Myticin, a novel cysteinerich antimicrobial ... - Wiley Online Library

Eur. J. Biochem. 265, 71±78 (1999) q FEBS 1999

Myticin, a novel cysteine-rich antimicrobial peptide isolated from haemocytes and plasma of the mussel Mytilus galloprovincialis Guillaume Mitta, Florence Hubert, Thierry NoeÈl and Philippe Roch DeÂfense et ReÂsistance chez les InverteÂbreÂs Marins (DRIM), IFREMER-CNRS-UM2, Montpellier, France

We report here the isolation of two isoforms of a novel cysteine-rich peptide from haemocytes (isoform A of 4.438 Da and B of 4.562 Da) and plasma (isoform A) of the mussel, Mytilus galloprovincialis. The two molecules display antibacterial activity against gram-positive bacteria, whereas only isoform B is active against the fungus Fusarium oxysporum and a gram-negative bacteria Escherichia coli D31. Complete peptide sequences were determined by a combination of Edman degradation, mass spectrometry and cDNA cloning using a haemocyte cDNA library. The mature molecules, named myticins, comprise 40 residues with four intramolecular disulfide bridges and a cysteine array in the primary structure different to that of the previously characterized cysteine-rich antimicrobial peptides. Sequence analysis of the cloned cDNAs revealed that myticin precursors consist of 96 amino acids with a putative signal peptide of 20 amino acids, the antimicrobial peptide sequence and a 36-residue C-terminal extension. This structure suggests that myticins are synthesized as preproproteins and then processed by various proteolytic events before storage of the active peptide in the haemocytes. Myticin precursors are expressed mainly in the haemocytes as revealed by Northern blot analysis. Keywords: antibacterial peptide; cDNA; mollusc immunity; Mytilus; precursor. Bivalve molluscs rely on both cellular and humoral components that interact to protect against potential pathogens. The cellular component of marine bivalve immunity is mediated by the blood cells (haemocytes), by which invading micro-organisms are encapsulated, phagocytosed [1] and/or subjected to associated oxygen-dependent microbicidal mechanisms [2]. The plasma contains various biologically active substances, including high molecular mass cytotoxic proteins capable of lysing various vertebrate erythrocytes [3] and recently discovered antimicrobial peptides. Two types of small, cationic, cysteine-rich peptides have been reported: mytilin (34 amino acid residues), a cyclic peptide containing eight cysteines [4] and several members of the large insect defensin family [4,5]. Mytimicin, a cysteine-rich antifungal peptide has also been partially characterized [4]. Nothing is known of the localization and regulation of antimicrobial peptide expression in molluscs although peptide expression is well known in arthropods: synthesis of insect antimicrobial peptides is induced upon injury [6] and in the horseshoe crab, the oldest existing marine arthropod, such peptides are stored in haemocyte granules and released by degranulation upon activation by bacterial endotoxin [7]. Here, we report the characterization of two isoforms of a novel cysteine-rich antimicrobial peptide purified from the haemocytes and the plasma of Mytilus galloprovincialis, and their activity spectrum, we also establish for the first time in molluscs the structure of the precursor.

Correspondence to Ph. Roch, UMR 219 DRIM, UniversiteÂ de Montpellier 2, CC 80, place EugeÁne Bataillon, F-34095 Montpellier cedex 5, France. Fax: + 33 46 714 4622, Tel.: + 33 46 714 4625, E-mail: [email protected] Abbreviations: c.f.u., colony forming units; DMEM, Dulbecco's modified Eagle's medium; MAS, modified Alsever solution; MBC, minimal bacteriacidal concentration; MHB, Mueller Hinton broth; MIC, minimal inhibitory concentration; PB, Poor-Broth nutrient medium; p.f.u., plaque forming units; UPW, ultra-pure water. (Received 26 April 1999, accepted 17 June 1999)

