Myticusin-beta, antimicrobial peptide from the marine bivalve, Mytilus coruscus

Journal Pre-proof Myticusin-beta, antimicrobial peptide from the marine bivalve, Mytilus coruscus Ryunkyoung Oh, Min Jeong Lee, Young-Ok Kim, Bo-Hye Nam, Hee Jeong Kong, JuWon Kim, Jung-yeon Park, Jung-Kil Seo, Dong-Gyun Kim PII:

S1050-4648(20)30106-6

DOI:

https://doi.org/10.1016/j.fsi.2020.02.020

Reference:

YFSIM 6819

To appear in:

Fish and Shellfish Immunology

Received Date: 6 September 2019 Revised Date:

7 February 2020

Accepted Date: 12 February 2020

Please cite this article as: Oh R, Lee MJ, Kim Y-O, Nam B-H, Kong HJ, Kim J-W, Park J-y, Seo J-K, Kim D-G, Myticusin-beta, antimicrobial peptide from the marine bivalve, Mytilus coruscus, Fish and Shellfish Immunology (2020), doi: https://doi.org/10.1016/j.fsi.2020.02.020. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.

1

Myticusin-beta, antimicrobial peptide from the marine bivalve,

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Mytilus coruscus

3

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Ryunkyoung Oha , Min Jeong Leea, Young-Ok Kima, Bo-Hye Nama, Hee Jeong Konga, Ju-

5

Won Kima, Jung-yeon Parka, Jung-Kil Seob and Dong-Gyun Kima *

6 a

7 8 9

Biotechnology Research Division, National Institute of Fisheries Science, Busan, 46083, Republic of Korea

b

Department of Food Science and Biotechnology, Kunsan National University, Kunsan

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54150, Korea

11 12 13

*

Corresponding author

E-mail address: [email protected] (D-G. Kim)

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1

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Abstract

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We isolated and purified an antimicrobial peptide (AMP) from the mantle of the hard-

18

shelled mussel, Mytilus coruscus. The peptide was purified through C18 reversed-phase high-

19

performance liquid chromatography, and displayed antibacterial activity. Total molecular

20

mass of 11,182 Da was determined using matrix-assisted laser desorption ionization time-of-

21

flight mass spectrophotometry. The N-terminal 23-amino acid sequence of its purified peak

22

was obtained through Edman degradation, revealing 82% identity with myticusin-1 of M.

23

coruscus. Complete sequence of the target peptide was determined through cDNA cloning

24

and rapid amplification of cDNA ends. The complete sequence comprised 574 bp with a 387-

25

bp open reading frame (ORF) encoding 24 amino acids of a signal peptide and 104 amino

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acids of a mature peptide, which was named myticusin-beta. Furthermore, we discovered two

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novel isoforms of myticusin-beta. We constructed and expressed recombinant myticusin-beta,

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which displayed antimicrobial activity against gram-positive (Bacillus cereus, Bacillus

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subtilis, Clostridium perfringens, Staphylococcus aureus, Streptococcus iniae, Streptococcus

30

mutans) and gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Vibrio

31

alginolyticus, Klebsiella pneumoniae). Purified recombinant myticusin-beta also showed anti-

32

parasitic activity at various concentrations. A short AMP analog was designed and

33

synthesized based on the sequence of myticusin-beta, with markedly improved antimicrobial

34

activity. Expression of myticusin-beta was detected in the mantle at the highest level,

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followed by hemocytes. The results obtained in this work suggest that myticusin-beta is an

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immune-related AMP of M. coruscus and an effective alternative to antibiotics.

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Key words: Antimicrobial peptide, Myticusin-beta, Mytilus coruscus, Purification 2

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1. Introduction

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Mollusks are a major phylum of marine invertebrates that include mussel, clam, oyster, and

40

scallop, which generally obtain nutrients from seawater through filter feeding. Among marine

41

mollusks, mussels attach to each other or to rocks with proteinaceous byssus in the middle of

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the foot. They take up plankton and microorganisms along with water through two siphons,

43

the exhalant and inhalant siphons, because they cannot move. Nutritious organic materials

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that enter the inhalant siphon are caught in the gill and aggregated with mucus, and then

45

move into the mouth. For these reasons, mollusks are exposed to microbe-rich conditions and

46

require effective immune systems to prevent microbial infections [1].

47

The acquired immune system of invertebrates such as mollusks is less developed, while

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their innate immune system is well developed [2]. Therefore, they depend on innate immunity,

49

including both the cellular and humoral components, to defend themselves against pathogenic

50

microorganisms [3]. Innate immunity is the first line of the host defense system and does not

51

have specificity, so it inhibits the growth of invading microorganisms in body fluids

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immediately after infection, before activation of the adaptive immune system or phagocytosis

53

[4]. Among defense systems, antimicrobial peptides (AMPs) act as the major components of

54

innate immunity in mollusks [5-8].

55

AMPs are host defense peptides and gene-encoded short oligopeptides (less than about 100

56

amino acids) with alpha-helix or beta-sheet folded structures, and are amphipathic molecules

57

containing hydrophobic and cationic amino acids that exhibit a broad spectrum of

58

antimicrobial activities against bacteria, fungi, parasites, and even certain enveloped viruses

59

[9-11]. The action of cationic AMPs depends on their secondary structure, overall net charge,

3

60

amphipathy, hydrophobicity, size, and the balance between hydrophobic and polar regions

61

[12,13]. AMPs are classified into membrane-targeting peptides and non-membrane-targeting

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peptides according to the locations of their target molecules, such as the cell wall, membrane,

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or cytoplasm [14].

64

Diverse AMPs have been isolated and characterized from different organisms. Despite

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reports of antimicrobial activity against a several of pathogens with more than 1,000 AMPs,

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studies on AMPs from marine mussels have included a few species. Identified AMPs from

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mussels have been divided into eight groups: defensing [15], mytilin [16], myticin [17],

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mytimycin [18], mytimacin [19], big defensing [19], mytichitin-CBD [20-22], and myticusin

69

[23]. Several AMPs have been purified and identified from marine mussels, particularly

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Mytilus edulis and Mytilus galloprovincialis; however, very little research has been conducted

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into the AMPs of Mytilus coruscus [1]. M. coruscus (also called hard-shelled mussel; family

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Mytilidae) has long been cultured as a nutrient food in East Asia, and is economically

73

important in South Korea. AMPs from M. coruscus, including mytichitin-CBD and myticusin,

74

have been identified in hemolymph and foot extracts from M. coruscus [20,23]. These

75

peptides show strong antimicrobial activity against gram-positive bacteria and constitute an

76

important component of the innate immune defense system.

