addition of cold diethyl ether. Crude peptides were

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purified by preparative HPLC with Prep. Nova-Pak Cartridge HRC18 (Waters Corporation,

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Milford, MA USA). Analytical HPLC analyses were performed on an Onyx Monolithic C18

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column (Phenomenex, Torrance, CA, USA) with a flow rate of 2 mL/min, and detection at 220 nm.

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The molecular peptide mass was confirmed by mass spectroscopy on a MALDI TOF–TOF mass

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spectrometer (model 4800, Applied Biosystems, Carlsbad, CA, USA). The peptide purity was in the

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range 95-98%, as evaluated by analytical HPLC.

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Oxidative Refolding. Following purification, the reduced peptides MytC[23-34] and MytC[19-

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40]S were dissolved at 1 mg/mL concentration in MQ water and allowed to spontaneously oxidize

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for 24 h. Disulphide bond formation was followed by HPLC and confirmed by mass spectroscopy

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using a MALDI TOF-TOF mass spectrometer for 48 h at room temperature. The resulting product

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was lyophilized. HPLC analysis was performed under the same conditions described above.


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Antibacterial Activity. Minimal Inhibitory Concentration (MIC) was determined on selected

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bacterial strains (Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853,

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Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212) using the broth

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microdilution susceptibility test24. Serial two-fold dilutions of each peptide (0.5-64 µM, final

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concentration) were prepared in 50 µL volume, using 96-well microtiter plates (Sarstedt, Germany)

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and 50% Muller-Hinton broth (MH; Difco Laboratories, USA) buffered at pH 5 or 7. Each dilution

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series included a control well without peptide. Fifty µL of bacterial culture (~5×105 CFU/mL in

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50% MH broth) were then added to each well. The MIC value was defined as the lowest peptide

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concentration that prevented visible bacterial growth after incubation for 24 h at 37 °C. The

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minimal bactericidal concentration (MBC) corresponded to the lowest peptide concentration that

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prevented any CFU after plating 50 µL from the wells onto nutrient agar plates. The reported MIC

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and MBC data result from three independent assays.

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Selected MytC fragments were tested on Micrococcus lysodeikticus ATCC 4698. Vibrio splendidus

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LGP32 and Vibrio aestuarianus, using suitable media: Poor Broth (5 g/L NaCl, 10 g/L

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bactotryptone, pH 7.5) for the standard G+ bacterium and Zobel medium modified for marine

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strains (artificial sea water at a third of the strength added with 4 g/L bactopeptone and 1 g/L yeast

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extract)25.

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The inhibition kinetics of MytC[19-40]SOX was comparatively evaluated on E. coli and E. faecalis

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at pH 5, using a greater bacterial inoculum (107 CFU/mL). The optical density was monitored every

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30 min at 620 nm for 24 h with the microplate reader Infinite 200 Pro (Tecan, Grödig, Austria).

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Antifungal Activity. The antifungal activity was evaluated on the sporogenous Aspergillus

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brasiliensis ATCC 16404 and the yeast Candida albicans ATCC 10231 strains using the broth

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microdilution assay26. Each peptide was prepared as indicated above (0.5-64 µM) and tested in

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Sabouraud broth (pH 5.8) inoculated with 50 µL of the yeast or spore suspension (no peptide in

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control wells). Yeast cells were cultivated overnight in liquid medium and their concentration 7 ACS Paragon Plus Environment


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adjusted to 2.5×105 CFU/mL, while the sporogenous strain was prepared by harvesting fungal

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spores and adjusting their concentration to 0.4–5×104 CFU/mL using a Bright Line™

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haemocytometer (Sigma, USA). The inoculated 96-well microplates were incubated at 35°C and

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MIC values were determined visually after 48 h. Minimal fungicidal concentration (MFC)

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corresponded to the lowest peptide concentration that prevented any CFU after plating 50 µL from

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the wells onto the corresponding nutrient agar plates.

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Permeability Assays. E. coli and E. faecalis bacteria were grown at 37°C in MH broth (pH 7) up to

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mid-logarithmic phase, washed and resuspended at 107 CFU/mL in 10 mM sodium phosphate

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buffer, pH 7.4. The cells were subsequently incubated for 15 min in the dark with 2 µM SYTOX ®

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Green Nucleic Acid Stain (Life Technologies), a dye that selectively permeates membrane-damaged

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bacteria. Then, the peptide to be tested was added at the MIC value and the fluorescence increase

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due to the binding of cationic dye to intracellular DNA was monitored every minute for 1 h

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(excitation and emission wavelengths: 485 nm and 520 nm, respectively). Melittin was used as

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positive control.

