Báo cáo khoa học: Novel L-amino acid oxidase with antibacterial activity against methicillin-resistant Staphylococcus aureus isolated from epidermal mucus of the flounder Platichthys stellatus pptx

Fish also produce such mucus Keywords antibacterial protein; methicillin-resistant Staphylococcus aureus MRSA; Platichthys stellatus; L -amino acid oxidase; mucus Correspondence T.. The

against methicillin-resistant Staphylococcus aureus

isolated from epidermal mucus of the flounder

Platichthys stellatus

Kosuke Kasai1,2, Takashi Ishikawa1, Takafumi Komata3, Kaori Fukuchi4, Mitsuru Chiba5,

Hiroyuki Nozaka1,2, Toshiya Nakamura1,2, Tatsusuke Sato1,2and Tomisato Miura1,2

1 Division of Medical Life Sciences, Hirosaki University Graduate School of Health Sciences, Japan

2 Research Center for Biomedical Sciences, Hirosaki University, Japan

3 Clinical Laboratory, Shichinohe Hospital, Japan

4 Clinical Laboratory, Suzuki Lady’s Hospital, Kanazawa, Japan

5 Graduate School of Comprehensive Human Sciences, University of Tsukuba, Japan

Introduction

The mucus layer covering the body surface of many

animal species plays a defensive role as both a physical

and chemical barrier against bacterial and viral

infection The mucus components are reported to vary widely and to have a number of biological functions for host defense [1–4] Fish also produce such mucus

Keywords

antibacterial protein; methicillin-resistant

Staphylococcus aureus (MRSA);

Platichthys stellatus; L -amino acid oxidase;

mucus

Correspondence

T Miura, Division of Medical Life Sciences,

Hirosaki University Graduate School of

Health Sciences, 66-1 Hon-cho, Hirosaki,

Aomori 036-8564, Japan

Fax: +81 172 39 5966

Tel: +81 172 39 5966

E-mail: tomisato@cc.hirosaki-u.ac.jp

(Received 26 August 2009, revised 26

October 2009, accepted 16 November

2009)

doi:10.1111/j.1742-4658.2009.07497.x

Fish produce mucus substances as a defensive outer barrier against envi-ronmental xenobiotics and predators Recently, we found a bioactive pro-tein in the mucus layer of the flounder Platichthys stellatus, which showed antibacterial activity against Staphylococcus epidermidis, Staphylococ-cus aureusand methicillin-resistant S aureus In this study, we isolated and identified the antibacterial protein from the mucus components of P stella-tususing a series of column chromatography steps We then performed gel electrophoresis and cDNA cloning to characterize the protein The antibac-terial protein in the mucus had a molecular mass of approximately 52 kDa with an isoelectric point of 5.3, and cDNA sequencing showed that it cor-responded completely with the peptide sequence of antibacterial protein from the gill A BLAST search suggested that the cDNA encoded an anti-bacterial protein sharing identity with a number of l-amino acid oxidases (LAAOs) and possessing several conserved motifs found in flavoproteins RT-PCR using a specific primer, and immunohistochemical analysis with anti-LAAO IgG, demonstrated tissue-specific expression and localization in the gill Moreover, the anti-LAAO IgG was able to neutralize the antibac-terial activity of the protein against methicillin-resistant S aureus Thus,

we demonstrated that this antibacterial protein, identified from P stellatus-derived epidermal mucus, is a novel LAAO-like protein with antibacterial activity, similar to snake LAAOs

Abbreviations

CFU, colony-forming units; GSP, gene-specific primer; HIO4⁄ Schiff, periodic acid ⁄ Schiff’s reagent; LAAO, L -amino acid oxidase; MRSA, methicillin-resistant Staphylococcus aureus; PSEM, Platichthys stellatus-derived epidermal mucus; psLAAO, LAAO sequence of

Platichthys stellatus; PVDF, poly(vinylidene difluoride); TSA, trypticase soy agar; 6 M urea ⁄ PAGE, PAGE in the presence of 6 M urea.

substances for defense, as their environment is rich in

microorganisms [5] Skin and gill mucus secretions of

fish are known to contain many substances that are

active against bacteria and viruses, including peptides,

lysozymes, lectins and proteases These also play an

important role in innate immunity [6,7]

Antibacterial peptides isolated from the epidermal

mucus of several species of fish have already been

characterized One type, the cathelicidins, act by

dis-rupting the bacterial cell membrane and are considered

to be important effectors of eukaryotic immunity [8]