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In this study, we isolated, purified, and characterized the AMP myticusin-beta from the

78

mantle of M. coruscus. Our results support myticusin-beta as an alternative material to

79

antibiotics and an immune system-related peptide.

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2. Materials and methods

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2.1. Tissue extraction

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Hard-shell mussels (M. coruscus) were collected from a fisheries market in Busan, South

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Korea, and gently washed with fresh water. We isolated and identified V. parahaemolyticus

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from ark shell (Sapharca broughtonii) in previous study [24]. We cultured Vibrio

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parahaemolyticus at 37℃ in Braine Heart Infusion (BHI) broth with 1% NaCl for overnight

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and diluted to 104-105 CFU/mL. M. coruscus was artificially infected with 0.1 mL of diluted

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Vibrio parahaemolyticus culture media injected into the adductor muscle through a sterile

89

needle. The mantle was isolated under aseptic conditions and boiled for 5 min with four

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volumes of pre-heated 1% acetic acid (HAc). The boiled tissues were centrifuged (4,000 rpm,

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30min, 4°C) and the supernatants were filtered through a 0.45-µm pore size syringe filter and

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stored at −70°C until use.

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2.2. Antimicrobial assessment via ultrasensitive radial diffusion assay

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The gram-positive and gram-negative bacteria and fungi listed in Table 1 were used as

96

reference virulence microorganisms to confirm antibacterial activity through the

97

ultrasensitive radial diffusion assay (URDA) [25]. In total, 19 bacterial strains were incubated

98

with the appropriate broth and temperature for 18 h (Table 1). Reference bacteria and fungi

99

were diluted to the McFarland turbidity standard of 0.5, corresponding to about 108 CFU/mL

100

or 106 CFU/mL. Then, 0.5 mL of diluted microbial culture medium was added to 9.5 mL of 5

101

underlay gel containing 1.2% type I agarose, which was poured to 1-mm thickness of

102

underlay gel with 2.2-mm diameter wells. Samples were added to the wells and the bacterial

103

or fungal suspensions were overlaid with 10 mL of overlay gel containing 1.2% agarose and

104

10 mM phosphate buffer (pH 6.5). The clear zone was measured after incubation at 25, 30, or

105

37°C for 18 h.

106

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2.3. High-performance liquid chromatography purification of peptides

108

The purification process was performed via reversed-phase (RP) high-performance liquid

109

chromatography (HPLC) using two types of RP columns: CapCell-Pak C18 RP (5 µm, 120Å,

110

4.6 x 250 mm; Shiseido, Japan) and TSK-gel ODS-80TM C18 RP columns (4.6 x 150 mm;

111

TOSOH BIOSCIENCE, Japan). First, acidified extract was injected into the CapCell-Pak C18

112

RP column with a gradient of 5–65% acetonitrile (ACN) in 0.1% trifluoroacetic acid (TFA)

113

over 60 min at a flow rate of 1.0 mL/min. Second, the collected fractions were applied to the

114

TSK-gel ODS-80TM C18 RP column with gradient of 5–65% ACN in 0.1% TFA over 1 h at a

115

flow rate of 1.0 mL/min. The eluted fractions were monitored at 220 nm and vacuum dried

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for further characterization. Each eluted peak was tested against Bacillus subtilis (KCTC

117

1021) via URDA to confirm its antibacterial activity.

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2.4. Digestion of purified peptide with protease

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Finally, a single purified peak was incubated with 2 µL (1,000 µg/mL) of trypsin at 37°C

121

for 1 h to confirm the proteinaceous nature of the compound. Trypsin hydrolyzed the peptide 6

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bonds and digested the protein into peptides at the carboxyl-terminal side of arginine and

123

lysine residues. After digestion, samples were incubated at 4°C to inactivate the trypsin. After

124

protease reaction, the single purified peak was subjected to URDA testing and its

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antimicrobial activity was compared to that of the peptide without trypsin treatment.

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2.5. Sequence identification of purified peptide

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Matrix-assisted laser desorption ionization time-of-flight mass spectrophotometry

129

(MALDI-TOF/MS) with an Ultraflex III instrument (Bruker Daltonik GmbH, USA) was used

130

to determine the molecular weight of the purified peptide. The purified peptide was dissolved

131

in 1% TFA and 50% ACN (1:1,v/v) mixed with α-cyano-4-hydroxycinnamic acid (CHCA)

132

matrix solution (10 mg/mL CHCA in 0.1% TFA/50% ACN, 1:1,v/v) and loaded onto a

133

MALDI plate. The N-terminal amino acid sequence was obtained through the Edman

134

degradation method using a pulse liquid automatic sequencer.

135

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2.6. RNA extraction, cDNA preparation, and nucleotide sequencing

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RNA was extracted and purified from the mussel mantle using TRI reagent and the purity

138

of the extracted RNA was determined with a spectrophotometer (Nanovue plus, USA). cDNA

139

of myticusin-beta from M. coruscus was synthesized using the SMARTer® RACE 5′/3′ Kit

140

(TaKaRa, Japan) to determine the complete DNA sequence. Then, first-strand cDNA was

141

synthesized with poly(A) polymerase. The 5′-RACE-Ready cDNA synthesis reactions were

142

performed in a final volume of 11 µL containing 1.0 µL of RNA and 1.0 µL of 5′-DNS Primer 7

143

A. Meanwhile, the 3′-RACE-Ready cDNA synthesis reactions were performed in a final

144

volume of 12 µL containing 1.0 µL of RNA and 1.0 µL of 3′-DNS Primer A. Tubes were

145

incubated at 72°C for 3 min, and then cooled to 42°C for 2 min. Next, 1.0 µL of

146

oligonucleotide was added to the 5′-RACE cDNA synthesis reactions. Master Mix was added

147

to denatured RNA from the 5′- and 3′-RACE-Ready cDNA synthesis reactions, which were

148

then incubated at 42°C for 90 min after treatment at 70°C for 10 min. The 3′- and 5′-RACE-

149

Ready cDNA samples were stored at −20°C until use. Two gene-specific oligonucleotide

150

primers were designed and applied. (Myticusin-3RACE: 5’-GATTACGCCAAGCTT GAC

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CAC

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GATTACGCCAAGCTT TAT TGC GGA TGC ATA TGC IGC GTC TTG IGC-3’)