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Circular Dichroism Spectroscopy of MytC [19-40]SOX. Samples were prepared at room

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temperature by adding increasing amounts of DPC or SDS (0.5-20 mM final detergent

180

concentration) to a 30 µM peptide solution in 20 mM phosphate buffer, pH 7.4. The peptide

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concentration was determined by UV-absorbance at 290 nm. All CD measurements were performed

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with a Jasco J-715 spectropolarimeter (Tokyo, Japan) using a HELLMA quartz cuvette with optical

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path length of 0.2 cm. The CD spectra were acquired from 192 nm to 250 nm, with 2 nm

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bandwidth, 2 s time constant, and 50 nm/min scan speed; they were then processed with the Jasco J-

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700 software (version 1.50.01). The signal/noise ratio was improved by accumulating ten scans per

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experimental condition.

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NMR Analysis of MytC[19-40]SOX. The lyophilized peptide was dissolved in MQ H2O to final

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concentration of 1 mM, and incubated for 240 h at 277 K in order to achieve complete solvation. To 8 ACS Paragon Plus Environment

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the peptide samples, DPC or SDS were added to a final concentration of 300 mM; 10% 2H2O was

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added and the pH was adjusted to 7.0. NMR experiments were performed in 5 mm tubes at 295 K

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using an Avance DMX600 spectrometer (Bruker, Germany) operating at 599.90 MHz for 1H and

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equipped with a 5 mm TXI xyz-triple gradient probe. COSY spectra were recorded with 1 s

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recovery delay whereas the TOCSY spectra were acquired using a DIPSI-2 spin-lock, 70 ms

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mixing-time and 1 s recovery delay. For the NOESY experiments, 150 ms mixing time and 1.4 s

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recovery delay were used. Data were processed using TopspinTM (Bruker, Germany) and analyzed

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with CARA 1.9.0 Beta 7 software (cara.nmr-software.org).

197 198 199

RESULTS AND DISCUSSION

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Structural features of MytC. We chose the consensus sequence of a MytC variant previously

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described11 and predominantly expressed in farmed Italian mussels7 to compute the model of

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mature MytC using the I-tasser algorithm for protein folding prediction18. The five resulting

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structures showed unequivocal pairing of eight conserved cysteines, though the disulfide bridges

204

were not entirely defined. Connectivity between cysteines 5-24, 10-33, 14-35 and 19-38 were set as

205

molecular constraints for model refinement by simulated annealing with GROMACS19, a flexible

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toolkit for molecular dynamics simulation (Figure 1A). Following a further simulation at 300 K for

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100 ns, we selected three convergent MytC structures which clearly reproduced a compact CSαβ

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scaffold and a γ-core motif common to various AMPs and to venom components27. These models

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showed the most variable AA residues clustered mainly on the surface in the α-helical region

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(Figure 1B). Both the invariant cysteine array and tolerated sequence changes are essential for the

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biological function while minimizing the risk of pathogen resistance8, hence useful for nature-

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guided drug design28.



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The γ-core motif of MytC does not present the typical GxC element and is likely stabilized by

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aromatic stacking interactions between two highly conserved tyrosines, as previously observed in

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tachyplesin I, a β-hairpin AMP of 17 AA isolated from horseshoe crab29. Once superimposed, the

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structural sub-domains of MytC appeared closely similar to those of other CSαβ peptides, such as

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defensin 1 (MGD1)30 and mytilin B31 from M. galloprovincialis and butantoxin, a scorpion venom

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peptide stabilized by three disulfide bridges32(Figure 2). MytC (PF10690) and PDB accession codes

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of the other peptides reported in the figure are available in public protein databases33,34.

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While maintaining the invariant cysteine array and stacking tyrosines, changes of the variable

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residues could be considered for the development of MytC-derived antimicrobial modules of

222

minimal size.

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Chemical synthesis of peptides mapping the mature MytC. We synthesized and purified several

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fragments and analogues, designed all along the 40 AA MytC sequence, aiming to explore their

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antimicrobial and antifungal activity (Table 1). Specifically, the β-turn suspected to play a role in

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the antimicrobial activity was covered with fragments of 12 and 22 residues: MytC[23-34],

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MytC[19-40], and MytC[19-40]S. In the latter, three Cys were replaced with Ser, to obtain a single

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intramolecular disulfide bridge after spontaneous oxidation for 24 h, yielding peptide MytC[19-

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40]SOX. According to MS analysis, the empirical mass values of the reduced and oxidized

230

fragments are consistent with the expected values reported in Table 1 (Figure 3: A, B). We also

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synthesized and purified the whole 40 AA peptide for comparative purposes (Figure 3C ). The

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sequence of MytC[1-40] makes its synthesis particularly difficult. Partial hydrophobicity and the

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tendency to adopt a beta structure lead to peptide aggregation on the resin. To overcome this issue,

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a strategy based on the coupling of Gly-Ser in positions 24-25 and Thr-Ser in positions 33-34 as

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pseudo-prolines (dimethyloxazolidine analogs) was followed. These building blocks have a

236

preference for cis-amide bonds resulting in a twisted conformation of the peptide backbone35. This


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strategy allowed us to greatly increase the global yield up to 20% after peptide purification (a

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promising result for the synthesis of other similar peptides).