Recently, it has been shown that infection with fish

pathogens causes up-regulation of cathelicidin mRNA

in various tissues such as the gill, spleen and head

kid-ney [9] A 22-residue antibacterial peptide,

moroneci-din, isolated from the skin and gill of hybrid striped

bass, exhibits a broad spectrum of antibacterial activity

[10] A lysozyme-like peptide from rainbow trout

(Oncorhynchus mykiss) demonstrates antibacterial

activity against gram-positive bacteria [11] Also, an

antibacterial protein with ion channel activity against

both gram-negative and gram-positive bacteria has

been found in mucus extract from carp (Cyprinus

carpio) [12] Pleurocidin, found in skin mucus

secre-tions of the winter flounder (Pleuronectes americanus),

has been shown to exhibit antibacterial activity against

both gram-negative and gram-positive bacteria [13]

In recent years, some reports have documented details

of high-molecular-mass antibacterial proteins in fish

mucus, such as that of the rockfish (Sebastes schegeli),

which demonstrates selective antibacterial activity

against gram-negative bacteria [14] A pore-forming

65-kDa glycoprotein isolated from the rainbow trout

(O mykiss, formerly Salmo gairdneri), has also been

found to have strong antibacterial properties [15]

Glycosylated proteins from the hydrophobic

superna-tant of mucus from tench (Tinca tinca), eel (Anguilla

anguilla) and rainbow trout (O mykiss) show strong

activity against both gram-negative and gram-positive

bacteria [16]

In the present study, we found an antibacterial

pro-tein in the epidermal mucus of the flounder

Platich-thys stellatus This species, which has a rich covering

of mucus on its body surface, inhabits brackish water

at the mouths of rivers This mucus protein was shown

to exert antibacterial activity against

Staphylococ-cus epidermidis, StaphylococStaphylococ-cus aureus and

methicillin-resistant S aureus (MRSA) Moreover, we identified

this antibacterial protein as a novel l-amino acid

oxidase (LAAO; EC.1.4.3.2) LAAOs catalyze the

oxidative deamination of an l-amino acid substrate

and have been reported to exert antibacterial activity

in a variety of animal fluids, such as snake venom [17]

The present communication describes the isolation and cloning of this LAAO-like antibacterial protein from

P stellatus

Results

Antibacterial activity of mucus

It is assumed that Platichthys stellatus-derived epider-mal mucus (PSEM) includes antibacterial substances, because the body surface, which is exposed to the external environment, functions as the first barrier to invasion by bacteria Therefore, we analyzed the anti-bacterial activity of PSEM against 19 different gram-positive and gram-negative clinically pathogenic bacte-ria using a growth-inhibition plate assay (Table 1) The PSEM inhibited the growth of all Staphylococcus spp (antibacterial score: 2+ to 3+) Proliferation of

S epidermidis in particular was strongly suppressed, the effect being most marked among all the bacteria

we studied (Fig 1A) The PSEM had intermediate

Table 1 Antibacterial activity spectra of Platichthys stellatus-derived epidermal mucus.

Species and strains

Diameter of clear zone (mm) Scorea Gram-positive bacteria

Staphylococcus aureus NIHJ JC-1 8.5 + + Staphylococcus aureus ATCC25923 6.3 + + Staphylococcus epidermidis 18.1 + + + Methicillin-resistant Staphylococcus

aureus 87-7920

8.2 + + Methicillin-resistant Staphylococcus

aureus 87-7927

8.3 + + Methicillin-resistant Staphylococcus

aureus 87-7928

8.1 + + Methicillin-resistant Staphylococcus

aureus 87-7931

8.2 + + Methicillin-resistant Staphylococcus

aureus 87-7958

8.1 + + Streptococcus pyogenes 5.5 + Streptococcus agalactiae 2.8 – Enterococcus faecalis ATCC33186 2.8 – Enterococcus faecium ATCC19434 2.8 – Enterococcus faecium BM4147 (VanA + ) 2.8 – Enterococcus faecalis V583 (VanB + ) 2.8 – Enterococcus gallinarum BM4174 (VanC1 + ) 2.8 – Gram-negative bacteria

Escherichia coli NIHJ JC-2 2.8 – Serratia marcescens 2.8 – Vibrio parahaemolyticus RIMD2210001 5.7 + Pseudomonas aeruginosa ATCC27853 2.8 –

a Clear zone £ 2.8 mm +, clear zone < 6.0 mm; + +, clear zone

< 10.0 mm; + + +, clear zone ‡ 10.0 mm.