CAG

ATG

GCA CAG

TCG

GCC

TGC-3

and

Myticusin-5RACE:

5’-

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Myticusin-beta was cloned into the pGEM® T-easy vector system (Promega, USA) and

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transformed into Escherichia coli XL-1 blue chemically competent cells. The plasmid was

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isolated and purified using the DNA-spin Plasmid Purification Kit (Intron, Korea), and its

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sequence was analyzed with the 3130XL Genetic Analyzer (Applied Biosystems, USA). The

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amino acid sequence obtained was submitted to the Basic Local Alignment Search Tool

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(BLAST)

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http://blast.ncbi.nlm.nih.gov/Blast). Sequences were aligned using BioEdit version 7.2.5 and

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GENETYX version 4.0 for comparison and analysis. The theoretical molecular weight and pI

161

value of the purified peptide were evaluated using the ExPASy online tool

162

(http://www.expasy.org/rools/protparam.html).

from

the

National

Center

for

163 8

Biotechnology

Information

(NCBI,

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2.7. Construction of recombinant plasmids

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To construct the expression plasmid, the target gene was amplified using a sense BamHI-

166

linker primer (5’-GGATCC ATG TCA GAC CAT CAG ATG GCA -3’) and an antisense XhoI-

167

linker primer (5’-CTCGAG TTA AAC GAT ACA ACA GCA GAA GTT-3’) via polymerase

168

chain reaction (PCR). PCR was performed in a total volume of 50 µL containing 1.0 µL of

169

DNA template, 1× PCR buffer containing MgCl2, 0.2 mmol/L of dNTP, 0.6 U of Taq

170

polymerase, and 1.0 pmol of each primer. The reaction included 25 cycles of 1 min at 94°C, 1

171

min at 55°C, and 1 min at 72°C, followed by a final extension at 72°C for 7 min, using a

172

TP600 PCR thermal cycler (TaKaRa, Japan). The amplicon was confirmed through 1.5%

173

agarose gel electrophoresis and purified using the QIAquick® PCR Purification kit (Qiagen,

174

Germany). The final product was cloned into pET-28a(+)-thioredoxin A (TrxA) fusion vector

175

containing BamHI and XhoI sites, and the N-terminus was 6X His-tagged [26]. The

176

recombinant plasmid encoding the myticusin-beta gene was isolated and transformed into E.

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coli DH5ɑ and BL21 (DE3) competent cells.

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2.8. Expression and purification of the recombinant protein

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Cells were incubated in Luria–Bertani (LB) liquid medium containing 50 µg/mL

181

kanamycin at 37°C until the optical density at 600 nm reached 0.4. The induction step was

182

initiated with isopropyl-D-1-thiogalactopyranoside (IPTG) at 37°C for 4 h. Cells were

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centrifuged at 4,000 rpm for 30 min, after which the pellet was suspended in 50 mM Tris-HCl

184

buffer (pH 8.0). Suspended cells were crushed through sonication in an ice-water bath and

185

centrifuged at 12,000 rpm for 10 min at 4°C. The supernatants were subjected to Ni-NTA His 9

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Bind® SuperflowTM purification (Millipore, USA). After purification, the eluted fraction was

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dialyzed in 20 mM Tris-HCl buffer and analyzed using an EzWay-PAG gel (Koma Biotech,

188

Korea) with 10% aspartate buffer. The Bradford assay was conducted for quantitative

189

analysis of the purified target proteins. The antimicrobial activity of the purified recombinant

190

protein was tested against the strains listed above using the URDA method.

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2.9. Western blotting

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Samples were heated for 10 min at 100°C and separated with the EzWay-PAG gel system

194

for western blots. Results were obtained using the WesternBreeze® chromogenic

195

Immunodetection Kit (Invitrogen, USA) according to the manufacturer’s protocol. Gels were

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washed and trans-blotted onto a nitrocellulose (NC) membrane at 15 V for 1 h. The NC

197

membrane was incubated with anti-His primary antibody (1:5000, Invitrogen, USA) and then

198

alkaline phosphatase-conjugated secondary antibodies for 1 h. Protein bands were developed

199

using a chromogenic substrate.

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2.10. Hemolytic activity

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Hemolytic activity was determined using olive flounder (Paralichthys olivaceus) blood

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cells [27]. Olive flounder blood cells were collected and diluted with sodium phosphate

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buffer (150 mM NaCl, 50 mM sodium phosphate pH 7.4) and then centrifuged at 5,000 rpm

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and 4°C for 1 min. Blood cells were washed to collect red blood cells, excluding the buffy

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coat and plasma. Next, 90 µL of 3% red blood cells in phosphate-buffered saline (PBS) was 10

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mixed with 10 µL of sample and incubated at 37°C for 1 h.

208

Then, 100 µL of 3% red blood cells in PBS and sample was centrifuged at 5,000 rpm and

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4°C for 1 min, after which 70 µL of the supernatant was injected into a 96-well plate.

210

Hemolysis was monitored by measuring the absorbance at 405 nm with a VICTOR3 1420

211

plate reader (PerkinElmer, USA). For this assay, 1% Triton X-100 and piscidin 1 (1 mg/mL)

212

were used as positive controls, and 0.01% HAc and PBS were used as negative controls. The

213

percentage of hemolytic activity was calculated as follows:

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Hemolysis (%) = [(Abs405nm test − Abs405nm buffer) / (Abs405nm 1% Triton X-100 − Abs405nm

215

buffer)] × 100

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2.11. Anti-scuticociliate assay

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An anti-scuticociliate assay was carried out in 96-well flat bottom plates containing 106

219

cells/well at 20°C using the Premix WST-1 Cell Proliferation Assay Kit (Clontech, USA).

220

Recombinant proteins at concentrations of 12.5, 25, 50, 100, and 200 µg/mL were filtered

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and evaluated in a final volume of 200 µL/well, and L-15 medium was used as a negative

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control. Treated and untreated scuticociliates were analyzed in triplicate. After treatment, the

223

plates were incubated at 20°C for 1 h and viability was determined. After 4 h at 20°C, the

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absorbance was read in an enzyme-linked immunosorbent assay reader (VICTOR3 1420,

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Perkin Elmer, USA) at 450 nm. Values are expressed as the percentage of viable

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scuticociliates after treatment. The morphology and motility of scuticociliates were

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monitored through a digital microscope camera (DFC425C Leica, Germany).