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MytC-derived peptide fragments show pH-dependent antimicrobial and antifungal activity.

240

AMPs can exhibit differential antimicrobial activity at different pH36,17.When dissolved and tested

241

at pH 7 on G+ and G- bacteria, the nine synthetic peptides of MytC were inactive or just weakly

242

active, with minimal inhibitory concentration (MIC) values above 64 µM, except for the G+

243

Enterococcus faecalis which was inhibited by 32 µM MytC[1-40], MytC[29-40], MytC[19-40],

244

MytC[19-40]S and by 16 µM MytC[19-40]SOX (Suppl. Table S1).

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The assays were repeated at pH 5 with the same peptides, this time evaluating both inhibitory and

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killing activity against bacterial, fungal and yeast strains (Table 2). At acidic pH, a greater number

247

of positive charges could improve the peptide interaction with bacteria37; moreover, the ionic

248

interactions are enhanced at the lower salt content of the medium. MytC[1-40] exhibited a better

249

MIC on G+ (4 µM, E. faecalis) than G- bacteria (32 µM, E. coli), similar to folded defensins from

250

M. galloprovincialis, Crassostrea gigas and Venerupis philippinarum5,25,38. Though partially

251

including α and β elements, MytC[9-28] inhibited only E. faecalis at 16 µM.

252

The three MytC fragments of 22 AA, covering the β elements, inhibited E. coli and E. faecalis at 8-

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16 µM and Staphylococcus aureus at 32 µM. MytC[19-40] was more effective than its substituted

254

and oxidised forms in killing E. coli (8 µM MBC). The tested peptides did not inhibit or kill

255

Candida albicans except MytC[19-40]SOX (16 µM) and MytC[19-40]S (32 µM). Overall, the

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activating effect of the acidic vs. neutral pH supports previous findings obtained in E. coli with a

257

synthetic MytC variant17 and the antimicrobial effectiveness of MytC in the endophagosome39. In

258

our experimental conditions, E. faecalis (G+) and Pseudomonas aeruginosa (G-) were the most and

259

least sensitive strains, respectively.

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In essence, the three 22 AA peptide fragments including two antiparallel β-strands and the putative

261

β-turn were bacteriostatic, bactericidal and somewhat fungistatic. Following a similar mapping 11 ACS Paragon Plus Environment


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approach, the antimicrobial activity of tenecin 1, an insect defensin, was also located in the β-sheet

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region40. Analogous peptide fragments from MGD1 inhibited Micrococcus lysodeikticus at 10-16

264

µM and E. coli at 22-62 µM when tested in poor broth5. Moreover, β-hairpins from insect defensins

265

killed G+ bacteria in the 1.1-14.9 µM range, with no evidence of antifungal activity41. Related to

266

these [19-40] fragments, the natively folded MytC with its putative helical element could present

267

increased stability as well as more efficient interaction/permeation of microbial membranes5,41.

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Besides, we tested the 22 AA peptides on M. lysodeikticus, Vibrio splendidus LGP32 and Vibrio

269

aestuarianus, the latter as opportunistic components of marine bivalve microbiomes42. Only

270

MytC[19-40]S and MytC[19-40]SOX inhibited M. lysodeikticus at 32 µM (Suppl. Table S2). Earlier,

271

a β-hairpin peptide designed on MytB and stabilized by two disulphide bonds inhibited M.

272

lysodeikticus (1 mM) more than V. splendidus LGP32 (125 µM)31 and defensin-like peptides were

273

found scarcely active on Vibrio spp. even in poor broth5,43,38.

274

At the MIC value of 8 and 16 µM, respectively, MytC[19-40]SOX caused a CFU reduction of 3 logs

275

on E. coli and 4 logs on E. faecalis (MBC: 32 µM and 64 µM, respectively) (Suppl. Table S3).

276

Additional kinetic absorbance measurements performed at pH 5 with the same G- and G+ strains

277

(107 CFU/mL, 0-64 µM range) highlighted a different bacteriostatic effect. MytC[19-40]SOX

278

partially inhibited E. coli at 8 µM (MIC) and likely caused immediate killing at 16-64 µM.

279

Conversely, the E. faecalis growth was delayed but not blocked in the 8-64 µM dose range. For

280

comparison, the pore-forming helical melittin tested at the MIC value on the same strains (2 µM on

281

E. coli; 16 µM on E. faecalis) produced immediate and total growth inhibition44,45 (Figure 4).

282

The pH dependency of the observed antimicrobial effects and the low micromolar MIC of the

283

unfolded 40 AA MytC support the hypothesis that MytC can act in multiple ways and persist in vivo

284

like human defensin 5 in the paneth cells (thioredoxin-dependent reduction and zinc-mediated

285

resistance to hydrolytic breakdown)46.