antibacterial activity for S aureus (Fig 1B), although

the antibacterial activity of PSEM against two strains

of S aureus was slightly different The growth of

MRSA was also inhibited by PSEM (Fig 1C) and

there was no marked difference in antibacterial activity

among the five MRSA strains tested (Table 1) Among

gram-positive cocci, except for the staphylococci,

PSEM weakly suppressed the growth of S pyogenes

(1+) Among gram-negative bacilli, the proliferation

of Vibrio parahaemolyticus was weakly suppressed by

PSEM However, PSEM showed no antibacterial

activ-ity against two strains of Streptococcus spp., five

strains of Enterococcus spp [including

vancomycin-resistant Enterococcus (VRE)], Escherichia coli,

Serra-tia marcescens and Pseudomonas aeruginosa In the

growth-inhibition plate assay, the agar medium in the

clear zone formed in the MRSA assay was collected

and cultured in trypticase soy agar (TSA) in order to

confirm the bactericidal activity of PSEM It was

clari-fied that the PSEM had bactericidal activity against

MRSA because MRSA did not proliferate in TSA

after 96 h of culture

Temperature sensitivity of PSEM for antibacterial activity

Generally, proteins lose their activity when subjected to heat treatment, and complement (which is a component

of blood) is inactivated by heating at 56C for 30 min Therefore, the antibacterial activity of PSEM was inves-tigated after incubation at various temperatures, in order to investigate the properties of the antibacterial components The antibacterial activity of PSEM for MRSA 87-7928 was lowered slightly at 45C, markedly

at 56C and completely at 70 C (Fig 1D), suggesting that the antibacterial component of PSEM is a protein

Purification of antibacterial protein from PSEM The antibacterial protein in PSEM was separated by ul-tracentrifugation and purified by hydrophobic chroma-tography (Fig 2A) Protein fractions were monitored by measuring the absorbance at 280 nm, and antibacterial activity was assayed using the growth-inhibition plate method Pooled antibacterial fractions were further purified by gel filtration chromatography (Fig 2B) and chromatofocusing (Fig 2C) In gel filtration chromato-graphy and chromatofocusing steps, the antibacterial activity was eluted as a single peak SDS⁄ PAGE of the fractions containing antibacterial activity that had been separated by chromatofocusing contained three main bands with molecular masses of 39, 40 and 52 kDa (Fig 2D) Because of irreversible denaturation of the protein, antibacterial activity was not detected in the gels after SDS⁄ PAGE Therefore, we performed PAGE

in the presence of 6 m urea (6 m urea⁄ PAGE) to sepa-rate the antibacterial protein as remaining bioactivity Interestingly, the purified PSEM retained its bioactivity after this step The antibacterial activity of gel extracts from the 6 m urea⁄ PAGE was analyzed using the growth-inhibition plate method, and the molecular mass

of the antibacterial protein was confirmed by SDS⁄ PAGE Antibacterial protein was detected only in fractions 19–22 (Fig 3A), and its molecular mass was estimated to be 52 kDa (Fig 3B) Two lower-molecular-mass proteins of 39 kDa (fractions 23–24) and 40 kDa (fractions 15–16) did not show antibacterial activity Moreover, 2D gel electrophoresis revealed a single spot

at 52 kDa with an isoelectric point of 5.3 (Fig 3C)

cDNA cloning and sequence analysis of antibacterial protein

For cloning, the antibacterial protein was blotted onto

a poly(vinylidene difluoride) (PVDF) membrane after 2D gel electrophoresis, and the spot corresponding to

Fig 1 Antibacterial activity of PSEM against (A) Staphylococcus

epidermidis, (B) Staphylococcus aureus NIHJ JC-1B and (C) MRSA,

clinical isolate 87-7928 Each bacterial strain was suspended in TSA

at a final concentration of 1 · 10 6

CFUÆmL)1 (c) Control buffer without PSEM and (mu) PSEM were applied to holes in the agar.

Antibacterial activity was measured after overnight incubation at

37 C (D) Heat sensitivity of PSEM against MRSA, clinical isolate

87-7928 (c) Control buffer without PSEM at 0 C PSEM was

exposed to temperatures of 0, 25, 37, 45, 56, 70 and 100 C for 1 h.

Each sample was applied to the holes in the agar, and antibacterial

activity was measured after overnight incubation at 37 C.