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2.12. Quantitative analysis of myticusin-beta expression

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M. coruscus was challenged with V. parahaemolyticus, and tissue-specific expression

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levels of myticusin-beta were determined through quantitative real-time PCR (qRT-PCR).

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Total RNA was extracted from the adductor muscle, foot, gill, hemocytes, hepatopancreas,

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mantle, and excurrent siphon using TRI Reagent. cDNA was synthesized with the

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Transcriptor First Strand cDNA Synthesis Kit (Roche Life Science, Germany) and used as the

235

template. Amplification reactions were performed in a final volume of 20 µL containing 1.0

236

µL of diluted cDNA, 10 µL of 2× Fast SYBR Green Master Mix (Applied Biosystems, USA),

237

0.5 µL of each primer (10 pmol/µL), and 8 µL of distilled water using the following cycle

238

profile: 1 period of 95°C for 10 min, followed by 40 cycles at 95°C for 15 s, 55°C for 1 min,

239

and 72°C for 30 s in the ABI 7500 Real-time PCR System (Applied Biosystems, USA). The

240

melting curve was measured and analyzed to verify the appropriate specific amplification.

241

Expression levels were compared with 18S rRNA expression levels using the 2−∆∆Ct method

242

to obtain differences, and results are presented as relative expression levels (mean ± standard

243

deviation) [28].

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2.13. Design and synthesis of peptide analogs of myticusin-beta

246

Analogs were designed to support the use of myticusin-beta as an antimicrobial compound,

247

which had reduced length for the rapid diffusion, enhanced activity and lower unit cost. Short

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AMP analogs were devised based on the original sequence of myticusin-beta. Analogs were

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manufactured through replacement of amino acids. Schiffer–Edmundson helical wheel 12

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projections were performed using EMBOSS Pepwheel (European Bioinformatics Institute,

251

UK) to analyze the properties of analogs.

252

We substituted amino acids with arginine (Arg) and leucine (Leu) to increase the

253

antimicrobial activity and cationic property of the peptides. Information about net charge,

254

molecular weight, Boman index (potential protein interaction index), pI value, and

255

hydrophobicity were calculated using the Antimicrobial Peptide Database version 2.34

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(APD2; http://aps.unmc.edu/AP/main.php) [29].

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3. Results and discussion

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3.1. Isolation of AMP from acidified crude extract of M. coruscus

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Mantle tissue from M. coruscus was collected, homogenized, and acidified for purification.

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Acidified mantle extracts were subjected to URDA and showed antibacterial activity against

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gram-positive bacteria including B. cereus, B. subtilis, and Streptococcus mutans, as well as

263

gram-negative bacteria including E. coli and Pseudomonas aeruginosa (data not shown).

264

Based on the initial screening results, B. subtilis, which showed high susceptibility to the

265

extract, was selected as the reference strain for the antimicrobial assay.

266

Acidified mantle extract was subjected to RP-HPLC for isolation and purification. Several

267

peaks exhibited antimicrobial activity against B. subtilis. The most active fraction was eluted

268

at 30–32 min from the CapCell-Pak C18 RP column (Fig. 1A). This fraction was collected,

269

concentrated, and applied to the TSK-gel ODS-80TM C18 RP column for further purification.

270

Finally, the purified active fraction was eluted at 31–33 min and treated with trypsin to

271

confirm its proteinaceous nature. Antimicrobial activity against B. subtilis disappeared

272

completely after trypsin reaction (Fig. 1B). These results suggest that the purified peak

273

represented a proteinaceous component with antimicrobial activity.

274

Antimicrobial peptides found in Mytilus species have mainly been isolated from

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hemocytes and hemolymph. Defensin from M. edulis and M. galloprovincialis, mytilin

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isoforms A and B from M. edulis, mytilin isoforms C, D, and G1 from M. galloprovincialis,

277

myticins A, B and mytimycin from M. galloprovincialis, mytichitin-CBD from M. coruscus

278

and myticusin-1 from M. coruscus have been reported [19-23,30-32]. Uniquely, the AMP

279

found in this investigation was purified from the mantle of M. coruscus, and the eluted 14

280

fraction exhibited antibacterial activity against gram-positive bacteria, such as B. subtilis, in

281

contrast to myticusin-1 of M. coruscus [23].

282

283

3.2. Identification of the target peptide

284

The molecular weight and N-terminal amino acid sequence of the purified peptide were

285

determined to enable identification. The molecular mass of the purified peptide was 11,182

286

Da based on MALDI-TOF/MS analysis, and 23 amino acid residues of the purified peptide

287

were obtained through Edman degradation: S-D-H-Q-M-A-Q-S-A-C-M-G-L-A-Q-D-A-A-Y-

288

A-S-A-I (Fig. 1C).

289

The identified partial sequence of 23 N-terminal residues matched 91% with a hypothetical

290

protein partial (OPL33655) sequence from M. galloprovincialis and 82% with the myticusin

291

alpha precursor (AFJ04410) of M. coruscus [23,33]. For these reasons, we proposed the name

292

of myticusin-beta for the purified peptide.

293

Diverse AMPs of various of phyla contain amino-terminal copper and nickel (ATCUN)-

294

containing peptides (ATCUN-AMPs), which have a tripeptide with the consensus sequence

295

Xaa-Xaa-His (where Xaa is an amino acid), have been reported [34]. AMPs in mollusks that

296

contain the ATCUN motif are considered to show antimicrobial activity, and the identification

297

of threonine-aspartic acid-histidine (TDH), histidine-proline-histidine (HPH), and histidine-

298

serine-histidine (HSH) motifs in mollusks can be expected. For example, the ATCUN motif

299

region sequence of myticusin-1 from M. coruscus is Thr-Asp-His (TDH), that of myticin B

300

from M. galloprovincialis is His-Pro-His (HPH), and that of myticin A from M.

301

galloprovincialis is His-Ser-His (HSH). From the results of this study, myticusin-beta has 15

302

serine-aspartic acid-histidine (SDH) in the N-terminus of the peptide. As reported for

303

ATCUN-AMPs, this peptide has a conserved sequence of three amino acid residues including

304

histidine at the N-terminus, whereas the hypothetical partial (OPL33655) protein sequence

305

from M. galloprovincialis contains serine-aspartic acid-histidine (SDH) in its N-terminus.

306

The spectrum of antimicrobial activity may be dictated by the ATCUN motif, and further

307

research on the correlation between the sequence of this motif and peptide activity is required.