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We further assessed the inhibition mechanism of MytC[19-40]SOX on E. coli and E. faecalis at the

287

MIC value by monitoring the uptake of SYTOX green, a nucleic acid binding molecule (Figure 5).

288

The positive control melittin (red line) induced immediate membrane damage on both bacterial

289

strains whereas the fluorescence increase starting five min after the addition of MytC[19-40]SOX

290

(black line) indicated a progressive damage of the E. coli membranes. Conversely, the fluorescence

291

values obtained with E. faecalis remained close to the baseline, suggesting a different mechanism of

292

action. The β-hairpin region is critical for the antimicrobial activity, even if the cysteine scaffold

293

with the α-helix is missing, and the selective membrane-disrupting action of MytC[19-40]SOX on G-

294

(E. coli) related to G+ bacteria (E. faecalis) likely depends on cell wall differences. Whether the

295

killing effect is due to direct membrane damage or consequent to events occurring in the reducing

296

cytosolic environment47 requires further study.

297

Direct accessibility to Lipid II in G+ bacteria (only reachable by perturbing the outer membrane in

298

G- bacteria) and subsequent inhibition of cell wall biosynthesis have been hypothesized to explain

299

the more pronounced antimicrobial effects of folded invertebrate defensins on G+48,39. Supposing a

300

similar behavior for MytC, our data suggest that the β-hairpin alone is able to damage the G-

301

membranes with only partial affinity to Lipid II. This could explain the lower MBC showed by the

302

MytC [19-40] fragments on E. coli compared to E. faecalis.

303

MytC[19-40]SOX adopts an unpredicted, atypical conformation in membrane-mimetic

304

environments. MytC[19-40]SOX was preliminary subjected to conformational analysis using far-

305

UV circular dichroism (CD) spectroscopy in different experimental conditions. In spite of one

306

disulfide bridge linking its N- and C-termini, the peptide did not show a defined secondary structure

307

in aqueous solution, as expected from the MytC model structure.

308

In the presence of dodecylphosphocholine (DPC) or sodium dodecyl sulfate (SDS) above the

309

critical micelle concentration, the CD spectra changed significantly, indicating at least a partially

310

structured peptide in the micellar environments. Specifically, the CD spectra displayed a positive 13 ACS Paragon Plus Environment


Page 14 of 32

311

peak below 200 nm and a negative one at around 217 nm in the presence of either detergent. Other

312

disulfide-stabilized β-hairpins displayed a CD spectrum with two positive bands at around 200 and

313

230 nm and a negative one between 205 and 210 nm49. Five aromatic residues and the disulfide

314

bridge might explain the atypical CD spectra of MytC[19-40]SOX (Suppl. Figure S1).

315

To get more insights on the structure adopted in micelle environments, we acquired sets of two-

316

dimensional homonuclear NMR spectra of MytC[19-40]SOX in the presence of SDS or DPC. The

317

dispersion of the amide protons was larger in both detergent solutions than in aqueous buffer,

318

confirming that the peptide is at least partially structured in the micellar environments (Figure 6A).

319

In both cases however, an excessive number of spin systems suggested structural heterogeneity. In

320

the presence of SDS, severe broadening of the peaks occurred during the acquisition of the NMR

321

dataset, most likely because of aggregation events. Nevertheless, the assignment was completed at

322

least for the most populated conformer, both in SDS and DPC. Due to extensive resonance overlap,

323

line broadening and lack of long-range NOEs, it was not possible to proceed with structure

324

calculation. Nevertheless, the small negative deviation of the Hα chemical shifts from typical

325

random coil values indicates a weak propensity to form helical segments. Moreover, the comparison

326

of Hα secondary chemical shifts in the two media suggests similar peptide conformation50 (Figure

327

6B). A consecutive stretch of α(i)-NH(i+2) NOE correlations in DPC indicates the presence of a 310

328

helix between Ser-2 and Leu-9. The signal between His-10 Hα and Lys-13 HN suggests a loop

329

between these two residues. Peptide flexibility from Ser-15 to Arg-22 could explain a localized

330

absence of medium range correlations and the general lack of long range peaks in the NOESY

331

spectrum (Figure 6C). In our experimental conditions, there is no evidence of a stable,

332

independently folded β-hairpin and, consistent with the CSαβ motif predicted for the mature MytC,

333

its formation would likely require two additional disulfide bonds with the preceding α-helix. Amino

334

acids substitutions designed to provide an additional disulfide could stabilize the hairpin

335

conformation. On the other hand, the conformation adopted by MytC[19-40]SOX in the presence of 14 ACS Paragon Plus Environment

Page 15 of 32


336

bacterial membranes could differ from that observed in micellar environments, thus explaining its

337

antimicrobial activity.