52 kDa was cut out Then, the N-terminal peptide

sequence was analyzed by Edman degradation and the

inner peptide sequences were determined using an

amino acid sequencer This showed that the N-terminal

peptide sequence was

Leu-Ser-Phe-Arg-Ala-His-Leu-Ser-Asp and that the internal peptide sequences were

Arg-Thr-Phe-Glu-Val-Asn-Ala-His-Pro-Asp-Ile-Leu,

Ser-Ala-Asp-Gln-Leu-Leu-Gln-Gln-Ala-Leu and

Ser-Glu-Gly-Arg-Leu-His-Phe-Ala-Gly-Glu-His-Thr To

deter-mine the cDNA encoding the antibacterial protein of

PSEM, mRNA was prepared from skin and gill PCR

was performed using degenerate primers based on the

N-terminal peptide sequence LSFRAHLSD and the

internal peptide sequence RTFEVNAHPDIL

Subse-quently, the full-length cDNA was amplified by

3¢-RACE and 5¢-RACE Sequence analysis identified

two genes, which completely corresponded to the

peptides of antibacterial protein from the gill (Fig 4),

and another highly homologous gene from skin (DDBJ accession number AB495361) The full-length cDNA found in the gill, which encodes an antibacterial pro-tein, consisted of 2002 bp plus poly (A) The N-termi-nal amino acid sequence of LSFRAHLSD was encoded

by nucleotides 183–209 The internal amino acid sequences RTFEVNAHPDIL, SADQLLQQAL and SEGRLHFAGEHT were found at positions 567–602, 636–675 and 1524–1559, respectively (Fig 4) The ini-tial codon, ATG, was found at positions 102–104, and the open reading frame was composed of a 1566-bp region, encoding a protein of 522 amino acid residues

A BLAST search demonstrated that the encoded anti-bacterial protein shared identity with a number of LAAO flavoproteins The gene encoding this antibacte-rial protein had 71% identity with the skin mucus antibacterial LAAO of S schlegeli (NCBI accession

no BAF43314) and 69% identity with the

Fig 2 Purification of epidermal mucus protein (A) Chromatography using a Phenyl Sepharose 6 Fast Flow high sub column One-hundred and thirty milliliters of PSEM was applied to the column at a flow rate of 30 mLÆh)1 The protein content of each fraction was monitored by measur-ing the absorbance at 280 nm (s) and antibacterial activity (d) was assayed usmeasur-ing the growth-inhibition plate method Pooled fractions indicated

by the bar (I) were used for gel filtration chromatography (B) Gel filtration chromatography using a Sephacryl S-100 HR column The fraction vol-ume was 2.5 mL and the flow rate was 8.0 mLÆh)1 The protein content of each fraction was monitored by measuring the absorbance at

280 nm (s) and antibacterial activity (d) was assayed using the growth-inhibition plate method Pooled fractions indicated by bar (II) were used for chromatofocusing (C) The antibacterial protein was further purified by chromatofocusing on a PBE94 column at pH 7–4 The fraction volume was 2.5 mL and the flow rate was 30 mLÆh)1 The protein content of each fraction was monitored by measuring the absorbance at 280 nm (s) and antibacterial activity (d) was assayed using the growth-inhibition plate method The pH of each fraction is indicated by a diamond Pooled fractions indicated by bar (III) were used for 6 M urea ⁄ PAGE (D) SDS ⁄ PAGE of the antibacterial fractions at each chromatography step C, crude mucus protein; I, pooled antibacterial fractions from Phenyl Sepharose chromatography; II, pooled antibacterial fractions from gel filtration chromatography; III, pooled antibacterial fractions from chromatofocusing The positions of the molecular mass markers are indicated.

inducing protein of Scomber japonicus (NCBI accession

no CAC00499) A domain search showed that the

gene detected in the gill of P stellatus contained a

dinucleotide-binding motif followed by a GG-motif

(R-x-G-G-R-x-x-T⁄ S), which is typical of flavoproteins

[18] RT-PCR using primers for the 5¢-UTR and

3¢-UTR regions of the LAAO sequence of P stellatus

(psLAAO) was performed to examine the tissue-specific

expression The results suggested that the psLAAO gene

was expressed in gill, but not in skin (Fig 5)

Localization of psLAAO by immunohistochemistry

To identify the localization of psLAAO protein in

the gill of P stellatus, immunohistochemistry was

performed with an anti-psLAAO IgG, obtained by

immunization of a Japanese white rabbit with insoluble

recombinant psLAAO purified from the E coli

expres-sion extracts The psLAAO cDNA sequence, without

the predicted signal peptide, was cloned into the

pET-20b vector and transformed into Rosetta2 (kDE3)

E coli competent cells In 5 L of Luria–Bertani (LB) broth, about 1.4 mg of insoluble recombinant psLAAO protein was expressed, but the protein was not detected

in soluble form by SDS⁄ PAGE or western blotting (Fig 6) The insoluble recombinant psLAAO protein was used for the preparation of antiserum Immunohis-tochemistry with the anti-psLAAO IgG showed a posi-tive reaction in the undifferentiated cells surrounding the vacuolated mucus-secreting cells of the gill (Fig 7B), principally within the epithelium of the primary lamellae and secondary lamellae The mucus-secreting cells stained positively with periodic acid⁄ Schiff’s reagent (HIO4⁄ Schiff), alcian blue and alcian blue-HIO4⁄ Schiff

Neutralization of antibacterial activity with anti-psLAAO IgG

In order to confirm whether the antibacterial protein was psLAAO, western blot analysis and a neutralization

Fig 3 Identification of antibacterial protein

by 6 M urea ⁄ PAGE and 2D gel

electrophore-sis (A) 6 M urea ⁄ PAGE after

chromatofo-cusing The antibacterial activity of each gel

extract from a 2-mm-wide strip was

measured using the growth-inhibition plate

method and is indicated as a diagram.