308

309

3.3. Cloning of cDNA encoding myticusin-beta

310

We screened the cDNA clones to determine the sequence of myticusin-beta. Specific

311

primers were designed to amplify the cDNA of myticusin-beta from M. coruscus and the full

312

cDNA sequence.

313

The complete sequence of myticusin-beta was 574 bp in length and contained a 387-bp

314

ORF sequence (Fig. 2). The purified peptide encoding 128 amino acids begins with 24 amino

315

acids of signal peptide, which was determined using the SIGNALP 4.0 online tool

316

(http://www.cbs.dtu.dk/services/SignalP). The mature peptide contains ten basic residues, 4

317

Lys, 4 Arg and 2 His, and its total hydrophobic ratio was 39%. The cleavage site for release

318

of the signal peptide is between glycine24 and serine25. Mature peptides of 104 amino acids

319

have 87% identity with myticusin-1 of M. coruscus and 88% identity with the hypothetical

320

protein of M. galloprovincialis.

321

Furthermore, we identified two other isoforms of myticusin-beta from the mantle of M.

322

coruscus and compared their amino acid sequences. We compared 128 amino acid sequences

323

of myticusin-beta and two isoforms, myticusin-beta 2 and myticusin-beta 3 were compared 16

324

with that of myticusin-1, revealing 74.2% amino acid identity (Fig. 3). Myticusin-beta had

325

very high amino acid sequence similarity with the two isoforms, showing 98.4% and 99.2%

326

identity with myticusin-beta 2 and myticusin-beta 3, respectively.

327

According to a comparison of 128 amino acid sequences of myticusin-1 with those of

328

myticusin-beta and its isoforms, threonine74 in myticusin-1 was substituted with alanine74 and

329

glycine74, whereas histidine118 in myticusin-1 was replaced with serine118 and asparagine118.

330

Consequently, two types of isoforms were identified for myticusin-beta. Differences in the

331

amino acid sequence can lead to diverse changes in protein functions; therefore, additional

332

analysis of the antimicrobial activity and structural properties of the synthetic peptide is

333

necessary.

334

A calculated molecular weight of 11,082 Da was obtained for the 104 amino acids of the

335

mature peptide, and the detected molecular weight was 11,182 Da based on mass

336

spectrometric analysis (Fig. 1C). The detected molecular weight was about 99 Da higher than

337

the calculated molecular weight and this phenomenon could be caused by various types of

338

post-translational modification, such as acetylation (shifted by 42 Da), Formylation (shifted

339

by 28 Da) allysine (loss of 1.03 Da), amidation (loss of 0.98 Da), 2,3-didehydroalanine (loss

340

of 18.02 Da), 2,3-didehydrobutyrine (loss of 18.02 Da), (Z)-2,3-didehydrotyrosine (loss of

341

2.02 Da), 3-oxoalanine cysteine (loss of 18.08 Da), 3-oxoalanine serine (loss of 2.02 Da), 2-

342

oxobutanoic acid (loss of 17.03 Da), pyrrolidone carboxylic acid (loss of 18.02 Da),

343

pyrrolidone carboxylic acid (loss of 17.03 Da), and pyruvic acid (loss of 33.10 Da) [35].

344

Therefore, modifications change the molecular weights and the difference between the

345

detected molecular weight and theoretical mass could be explained the characteristic of the

346

type of post-translational modification. 17

347

3.4. Expression and purification of the protein

348

Based on the cDNA sequence determined using the RACE method, the myticusin-beta

349

gene was amplified and cloned into the TrxA-fused pET-28a(+) vector [25]. The pET-28a(+)-

350

TrxA fusion vector comprising an N-terminal 6X His-tag fused with TrxA protein from E.

351

coli was used as the expression vector, because it is useful for high-level production of

352

soluble protein in the E. coli cytoplasm [25]. The constructed plasmid was transformed into E.

353

coli BL21 for expression.

354

Induced recombinant protein expression was analyzed using an EzWay-PAG gel and

355

protein expression was detected in the supernatant as well as in the pellet. The target protein

356

with 12 kDa of TrxA was purified, and the result was in agreement with the calculated

357

molecular weight of the target protein with TrxA (Fig. 4). Western blot analysis was

358

performed to confirm expression of the recombinant protein and a single clear band

359

corresponding to the molecular weight of the expected protein was observed. Finally, the

360

purified recombinant peptide was desalted and freeze-dried for evaluation of its antimicrobial

361

activity.

362

Expression systems using E. coli as the host pose several obstacles to the expression of

363

small cationic peptides. The target recombinant protein gene may be recognized as a harmful

364

or unnecessary protein introduced from the outside, and thus would be unproduced or

365

expressed as an insoluble aggregate. Insoluble aggregation leads to the need for refolding into

366

the correct form through a refolding process to obtain activity [36]. Various methods have

367

been investigated to overcome the shortcomings of the E. coli expression method, among

368

which fusion protein tactics have been reported as the most suitable for high-yield expression 18

369

of target proteins. Therefore, we ligated myticusin-beta into the pET-28a(+)-TrxA fusion

370

vector to make it suitable for cationic peptide expression [26,37].

371

Several fusion protein methods have been developed using glutathione S-transferase (GST),

372

N-utilization substance A (NusA), maltose-binding protein (MBP), and TrxA protein [38].

373

These fusion partners improved the production and solubility of target proteins. Among these

374

fusion partners, GST (20 kDa), NusA (55 kDa), and MBP (43 kDa) are large tags and their

375

co-expression may lead to erroneous assessment. However, TrxA protein is desirable due to

376

its small size of only 12 kDa and strong solubility-enhancing effects [38].

377

In this study, myticusin-beta was produced through induced expression, and the trxA fusion

378

expression system was used for expression of a soluble form of the AMP in the cytoplasm of

379

E. coli, which was suitable for subsequent purification steps.

380

381

3.5. Hemolytic activity

382

To determine the toxicity of the recombinant peptide, hemolytic activity was measured

383

against erythrocytes from olive flounder. Piscidin 1 and Triton X-100, used as positive

384

controls, showed strong hemolytic activity, but recombinant myticusin-beta peptide did not

385

show hemolytic activity (Fig. 5A). Furthermore, Piscidin 1 showed very high hemolytic

386

activity at a high concentration that decreased sharply at a low concentration, whereas

387

myticusin-beta peptide showed no dose-dependent hemolytic activity (Fig. 5B). These results

388

demonstrate that the recombinant peptide has no hemolytic activity.