338

In agreement to the nature-guided development of bioactive molecules, rigorous structural and

339

biological data are an essential step to understand the biological roles of MytC. In perspective,

340

MytC-derived peptides acting at low or neutral pH could be validated to protect acidic mucosal

341

tissues, cosmetic and food products and possibly used as adjuvants in aquaculture.

342

Abbreviations

343

AA, amino acid

344

AMPs, antimicrobial peptides

345

CD, circular dicroism

346

COSY, correlation spectroscopy

347

CSαβ, cysteine-stabilized alpha beta

348

DPC, dodecylphosphocholine

349

EST, expressed sequence tag

350

GFP, green fluorescence protein

351

G+/G-, Gram positive/Gram negative

352

HPLC, high performance liquid chromatography

353

MIC, minimal inhibitory concentration

354

MBC, minimal bactericidal concentration

355

MS, mass spectrometry

356

MQ, MilliQ [water]

357

Myt, myticin [MytC, myticin C] 15 ACS Paragon Plus Environment


Page 16 of 32

358

NMR, nuclear magnetic resonance

359

NOE/NOESY, Nuclear Overhauser Effect/ Nuclear Overhauser Effect Spectroscopy

360

SDS, sodium dodecyl sulfate

361

TOCSY, total correlation spectroscopy

362

vdW, van der Waals

363

ACKNOWLEDGMENTS

364

We thank Umberto Rosani (INNOV-H20 network for technological innovation in aquaculture) for

365

updating the MytC ESTs analysis.

366

ASSOCIATED CONTENT

367

S Supporting Information

368

Tables S1, S2, S3 and Figure S1. This material is available free of charge via the Internet at

369

http://pubs.acs.org.

370

AUTHOR INFORMATION

371

Corresponding Author

372

PV. E-mail: [email protected]. Address: Department of Biology, Via U. Bassi 58/B, 35131

373

Padova, Italy

374

SD, MF and ND did the experimental work and critically contributed to its development. RN and

375

NMEH contributed to the synthesis and antifungal assays, respectively. MB and SM, OM, and PV

376

supervised selected parts of work. PV designed the work and drafted the manuscript. All Authors

377

contributed to the manuscript and approved its final version.

378

Funding


Page 17 of 32


379

Work primarily funded by PRAT 2012 (CPDA128951) to PV and, in part, by PRIN 2008-2010

380

(20109XZEPR) to PV and SM. According to the objectives of the mentioned projects, SD and RN

381

were contracted as post-doc and graduated fellows, respectively. We thank the Ministry of Higher

382

Education (MoHE) and Missions Sector-Egypt for granting a visiting research scholarship to

383

NMEH.

384 385

Notes

386

The authors declare no competing financial interest.

387



388 389

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390

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[Erratum in: Eur. J. Biochem. 240, 815].

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3. Mitta G.; Hubert F.; Noël T.; Roch P. Myticin, a novel cysteine-rich antimicrobial peptide

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4. Mitta G.; Vandenbulcke F.; Roch P. Original involvement of antimicrobial peptides in mussel innate immunity. FEBS Lett. 2000a, 486, 185-90.

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7. Rosani, U.; Varotto, L.; Rossi, A.; Roch, P.; Novoa, B.; Figueras, A.; Pallavicini, A.;

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8. Padhi, A.; Verghese, B. Molecular diversity and evolution of myticin-C antimicrobial

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415 416 417

10. Tassanakajon, A.; Somboonwiwat, K.; Amparyup, P. Sequence diversity and evolution of antimicrobial peptides in invertebrates. Dev. Comp. Immunol. 2014, 48, 324-41. 11. Venier, P.; De Pittà, C.; Bernante, F.; Varotto, L.; De Nardi, B.; Bovo, G.; Roch, P.; Novoa,

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12. Gerdol, M.; De Moro, G.; Manfrin, C.; Venier, P.; Pallavicini, A. Big defensins and

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mytimacins, new AMP families of the Mediterranean mussel Mytilus galloprovincialis. Dev.

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Comp. Immunol. 2012, 36, 390-9.

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13. Liao Z.; Wang X.C.; Liu H.H.; Fan M.H.; Sun J.J.; Shen W. Molecular characterization of a

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novel antimicrobial peptide from Mytilus coruscus. Fish Shellfish Immunol. 2013, 34, 610-6.

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14. Gerdol M.; Puillandre N.; De Moro G.;, Guarnaccia C.; Lucafo M.; Benincasa M.; Zlatev

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V.; Manfrin C.; Torboli V.; Giulianini P.G.; Sava G.; Venier P.; Pallavicini A. Identification

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and characterization of a novel family of cysteine-rich peptides(MgCRP-I) from Mytilus

428

galloprovincialis. Genome Biol. Evol. 2015, 7(8):2203-19.

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15. Balseiro, P.; Moreira, R.; Chamorro, R.; Figueras, A.; Novoa, B. Immune responses during

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the larval stages of Mytilus galloprovincialis: metamorphosis alters immunocompetence,

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body shape and behavior. Fish Shellfish Immunol. 2013, 35, 438-47.