(B) SDS ⁄ PAGE after 6 M urea⁄ PAGE Each

of the gel extracts (slice numbers 8–29) was

subjected to determination of the molecular

mass of the antibacterial protein

Antibacte-rial fractions correspond to the upper

diagram and are indicated by ‘+’ The

asterisk indicates the specific band of the

antibacterial protein (C) 2D gel

electrophore-sis shows a single spot of antibacterial

protein indicated by a circle The positions

of the molecular mass markers are

indicated.

assay of antibacterial activity were performed using the

anti-psLAAO IgG In the western blot analysis,

psLAAO was detected in mucus and gill extract

(Fig 8A) In the neutralization assay, an apparent

dis-tinction was not found between the anti-psLAAO IgG

free control and the normal rabbit immunoglobulin

con-trol (Fig 8B) The neutralization activity of the

anti-psLAAO IgG increased in an antibody

concentration-dependent manner

Discussion

In the present study, we showed that the epidermal

mucus of P stellatus contains a protein with activity

against various pathogenic species and strains of

bacteria We isolated this antibacterial protein by col-umn chromatography through three different matrices and gel electrophoresis Furthermore, we detected the

Fig 4 The cDNA and amino acid sequences of Platichthys stellatus antibacte-rial protein The nucleotide sequence of cDNA encoding the PSEM antibacterial protein (DDBJ accession number AB495360) and the derived amino acid sequence are shown The N-terminal and internal peptide sequences of antibacterial protein detected by amino acid sequencing analysis are indicated by boxes The predicted dinucleotide-binding motif and the GG-motif are indicated by a straight line and

a broken line, respectively.

Fig 5 Tissue-specific expression of psLAAO mRNA by RT-PCR Tissues were collected from the same fish Lane 1, total RNA from gill; lane 2, total RNA from skin.

N-terminal and internal peptide sequences of this pro-tein and elucidated its complete mRNA sequence by cDNA cloning Because a BLAST search demonstrated that the encoded antibacterial protein shared identity with a number of LAAO flavoproteins, and a domain search showed that the gene contained typical flavo-protein motifs, the flavo-protein was suggested to be a new member of the LAAO family RT-PCR and immuno-histochemical analysis demonstrated tissue-specific expression and localization in the gill Western blot analysis with an anti-psLAAO IgG detected the pro-tein in mucus and gill extract Moreover, a neutraliza-tion assay of antibacterial activity against MRSA demonstrated that the clear zone was slightly reduced

employed Thus, we confirmed that the protein present

in PSEM was a novel LAAO-like antibacterial protein LAAOs are flavoenzymes that catalyze the oxidation

of l-amino acids, resulting in the production of a-keto acids, ammonia and hydrogen peroxide [19] It has

Fig 6 Recombinant protein expression in the transfected bacteria.

(A) SDS⁄ PAGE and (B) western blot analysis of the bacterial

extracts Lane 1, soluble cytoplasmic fraction; lane 2, insoluble

cytoplasmic fraction The positions of the molecular mass markers

are indicated M, positions of the molecular mass markers.

Fig 7 Immunohistochemical analysis of

Platichthys stellatus gill tissues with

anti-psLAAO IgG Gill sections of P stellatus

were stained with (A) nonimmune control

immunoglobulin, (B) anti-psLAAO IgG

(C) hematoxylin & eosin, (D) HIO4⁄ Schiff,

(E) alcian blue and (F) alcian

blue-HIO 4 ⁄ Schiff Arrows denote the mucous

cells Scale bar, 50 lm.

been reported that LAAOs have bioactivities as

anti-bacterial, antiviral and cytotoxic agents in a variety of

animal fluids, such as snake venom [20–25], mouse

milk [26,27], fish epidermal mucus and extract [28,29], body surface mucus of the giant African snail [30] and the ink of the sea hare [31,32] Previous studies have suggested that the bioactivity of LAAO is elicited by hydrogen peroxide generated from l-amino acid oxida-tion [25,32] and the binding of LAAO to bacterial cells and viruses [33,34] Achacin, an antibacterial protein

in the mucus of the giant African snail, also shows significant bacterial-binding and LAAO activity against S aureus and E coli [33] Escapin, from the ink of the sea hare, has an l-lysine-dependent antibac-terial effect and a broad antimicrobial spectrum, being most effective against S aureus [32] Moreover, the antimicrobial and antiparasitic LAAO isolated from Bothrops jararaca has the highest effectiveness against