389

Several AMPs have disadvantages, including hemolytic activity toward host cells. AMPs 19

390

bind indirectly to bacterial surfaces via electrostatic interactions or interact directly with host

391

cells, increasing the permeability of the cell membrane and causing membrane disruption

392

[39]. Therefore, the non-hemolytic property is necessary for application of AMPs as

393

alternative antibiotics. In this investigation, we showed that the recombinant protein

394

myticusin-beta showed low hemolytic activity, and thus could be applied as a substitute for

395

antibiotics.

396

397

3.6. Anti-scuticociliate activity

398

Scuticociliates are parasites regarded as the causative pathogen of the disease

399

scuticociliatosis in the marine aquaculture industry [40]. They cause mass mortality and

400

significant economic losses in aquaculture. Frequent outbreaks of scuticociliatosis in

401

aquaculture fish have been increasing worldwide, and development of effective anti-

402

scuticociliate technologies is required to solve this problem. Some marine AMPs show

403

activity against protozoa. For example, the antimicrobial cationic peptide Pc-Pis from

404

Psuedosciaena corcea is genetically related to the piscidin family and shows antimicrobial

405

activity against bacteria, fungi, and parasites [41].

406

We investigated the anti-scuticociliate activity of the recombinant protein against a

407

scuticociliate using the WST-1 solution assay [42]. The anti-scuticociliate activity of the

408

recombinant protein was evaluated based on the mortality, morphology, and number of living

409

parasites. The highest concentration of recombinant protein, 200 µg/mL, diminished parasite

410

numbers by approximately 50% after 24 h (Fig. 6). Damaged scuticociliates were observed

411

under a microscope, and the spindle shape of scuticociliate cells were swollen or burst (Fig. 20

412

6A). In addition, the fast-moving parasites slowed or stopped their movement. Scuticociliates

413

treated with recombinant protein had lower viability than untreated scuticociliates. In this

414

investigation, the viability of scuticociliates decreased with increasing concentration of the

415

recombinant protein through the WST-1 cell proliferation assay (Fig. 6B). These data indicate

416

that recombinant myticusin-beta has antiparasitic activity against scuticociliates.

417

418

3.7. Antimicrobial activities

419

In the present study, we verified the antibacterial activity of the expressed myticusin-beta

420

protein using the URDA method with various pathogenic reference strains (Table 1).

421

Recombinant myticusin-beta showed antibacterial activity against both gram-positive and

422

gram-negative bacteria (Fig. 7). Myticusin-beta displayed strong activity against gram-

423

positive strains, including B. subtilis, Clostridium perfringens, Staphylococcus aureus, and S.

424

mutans, as well as gram-negative strains including P. aeruginosa and Vibrio alginolyticus,

425

moderate activity against B. cereus and Klebsiella pneumoniae, and weak activity against

426

Streptococcus iniae and E. coli. Interestingly, gram-positive strains showed greater

427

susceptibility to myticusin-beta than gram-negative bacteria.

428

The antimicrobial spectrum of an AMP describes whether it has a wide range of activity or

429

narrow specificity to certain strains. Various AMPs such as defensins, mytilins, mytichitin-

430

CBD, myticusin-1, and myticins from Mytilus species have activity against bacteria and fungi,

431

but mytimycin has activity only against fungi [17-23,32]. Among reported AMPs, myticin,

432

mytichitin-CBD, and myticusin-1 are much less active against gram-negative bacteria and

433

fungi than other AMPs [17-23]. Myticusin-beta also showed antibacterial activity against 21

434

gram-positive and gram-negative bacteria, but no activity against fungi. Activity that was

435

stronger against gram-positive than gram-negative bacteria has been reported for most

436

mollusk AMPs.

437

The multiple modes of action of AMPs against gram-positive and gram-negative bacteria

438

and fungi have been described in recent years. Differences in membrane composition have

439

also been well investigated in gram-positive and gram-negative bacteria and fungi. Gram-

440

positive bacteria contain several layers of peptidoglycan in the cell wall, whereas the bacterial

441

membrane of gram-negative bacteria includes a single layer of peptidoglycan surrounded by

442

an outer membrane containing lipopolysaccharides, which protects bacteria as a permeability

443

barrier [13]. AMPs may interact with or pass through the cell envelope to reach their cellular

444

target. Fungal cell walls contain complex polysaccharides, such as chitin and glucan, that

445

inhibit passage through the cytoplasmic membrane. Therefore, a broad range of antimicrobial

446

activity of AMPs can be explained based on functional mechanisms and microbial cellular

447

structures.

448

As noted above, AMPs may have broad or specific antimicrobial activity spectra, and must

449

be carefully considered for use alone or in combination with other AMPs for their intended

450

purpose. Therefore, continuing research to identify novel AMPs is necessary for industrial

451

application of AMPs to replace antibiotics.

452

453

3.8. Quantitative analysis of myticusin-beta expression

454

As shown in Fig. 8, mRNA expression of myticusin-beta was measured via real-time

455

quantitative PCR as the transcriptional levels of myticusin-beta gene in various tissues, 22

456

including the adductor muscle, foot, gill, hemocytes, hepatopancreas, mantle, and excurrent

457

siphon. The mRNA levels were quantified after normalization to the internal reference gene

458

β-actin, and the amplification specificity for myticusin-beta and β-actin was determined and

459

analyzed from the melting curve. Gene expression of myticusin-beta was detected in all

460

tissues (Fig. 8). Myticusin-beta mRNA expression was highest in the mantle, followed by

461

hemocytes, moderate in the adductor muscle and gill, and low in the foot, hepatopancreas,

462

and excurrent siphon. We checked the control groups without injection and saline injected

463

samples. Control groups did not exhibit physical changes, and showed low gene expression

464

level compared to the infected group (Data not shown).

465

Mytilus species have an open circulatory system characteristic of mollusks, and AMPs are

466

produced and transported through the hemolymph to all tissues. Furthermore, abundant

467

myticusin-1 from M. coruscus was isolated from hemocytes [23]. However, the greatest

468

enhancement of myticusin-beta mRNA expression was detected in mantle tissue after

469

bacterial infection (Fig. 8). These results indicate that myticusin-beta is produced in a tissue-

470

specific manner and is related to the innate immune system of M. coruscus.