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16. Balseiro, P.; Falcó, A.; Romero, A.; Dios, S.; Martínez-López, A.; Figueras, A.; Estepa, A.;

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Novoa, B. Mytilus galloprovincialis myticin C: a chemotactic molecule with antiviral

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activity and immunoregulatory properties. PLoS One 2011, 6, e23140.



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17. Martinez-Lopez, A.; Encinar, J.A.; Medina-Gali, R.M.; Balseiro, P.; Garcia-Valtanen, P.;

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Figueras, A.; Novoa, B.; Estepa, A. pH-dependent solution structure and activity of a

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reduced form of the host-defense peptide myticin C (Myt C) from the mussel Mytilus

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galloprovincialis. Mar. Drugs 2013, 11, 2328-46.

439 440 441

18. Yang, J.; Yan, R.; Roy, A.; Xu, D.; Poisson, J.; Zhang, Y. The I-TASSER Suite: Protein structure and function prediction. Nat. Methods 2015, 12, 7-8. 19. Pronk, S.; Páll, S.; Schulz, R.; Larsson, P.; Bjelkmar, P.; Apostolov, R.; Shirts, M.R.; Smith,

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J.C.; Kasson, P.M.; van der Spoel, D.; Hess, B.; Lindahl, E. GROMACS 4.5: a high-

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throughput and highly parallel open source molecular simulation toolkit. Bioinformatics

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2013, 29, 845-54.

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20. van der Spoel, D.; Lindahl, E.; Hess, B. and the GROMACS development team GROMACS User Manual version 4.6.7. 2014, (accessed at www.gromacs.org). 21. Holm, L.; Rosenström, P. Dali server: conservation mapping in 3D. Nucleic Acids Res. 38(Web Server issue), 2010, W545-9. 22. Fields, G.B.; Noble, R.L. Solid phase peptide synthesis utilizing 9fluorenylmethoxycarbonyl amino acids. Int. J. Pept. Protein Res. 1990, 35, 161–214. 23. Carpino, L.A.; Ferrer, F.J. The 5,6- and 4,5-benzo derivatives of 1-hydroxy-7azabenzotriazole. Org Lett. 2001, 3, 2793-5.

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24. Wiegand, I.; Hilpert, K.; Hancock, R.E.W. Agar and broth dilution methods to determine the

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minimal inhibitory concentration (MIC) of antimicrobial substances. Nat. Protoc. 2008, 3,

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163-75.

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25. Schmitt, P.; Wilmes, M.; Pugnière, M.; Aumelas, A.; Bachère, E.; Sahl, H.G.; Schneider, T.;

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Destoumieux-Garzón, D. Insight into invertebrate defensin mechanism of action: oyster


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defensins inhibit peptidoglycan biosynthesis by binding to lipid II. J. Biol. Chem. 2010, 285,

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29208-16.

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26. CLSI Reference method for broth dilution antifungal susceptibility testing of filamentous

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fungi; approved standard CLSI document M38-A2. Clinical and Laboratory Standards

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Institute, Wayne. 2008 (accessed at www.springer.com).

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27. Yeaman, M.R.; Yount, N.Y. Unifying themes in host defence effector polypeptides. Nat. Rev. Microbiol. 2007, 5, 727-40. 28. Zhu, S.; Gao, B.; Tytgat, J. Phylogenetic distribution, functional epitopes and evolution of the CSalphabeta superfamily. Cell Mol. Life Sci. 2005, 62, 2257-69. 29. Laederach, A.; Andreotti, A.H.; Fulton, D.B. Solution and micelle-bound structures of

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tachyplesin I and its active aromatic linear derivatives. Biochemistry 2002, 41, 12359-68.

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30. Gueguen, Y.; Herpin, A.; Aumelas, A.,; Garnier, J.; Fievet, J.; Escoubas, J.M.; Bulet, P.;

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Gonzalez, M.; Lelong, C.; Favrel ,P.; Bachère, E. Characterization of a defensin from the

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oyster Crassostrea gigas. Recombinant production, folding, solution structure, antimicrobial

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activities, and gene expression. J. Biol. Chem. 2006, 281, 313-23.

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31. Roch, P.; Yang, Y.; Toubiana, M.; Aumelas, A. NMR structure of mussel mytilin, and

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antiviral-antibacterial activities of derived synthetic peptides. Dev. Comp. Immunol. 2008,

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32, 227-38.

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32. Holaday, S.K. Jr.; Martin, B.M.; Fletcher, P.L. Jr.; Krishna, N.R. NMR solution structure of butantoxin. Arch. Biochem. Biophys. 2000, 379, 18-27. 33. Berman H.M.; Westbrook J.; Feng Z., Gilliland G.; Bhat T.N., Weissig H., Shindyalov I.N., Bourne P.E. The Protein Data Bank Nucleic Acids Res. 2000, 28, 235-42.