S aureus [25] These findings suggest that the anti-bacterial effect is dependent on hydrogen peroxide production, because the antibacterial activity was abolished by catalase In the present study, PSEM also showed specific antibacterial activity against S aureus, and MRSA was significantly suppressed depending on the dose of catalase employed (data not shown) Thus, psLAAO in PSEM exerts antibacterial activity through hydrogen peroxide generated from the catalytic oxida-tion of l-amino acid, although details of the selective effect against bacteria are still unclear

In the cloning analysis, we identified a cDNA corre-sponding to the peptide sequence of the antibacterial protein RT-PCR analysis suggested that psLAAO mRNA was specifically expressed in the gill, and immunohistochemistry with anti-psLAAO IgG also showed that psLAAO-positive cells were present in the gill These results suggest that psLAAO has tissue-specific expression and is localized in gill Interestingly, using cloning analysis, we identified a highly homolo-gous gene that was expressed in the skin A domain search analysis suggested that this homologous gene also has a dinucleotide-binding motif and a GG motif, which are characteristic of the LAAO family Further-more, a BLAST search demonstrated high identity with the antibacterial protein of S schlegeli and other members of the LAAO family Immunohistochemical staining also showed a positive reaction with anti-psLAAO IgG in skin tissue (data not shown) because the anti-psLAAO IgG was cross-reactive with highly homologous LAAO extracted from skin mucus These results suggest that some types of LAAO are expressed

in different tissues of fish epidermis

The gill has a very important function as the main respiratory organ of fish and it also has an additional role in defense by secreting a mucus layer, which includes antibacterial proteins, as it is constantly exposed to bacteria in the external environment [6,7]

Fig 8 Reaction of anti-psLAAO IgG with antibacterial protein (A)

SDS ⁄ PAGE and western blot analysis Lanes 1 and 3, PSEM; lanes 2

and 4, gill extract The 52 kDa band is indicated by an asterisk (B)

Neu-tralization of antibacterial activity with anti-psLAAO IgG The MRSA

clinical isolate 87-7928 was suspended in TSA at a final concentration

of 1 · 10 6 CFUÆmL)1 Ten microliters of PSEM (upper panel) or gill

extract (lower panel) with the indicated volume (0–10 lL) of

anti-psLAAO IgG were applied to each hole in the agar after incubation at

37 C for 1 h Control immunoglobulin (10 lL) was applied with 10 lL

of PSEM or gill extract to the holes, as indicated by the hole labelled

‘C’ The total volume was adjusted with NaCl ⁄ P i to 20 lL PSEM and

gill extract protein in the clear zone on the growth-inhibition plate are

indicated as a diagram M, positions of the molecular mass markers.