471

472

3.9. Antimicrobial activity of peptide analogs of myticusin-beta

473

In this study, we considered the length of the peptide, charge, hydrophobicity, Boman

474

index, and amino acid side chain in development of a short antibacterial peptide as a

475

substitute for antibiotics. We selected and synthesized several analog peptides, and selected

476

the peptide fragment VDAFHIYSRR in the mature peptide of myticusin-beta (Table 2).

477

Based on these results, amino acids in the selected region were replaced and rearranged to 23

478

increase antimicrobial activity. The net charge, molecular weight, Boman Index, and pI were

479

predicted using APD2 and the ExPASy online tool (Table 2). Schiffer–Edmundson helical

480

wheel projections were performed to predict hydrophobic and hydrophilic regions in the

481

secondary structures of synthetic peptides (Fig. 9).

482

The EMBOSS Pepwheel program was used to identify hydrophobic and hydrophilic

483

regions in the secondary structure of the native and analog peptides. With this program, we

484

could predict interactions and effects between adjacent amino acids and side chains. We

485

considered amino acids not only at the front and back, but also at the top and bottom of the

486

helical structure, and designed cationic AMPs to increase antibacterial activity. Arginine

487

(Arg), tyrosine (Tyr), aspartic acid (Asp), and serine (Ser) residues in the native form of

488

myticusin-beta were modified with Arg or Leu in the analog peptide (Fig. 9). Re-modeling of

489

the analog peptide showed opposite positions of hydrophobic and hydrophilic regions,

490

contributing to the antimicrobial activity.

491

Cationic AMPs are amphipathic peptides with an overall positive charge that interact with

492

negatively charged bacterial membranes and form transmembrane pores [43-47]. Given these

493

characteristics, we attempted to improve the related values of various factors. The

494

antimicrobial activities of the native and analog peptides are shown in Table 3. Interestingly,

495

increased net charge (+1 to +4), basic pI value (> 10), reduced Boman index value (≤ 1), and

496

converted structure increased the spectrum of antimicrobial activity (Table 3). The analog

497

showed increased antimicrobial activity against all strains tested (nine gram-positive bacteria,

498

nine gram-negative bacteria, and one fungus, Candida albicans).

499

The development of AMPs with increased activity in this manner was previously reported 24

500

as a valid method, and the synthesized analog peptide showed increased antibacterial activity

501

against gram-positive and gram-negative bacteria and fungi due to its low Boman index,

502

increased net charge, and total hydrophobic ratio. This study of synthetic analog peptides

503

shows that AMPs can be activated and can contribute greatly to research into new antibiotic

504

substitutes.

505

25

506

4. Conclusion

507

We isolated and purified 11,182 Da of a novel AMP from acidified mantle tissue extract of

508

M. coruscus. The purified AMP was composed of 24 amino acids of signal peptide and 104

509

amino acids of mature peptide based on the complete sequence of the target protein. The

510

sequence identity was similar to the reported myticusin-1 of M. coruscus. Hence, we

511

proposed naming the purified peptide myticusin-beta and identified two other isoforms based

512

on the sequence.

513

Constructed TrxA-fused recombinant myticusin-beta was expressed in a soluble form, and

514

the purified single peptide exhibited a broad spectrum of antibacterial activity against gram-

515

positive and gram-negative strains. Furthermore, recombinant myticusin-beta revealed anti-

516

scuticociliate activity in a dose-dependent manner without causing hemolysis. We designed a

517

short AMP analog derived from myticusin-beta based on its amino acid sequence, secondary

518

structure, charge, hydrophobicity, and correlation of the amino acid side chain. The analog

519

was rearranged with substituted amino acids and displayed strongly increased activity on the

520

URDA plate.

521

The highest transcriptional level of the myticusin-beta gene was detected in mantle tissue

522

after infection. These results suggest that myticusin-beta is an immune-related AMP of M.

523

coruscus and a promising template for the development of novel AMPs as a substitute for

524

antibiotics. This report contributes to elucidating AMPs and the immune system of mollusks,

525

although more studies are needed on the structural features and functional mechanisms of

526

AMPs.

527 26

528

Acknowledgments

529

This research was supported by a grant from the National Institute of Fisheries Science

530

(R2019016), Korea. This research was also supported by the Collaborative Genome

531

Program of the Korea Institute of Marine Science and Technology Promotion (KIMST)

532

funded by the Ministry of Oceans and Fisheries (MOF) (No. 20180430).

533

27

534

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698

pacific oyster, Crassostrea gigas, Fish Shellfish Immunol. 67 (2017) 675-683.

699 700

[47] B.H. Nam, J.Y. Moon, E.H. Park, H.J. Kong, Y.O. Kim, D.G. Kim, W.J. Kim, C.M. An,

701

J.K. Seo, Antimicrobial and antitumor activities of novel peptides derived from the

702

lipopolysaccharide- and ß-1,3-glucan binding protein of the Pacific abalone Haliotis discus

703

hannai, Mar. drugs 14 (2016) 227.

704 705 33

706

Table 1. Pathogenic strains used in this study.

Strain

Reference

Medium

Temperature (°C)

Bacillus cereus

KCTC 1012

NB

30

Bacillus subtilis

KCTC 1021

TSB

37

ECa

TSB

37

Enterococcus faecalis

KCTC 3206

BHI

37

Staphylococcus aureus

KCCM 11335

TSB

37

Streptococcus iniae

KCTC 3657

BHI+1% NaCl

37

Streptococcus mutans

KCTC 3065

BHI

37

Streptococcus vestibularis

KCTC 3650

TSB+0.3%YE

37

Streptococcus parauberis

KCTC 3651

TSB+0.3%YE

37

Lactococcus garvieae

KCTC 3772

MRS

30

KCCM 40271

NB

37

Enterobacter cloacae

KCTC 2361

NB

37

Klebsiella pneumoniae

KCTC 12385

NB

37

Proteus mirabilis

KCTC 2510

NB

37

Providencia stuartii

KCTC 2569

NB

37

Pseudomonas aeruginosa

KCTC 1636

NB

37

Vibrio alginolyticus

KCTC 2472

BHI

37

Vibrio harveyi

KCCM 40866

BHI+1% NaCl

25

Vibrio parahaemolyticus

KCCM 41664

BHI+1% NaCl

37

KCCM 11282

YM

25

Gram positive

Clostridium perfringens

Gram negative Escherichia coli

Fungus Candida albicans 707

a

EC; Environmental collection

34

708

Table 2. Sequences and physicochemical properties of the peptide variants used in this study.