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34. Finn R.D.; Bateman A.; Clements J.; Coggill P.; Eberhardt R.Y.; Eddy S.R.; Heger A.;

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Hetherington K.; Holm L.; Mistry J.; Sonnhammer E.L.L.; Tate J.; Punta M. Pfam: the

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protein families database. Nucleic Acids Res. 2014, 42(Database issue), D222-30.

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35. Cremer, G.A.; Tariq, H.; Delmas, A.F. Combining a polar resin and a pseudo-proline to

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optimize the solid-phase synthesis of a 'difficult sequence'. J Pept Sci. 2006, 12, 437-42.

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36. Park, S.C.; Kim, J.Y.; Lee, J.K.; Hahm, K.S.; Park, Y. Antibacterial action of new

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antibacterial peptides, Nod1 and Nod2, isolated from Nordotis discus discus. J. Agric. Food

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Chem. 2012, 60, 6875-81.

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37. Huang, X.X.; Gao, C.Y.; Zhao, Q.J., Li, C.L. Antimicrobial characterization of site-directed mutagenesis of porcine beta defensin 2. PLoS One 2015, 10, e0118170. 38. Zhang L.; Yang D.; Wang Q.; Yuan Z.; Wu H.; Pei D.; Cong M.; Li F.; Ji C.; Zhao J. A

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defensin from clam Venerupis philippinarum: Molecular characterization, localization,

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antibacterial activity, and mechanism of action. Dev. Comp. Immunol. 2015, 51, 29-38.

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39. Bachère, E.; Rosa R.D.; Schmitt, P.; Poirier, A.C.; Merou, N.; Charrière, G.M.;

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Destoumieux-Garzón, D. The new insights into the oyster antimicrobial defense: Cellular,

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molecular and genetic view. Fish Shellfish Immunol. 2015, 46, 50-64.

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40. Lee, K.H.; Hong, S.Y.; Oh, J.E.; Kwon, M.; Yoon, J.H.; Lee, J.; Lee, B.L.; Moon, H.M.

497

Identification and characterization of the antimicrobial peptide corresponding to C-terminal

498

beta-sheet domain of tenecin 1, an antibacterial protein of larvae of Tenebrio molitor.

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Biochem J. 1998, 334, 99-105.

500 501 502 503

41. Gao, B.; Zhu, S. An insect defensin-derived β-hairpin peptide with enhanced antibacterial activity. ACS Chem. Biol. 2014, 9, 405-13. 42. Domeneghetti S.; Varotto L.; Civettini M.; Rosani U.; Stauder M.; Pretto T.; Pezzati E.; Arcangeli G.; Turolla E.; Pallavicini A.; Venier P. Mortality occurrence and pathogen 22 ACS Paragon Plus Environment

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detection in Crassostrea gigas and Mytilus galloprovincialis close-growing in shallow

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waters (Goro lagoon, Italy). Fish Shellfish Immunol. 2014, 41, 37-44.

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43. Schmitt, P.; Rosa, R.D.; Duperthuy, M.; de Lorgeril, J.; Bachère, E.; Destoumieux-Garzón,

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D. The Antimicrobial defense of the Pacific oyster, Crassostrea gigas. How diversity may

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compensate for scarcity in the regulation of resident/pathogenic microflora. Front.

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Microbiol. 2012, 3, 160.

510

44. Chen, C.H.; Wiedman, G.; Khan, A.; Ulmschneider, M.B. Absorption and folding of

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melittin onto lipid bilayer membranes via unbiased atomic detail microsecond molecular

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dynamics simulation. Biochim. Biophys. Acta 2014, 1838, 2243-9.

513

45. Upadhyay, S.K.; Wang, Y.; Zhao, T.; Ulmschneider, J.P. Insights from Micro-second

514

Atomistic Simulations of Melittin in Thin Lipid Bilayers. J Membr Biol. 2015, 248, 497-

515

503.

516

46. Zhang, Y.; Cougnon, F.B.; Wanniarachchi, Y.A.; Hayden, J.A.; Nolan, E.M. Reduction of

517

human defensin 5 affords a high-affinity zinc-chelating peptide. ACS Chem. Biol. 2013, 8,

518

1907-11.

519

47. Chileveru, H.R.; Lim,S.A.; Chairatana, P.; Wommack, A.J.; Chiang, I.L.; Nolan, E.M.

520

Visualizing attack of Escherichia coli by the antimicrobial peptide human defensin 5.

521

Biochemistry. 2015, 54, 1767-77.

522 523

48. Dias, R.O.; Franco, O.L. Cysteine-stabilized defensins: From a common fold to antibacterial activity. Peptides 2015, doi: 10.1016/j.peptides.2015.04.017 [Epub ahead of print]

524

49. Powers, J.P.; Rozek, A.; Hancock, R.E. Structure-activity relationships for the beta-hairpin

525

cationic antimicrobial peptide polyphemusin I. Biochim. Biophys. Acta 2004, 1698, 239-50.