The biological importance of the mucus interface

between the body and the aqueous environment

includes functions such as physiological and chemical

protection In the present study, the N-terminal peptide

sequence of psLAAO was found to start with a leucine

residue, not a methionine residue Moreover, the

complete psLAAO sequence was 1566 bp in length,

encoding a protein of 522 amino acid residues and with

an expected molecular mass of higher than 52 kDa The

antibacterial protein we isolated was estimated to have

a molecular mass of approximately 52 kDa Therefore,

psLAAO may be cleaved at Ala27 to become a mature

protein and secreted from the gill into the extracellular

matrix, and the antibacterial protein starting at Leu28

may be a component of the mucus covering the body

surface and acting as a barrier against bacteria

We found that psLAAO is effective against various

species of bacteria, suggesting its potential use against

clinical pathogens MRSA is a major cause of

hospi-tal-acquired infections and a matter of serious

public-health concern worldwide [35], including the UK [36],

Japan [37] and the USA [38] The appearance of such

multidrug-resistant bacteria has made it imperative to

develop effective and novel antimicrobial agents that

could be used to treat infection with these pathogens

We speculate that the psLAAO included in PSEM

could be one such agent because it has activity against

MRSA Our future work will be aimed at improving

the expression of bioactive recombinant psLAAO and

evaluating the mechanism of its antibacterial effect

Experimental procedures

Collection of epidermal mucus

P stellatus was caught in the brackish-water region of

Jusanko Lake, in Goshogawara City, Aomori, Japan After

rinsing the body surface with distilled water, the epidermal

mucus was scraped off with a rubber spatula and frozen at

) 80 C The PSEM was then thawed and centrifuged at

105 000 g for 1 h The supernatant was stored at) 80 C

Bacterial species and strains

Nineteen species or strains of bacteria were used to test the

antibacterial activity of PSEM: the gram-positive bacteria

S aureus (ATCC25923 and NIHJ JC-1), S epidermidis

(community isolate), MRSA (clinical isolates 87-7920,

87-7927, 87-7928, 87-7931 and 87-7958), Streptococcus

pyogenes (clinical isolate), Streptococcus agalactiae (clinical

isolate), Enterococcus faecalis ATCC33186, Enterococcus

faecium ATCC19434, E faecium BM4147 (VanA+, clinical

isolate), E faecalis V583 (VanB+, clinical isolate) and

Entero-coccus gallinarumBM4174 (VanC1+, clinical isolate); and the gram-negative bacteria E coli NIHJ JC-2, S marcescens (clinical isolate), V parahaemolyticus RIMD2210001 and

P aeruginosaATCC27853 All clinical isolates were provided

by Hirosaki University School of Medicine and Hospital

Antibacterial assay

The antimicrobial effects of PSEM were determined using a growth-inhibition plate assay The various bacterial species and strains were cultured in TSA (Difco, Detroit, MI, USA) for 16 h at 37C, except for V parahaemolyticus, which was cultured in trypticase soy broth supplemented with 0.5% NaCl The cell culture density was measured at

655 nm in a spectrophotometer and then adjusted to approximately 1· 108

colony-forming units (CFU)ÆmL)1 with phosphate-buffered saline (NaCl⁄ Pi), based on the standard curve In order to prepare pour plates, bacteria were suspended in TSA at a final concentration of

1· 106

CFUÆmL)1 Next, a hole of 2.8 mm in diameter was punched in the pour plate and filled with 12 lL of mucus or fractions from each of the purification steps After overnight incubation at 37C, the clear zone around the hole was measured To examine heat resistance, the PSEM was incubated for 1 h at 25, 37, 45, 56, 70 and

100C Each PSEM sample that had been subjected to the heating treatment was then applied to each hole After incubation overnight at 37C, the diameter of the clear zone around each spot was then measured

Purification of antibacterial protein from epidermal mucus

Unless indicated otherwise, all procedures were performed at

4C One-hundred and thirty milliliters of PSEM was thawed and dialyzed against 1 m (NH4)2SO4in 50 mm phos-phate buffer (pH 7.0), then applied to a column of Phenyl Sepharose 6 Fast Flow high sub (1.0· 25 cm; GE Health-care UK Ltd., Little Chalfont, Bucks, UK), equilibrated pre-viously with the same buffer, and the column was then washed with the buffer The flow rate of the column was

30 mLÆh)1and the fraction volume was 10 mL The protein concentration in each fraction was monitored by measuring the absorbance at 280 nm Adsorbed proteins were eluted from the column using a linear gradient of 1–0 m (NH4)2SO4

in 50 mm phosphate buffer, followed by elution with 50 mm phosphate buffer and 10 mm phosphate buffer Antibacterial activity was assayed using the growth-inhibition plate method The fractions with antibacterial activity were col-lected and the solution was subjected to 80% ammonium sulfate fractionation After centrifugation, the resulting pre-cipitate was dissolved in a small quantity of 0.1 m NaCl in

20 mm Tris⁄ HCl buffer (pH 7.5) and dialyzed against the same buffer The collected proteins were subjected to gel