MW Sequence

Native

VDAFHIYSRR

Length

10

Boman index

Charge

+1

Hydrophobic

Hydrophobicity

pI (Da)

(kcal/mol)

1,263.42

3.3

8.72

Region Modifications ratio (%)

(kcal/mol)

40

1.01

84–93 D→R, Y→L

Analog

VRAFHILLRL

10

+2

1,237.56

0.59

11.8

70

−1.09 S→L, R→L

35

709

Table 3. Antimicrobial activities of peptides against pathogenic bacteria based on the URDA

710

method. Native

Analog

Diameter of zone of inhibition (mm) Gram positive Bacillus cereus

-

4.9

Bacillus subtilis

4.5

7.9

Enterococcus faecalis

-

5.1

Streptococcus mutans

-

8.0

Streptococcus iniae

-

5.1

2.5

7.2

Streptococcus vestibularis

-

2.5

Streptococcus parauberis

-

13.1

Lactococcus garvieae

-

7.9

Vibrio alginolyticus

-

6.6

Vibrio parahaemolyticus

-

7.6

Escherichia coli

-

11.7

Enterobacter cloacae

-

8.6

Klebsiella pneumoniae

-

9.9

Edwardsiella tarda

-

4.3

Proteus mirabilis

-

6.6

Providencia stuartii

-

9.1

Pseudomonas aeruginosa

-

9.2

2.5

9.3

Staphylococcus aureus

Gram negative

Fungus Candida albicans 711

Well diameter: 2.2 mm 36

712

Figure legends

713

Fig. 1. Purification and antimicrobial activity of purified peptide from mantle extract of

714

Mytilus coruscus.

715

(A) Antimicrobial activity of the purified peak (30–32 min) against Bacillus subtilis.

716

Acidified extract was injected into a CapCell-Pak C18 RP column with a gradient of 5–65%

717

ACN in 0.1% TFA over 60 min at a flow rate of 1.0 mL/min. (B) Antimicrobial activity of the

718

purified peak (31–33 min) and trypsin-treated purified peak against B. subtilis. The active

719

fraction was applied to a TSK-gel ODS-80TM C18 RP column. Elution was performed with a

720

linear gradient of 5–65% ACN (pH 2.2) in 0.1% TFA for 60 min at a flow rate of 1.0 mL/min.

721

The eluate was monitored at 220 nm. The fraction with the absorbance peak (indicated by an

722

arrow) showed antimicrobial activity before and after trypsin treatment against B. subtilis.

723

Scale represents 5 mm. (C) Mass spectrometric analysis showing a mass of 11,182.003 Da

724

and the N-terminal sequence (23 amino acids) determined through Edman degradation.

725 726

Fig. 2. cDNA sequence and derived amino acid sequence of myticusin-beta.

727

Deduced amino acid sequence of the ORF is shown below the nucleotide sequence. Asterisk

728

indicates the stop codon and outline indicates the signal peptide of myticusin-beta.

729 730

Fig. 3. Multiple sequence alignment of myticusin-1 with myticusin-beta and its isoforms.

731

Myticusin-beta is indicated by MC1 and its two isoforms by MC2 and MC3. Myticusin-beta

732

and two of its isoforms were identified and compared to myticusin-1. Residues are shaded

733

based on their degree of conservation.

734

Fig. 4. Overexpression and purification of recombinant myticusin-beta. 37

735

TrxA-fused recombinant protein was purified using Ni-NTA affinity chromatography. Lane

736

M, molecular mass marker; lane 1, non-induced protein; lane 2, induced protein; lane 3,

737

soluble protein; lane 4, inclusion body; lane 5, purified myticusin-beta protein (denoted by

738

red arrow); lane 6, product of western blot with purified myticusin-beta protein.

739

Fig. 5. Hemolytic activity of myticusin-beta and piscidin 1 against erythrocytes of flounder

740

(Paralychthys olivaceus).

741

(A) The concentration of each sample was 100 µg/mL. (B) The effect of hemolysis depending

742

on concentration (100, 50, 25, 12.5, and 6.25 µg/mL).

743

744

Fig. 6. Anti-scuticociliate activity of recombinant myticusin-beta based on WST-1 reagent.

745

(A) Agglutination of parasites treated with various concentrations of recombinant myticusin-

746

beta (0–200 µg/mL). (B) Percent viability of parasites (scuticociliates) by concentration of

747

recombinant myticusin-beta (0–200 µg/mL). Error bars represent the mean ± standard

748

deviation of three technical replicates.

749

Fig. 7. Antimicrobial activity and spectrum of purified recombinant myticusin-beta.

750

Antimicrobial activity of recombinant myticusin-beta against various strains. The

751

antimicrobial activity value represents the diameter of the clearing zone. Well diameter: 2.2

752

mm.

753

Fig. 8. Quantitative analysis of myticusin-beta expression in various tissues.

754

Tissue-specific expression of myticusin-beta. Error bars represent the mean ± standard

755

deviation of three technical replicates. ADD, adductor muscle; FOO, foot; GIL, gill; HEM,

756

hemocytes; HEP, hepatopancreas; MAN, mantle; SIO, excurrent siphon. 38

757 758

Fig. 9. Helical wheel depiction of myticusin-beta synthetic peptide.

759

Arrows indicate the amino acid residues substituted in the peptide. Amino acid residues were

760

substituted to increase net charge and hydrophobic ratio. Blue, hydrophobic residues; red,

761

acidic residues; orange, basic residues; purple, uncharged residues.

762 763

39

764

Fig. 1.

765 766

(A)

767 768

(B)

769

40

770

(C)

771 772

773

41

774 775

Fig. 2.

776

777

42

778 779

Fig. 3.

780

43

781 782

Fig. 4.

783

784 785

44

786 787

Fig. 5.

788

(A)

789 790

791

(B)

792 793

45

794

Fig. 6.

795

(A)

796 797

(B)

798 46

799

Fig. 7.

800 801

47

802

Fig. 8.

803 804

48

805

Fig. 9.

806

807 808 809 810 811 812 813 814 815 816

49

- We isolated and purified 11,182 Da of a novel antimicrobial peptide from acidified mantle tissue extract of Mytilus coruscus. - Constructed TrxA-fused recombinant myticusin-beta exhibited spectrum of antibacterial activity and anti-scuticociliate activity without causing hemolysis. - Designed and synthesized myticusin-beta analog revealed markedly improved antimicrobial activity. - Myticusin-beta is an immune-related antimicrobial peptide of Mytilus coruscus and an effective alternative to antibiotics.