526

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50. Ulrich, E.L.; Akutsu, H.; Doreleijers, J.F.; Harano, Y.; Ioannidis, Y.E.; Lin, J.; Livny, M.;

527

Mading, S.; Maziuk, D.; Miller, Z.; Nakatani, E.; Schulte, C.F.; Tolmie, D.E.; Kent Wenger,

528

R.; Yao, H.; Markley, J.L. BioMagResBank. Nucleic Acids Res. 2008, 36, D402-8.

529

CAPTIONS FOR FIGURES AND TABLES

530

Figure 1. (A) Superimposed ribbon structures of three converging models of the mature MytC

531

obtained by I-tasser18 and refined with GROMACS19. Arrows indicate two stacking tyrosines in the

532

two antiparallel β-strands; N- and C- termini are also indicated. (B) Mesh representation of MytC

533

highlighting cysteine (blue) and variable (red) residues. Most of the latter are clustered in the helix

534

region.

535

Figure 2. Left. Multiple alignments illustrating the primary sequence (top) and secondary structure

536

elements (bottom) of MytC and selected CSαβ peptides. Right. Superimposed structures of MytC

537

(green) with mytilin B (2eemA, top), MGD1 (1fjnA, center), and butantoxin (1c56A, bottom).

538

Despite general similarities in the molecular organization and cysteine array, the superimposition

539

reveals topological differences in terms of loop length and β-sheet twist (the γ-core region).

540

Figure 3. Analytical RP-HPLC and MS analysis of MytC[19-40]S (A), MytC[19-40]SOX (B) and

541

MytC[1-40] (C). The chromatographic profiles, indicating the high peptide purity, were obtained on

542

a monolithic 100x4.6 mm, C18 column (OnyxTM, Phenomenex, USA) with appropriate acetonitrile

543

gradients (dashed lines). Though the peptides MytC[19-40]S and MytC[19-40]SOX differ for an

544

intramolecular disulfide bridge, no significant difference in their retention time was observed. Insets

545

show the related MS spectra. The experimental m/z values are consistent with the sequences of the

546

peptides, based on their theoretical molecular masses in Table 1.

547

Figure 4. Effect of 0-64 µM MytC[19-40]SOX on the growth of the E. coli (A) and E. faecalis (B) at

548

pH 5 (inoculum: 107 CFU/mL; absorbance at 620 nm). Black lines: negative control. Red lines:

549

melittin tested at MIC (2 µM on E. coli; 16 µM on E. faecalis).


Page 25 of 32


550

Figure 5. Evaluation of membrane permeabilization by SYTOX green intracellular uptake in E.coli

551

(A) and E. faecalis (B). Lines indicate melittin (red), MytC[19-40]SOX (black) or no peptide (dashed

552

line). Following 15 min incubation of bacterial cells at pH 7 with 2 µM SYTOX green, each peptide

553

was added at 1x MIC and the fluorescence values were recorded every minute for 1 h (Ex. 485 nm

554

and Em. 520 nm).

555

Figure 6. (A) Amide region of the 1H-NMR spectrum of MytC[19-40]SOX in SDS (blue), DPC

556

(red) and water (green), Peak dispersion and width suggest partial structuring of the peptide

557

fragment in both membrane-mimetic environments. (B) Difference between random and

558

experimental Hα chemical shifts of assigned amino acids in SDS and DPC50. In both cases, the data

559

suggest two helical regions separated by a loop. (C) NOE signals of MytC[19-40]SOX in DPC:

560

strong peaks (black), weak signals (grey) and inferred correlations partially masked by other signals

561

(empty bars).

562

Graphic for Table of Contents. The mature myticin C peptide is stabilized by 4 disulfide bonds

563

(in yellow) within the Csαβ motif (in green and blue) and is characterized by two stacking tyrosines

564

(in background a mussel hemocyte activated by heat-killed bacteria).The sequence logo with

565

residue conservation probability is also reported. Synthetic MytC and related fragments were tested

566

on reference Gram positive/negative bacteria and fungal strains, with only weak activity on these

567

latter ones.

568

Table 1. Name, sequence, length and monoisotopic mass of the mature myticin C and related

569

peptide fragments obtained by chemical synthesis.

570

Table 2. Minimal Inhibitory Concentration (MIC) and Minimal Bactericidal or Fungicidal

571

Concentration (MBC or MFC) of the MytC peptide fragments at acidic pH.



Page 26 of 32


Page 27 of 32


FIGURE GRAPHICS

Figure 1



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Figure 2


Page 29 of 32


Figure 3



Page 30 of 32

Figure 4


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Figure 5

Figure 6



Page 32 of 32

Graphic for Table of Contents


Structural and Antimicrobial Features of Peptides Related to Myticin C

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