filtration chromatography on a column of Sephacryl S-100

HR (1.2· 147cm; GE Healthcare) equilibrated with the

same buffer The fraction volume was 2.5 mL and the flow

rate was 8.0 mLÆh)1 Antibacterial protein was further

purified by chromatofocusing at pH 7–4 The protein in the

antibacterial activity fraction was concentrated by 80%

ammonium sulfate fractionation, as described above The

resulting precipitate was dialyzed against 25 mm

imidazole-HC1 (pH 7.4) and applied to a column of PEB94 polybuffer

exchanger (1.0· 27 cm; GE Healthcare) equilibrated with

25 mm imidazole-HC1 (pH 7.4) The fraction was eluted

with polybuffer 74 (pH 4.0), diluted 12-fold with de-aerated

water and further eluted with 0.5 m NaCl The fraction

volume was 2.5 mL and the flow rate was 30 mLÆh)1

Tissue collection and purification of antibacterial

protein from gill

After rinsing P stellatus in distilled water, the gill tissue was

harvested and ground into powder using a mortar and

pestle under liquid nitrogen Proteins were extracted in

the CytoBuster Protein Extraction Reagent (Novagen,

Madison, WI, USA) containing the protease inhibitor by

incubation at room temperature for 5 min After

centrifuga-tion, the supernatants were collected Extracted protein

from the gill was thawed and dialyzed against 1 m

(NH4)2SO4 in 50 mm phosphate buffer (pH 7.0), then

applied to a column of HiTrap Phenyl FF high sub

(1.6· 2.5 cm; GE Healthcare) equilibrated with the same

buffer, and the column was then washed with the buffer

The flow rate of the column was 1 mL⁄ min and the fraction

volume was 1 mL Proteins were eluted stepwise from the

column using 1–0 m (NH4)2SO4in 50 mm phosphate buffer,

followed by elution with 50 mm phosphate buffer

Antibac-terial activity was assayed using growth-inhibition plates

The fractions with antibacterial activity were collected

Electrophoresis

SDS⁄ PAGE was performed according to the method of

Laemmli [39] The samples were heated in 10% glycerol,

2% SDS, 6% 2-mercaptoethanol and 0.05 m Tris⁄ HCl

buf-fer (pH 6.8) for 3 min in a boiling water bath and subjected

to SDS⁄ PAGE with a 10% polyacrylamide gel Protein was

stained with Coomassie Brilliant Blue R-250 The

antibacte-rial protein fraction separated by chromatofocusing was

subjected to 6 m urea⁄ PAGE at room temperature The

lower gel consisted of 7.5% acrylamide, 6 m urea, 0.06%

ammonium persulfate, 0.15% N,N,N¢,N¢-tetramethyl

ethy-lenediamine (TEMED) and 0.3 m acetate buffer (pH 4.8),

while the upper gel consisted of 5.0% acrylamide, 6 m urea,

0.002% riboflavin 0.015% TEMED and 0.2 m acetate

buf-fer (pH 5.0) The reservoir bufbuf-fer was composed of 0.35 m

b-alanine and 0.136 m acetate buffer (pH 4.8) The upper

gel was polymerized by illumination with a fluorescent

light After electrophoresis, the lower gel was cut into strips

2 mm wide Then, 40 lL of 10 mm phosphate buffer was added and the gel was broken into small pieces The super-natant obtained by centrifugation was then used to measure antibacterial activity or to determine the molecular mass of antibacterial protein by SDS⁄ PAGE 2D gel electrophoresis was performed according to the method of O’Farrell [40],

as modified by Hirsch et al [41] Protein was stained with Coomassie Brilliant Blue R-250 The second-dimension electrophoresis was carried out on a 10% acrylamide gel

Amino acid sequencing

After 2D gel electrophoresis, proteins in the gel were blotted onto a PVDF membrane (Millipore Corp., Bedford,

MA, USA) using a semidry-type blotting apparatus, and the target protein spot was cut out The N-terminal amino acid sequence was analyzed using the Edman degradation method An inner peptide amino acid sequence analysis was also performed Peptidase digestion using lysyl end-peptidase, separation of the fragments by RP-HPLC and amino acid sequence analysis were assigned to the APRO Life Science Institute Inc (Naruto, Tokushima, Japan)

mRNA extraction and degenerate PCR

Total RNA was extracted from the epidermis and gill tissues

of P stellatus using an RNeasy Mini kit (Qiagen, Valencia,

CA, USA) in accordance with the manufacturer’s instruc-tions Total RNA was transcribed to cDNA at 42C for

60 min in the presence of the oligo (dT)15Primer (Promega, Madison, WI, USA) and Primescript Reverse Transcriptase (Takara, Tokyo, Japan) Degenerate oligonucleotide primers were designed on the basis of the determined amino acid sequences of the peptide fragments The forward degen-erate primers were 5¢-YTITCITTYCGIGIGCNCAY-3¢, 5¢-YTIAGYTTYCGIGCNCAY-3¢, 5¢-YTITCITTYAGRG CNCAY-3¢ and 5¢-YTIAGYTTYAGRGCNCAY-3¢ (corre-sponding to LSFRAHLSD) The reverse degenerate primer was 5¢-RTGIGCRTTIACYTCRAANGT-3¢ (corresponding

to RTFEVNAHPDIL) Amplification was carried out using

Ex Taq polymerase (Takara) under the following condi-tions: 95C for 5 min; 35 cycles of 95 C for 1 min, 48 C for 1 min and 72C for 1 min; 72 C for 9 min All PCR products were subcloned into the T-vector prepared by dT addition on EcoRV-digested blunt ends of pBluescript II SK+ (Stratagene, LA Jolla, CA, USA) DNA sequences were determined using an abi prism 310 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA)

5¢-RACE and 3¢-RACE

5¢-RACE was carried out according to the procedure of the 5¢-RACE System for Rapid Amplification of cDNA Ends