M AT E R I A L S A N D M E T H O D S Haemolymph collection Adult mussels (M. galloprovincialis) were obtained from a commercial shellfish farm (Palavas, Gulf of Lion, France) during winter. The haemolymph (< 0.5 mL per animal) was extracted via a 23 gauge needle plus syringe, directly into an equal volume of the anti-aggregation buffer, modified Alsever solution (MAS; [8]), and immediately centrifuged at 800 g for 15 min at 4 8C. Plasma (cell-free haemolymph) was then supplemented with aprotinin (5 mg´mL21) and kept at 280 8C until use, while the cell pellet was air dried and stored at 280 8C until required. None of the animals used had been challenged with bacteria prior to haemolymph extraction. Peptide extractions The pellets of haemocytes were thawed and then suspended (5 : 1, v/v) in 50 mm Tris buffer, pH 8.7, containing 50 mm NaCl, and homogenized (30 strokes) using Dounce apparatus (100 mm). After centrifugation (10 000 g, 20 min, 4 8C), cellular organelles contained in the pellet were extracted (3 : 1, v/v) in 2 m acetic acid by sonication (three times for 30 s each) in an ice-cold water bath. Debris were eliminated by centrifugation (10 000 g, 20 min, 4 8C) and the organelle acid extract was kept at 4 8C until use. The plasma was first diluted (1 : 1, v/v) in ultra-pure water (UPW) containing 0.1% trifluoroacetic acid. The pH was brought to 3.9 with 1 m HCl in an ice-cold water bath under gentle stirring for 30 min. Centrifugation (10 000 g, 20 min, 4 8C) was used to clarify the supernatant, which was kept at 4 8C until use. Solid-phase extraction Organelle acid extracts and plasmatic supernatants were loaded onto Sep-Pak C18 Vac cartridges (Waters Associates)

72 G. Mitta et al. (Eur. J. Biochem. 265)

equilibrated in acidified water (0.05% trifluoroacetic acid in UPW). After washing with acidified water, two successive elutions were performed with 5 and 40% acetonitrile in acidified water. The fractions obtained were lyophilized and reconstituted with 14 mL UPW and tested for antimicrobial activity as described below. Only 40% fractions were active and submitted to reverse-phase HPLC. HPLC purification All HPLC purification steps were carried out on a Beckman Gold HPLC system equipped with a Beckman 168 photodiode array detector. Column effluent was monitored by UV absorption at 225 nm. Step 1. Aliquots of 1 mL of the 40% Sep-Pak fractions were subjected to reverse-phase HPLC on a Sephasil C18 column (250 4.1 mm, Pharmacia). Elution was performed with a linear gradient of 5±50% acetonitrile in acidified water over 90 min at a flow rate of 0.9 mL´min21. Fractions corresponding to absorbance peaks were collected in polypropylene tubes, freeze dried, reconstituted in 0.2 mL UPW and tested for antimicrobial activity as described below. Step 2. Active fractions were further loaded onto a Sephasil C8 column (250 4.1 mm, Pharmacia). Elution was performed with a linear gradient of 20±30% acetonitrile in acidified water over 40 min at a flow rate of 0.9 mL´min21. Fractions were collected and treated as above. Step 3. The peptides were purified on the same reversed-phase column as in step 1 using the acetonitrile gradient described in step 2. Step 4. To ascertain the purity of the peptides, one additional step was performed on a narrow bore C18 reverse-phase column (150 2 mm, Waters Associates) at a flow rate of 0.3 mL´min21 using the acetonitrile gradient described in step 2. Mass spectrometry and sequencing Ten microlitres of purified peptides, corresponding to 50 pmol, were dissolved in UPW and analysed by electrospray ionization in a Fisons mass spectrometer equipped with a quadrupol mass analyser (mass range of 1±5000 Da). Molecular mass was calculated from a series of multiple-charged protonated molecular ions. Purified peptides (0.5±1.5 nmol) were submitted to reduction and alkylation as described previously [5] before automated Edman degradation on a pulse-liquid automatic peptide sequencer (Applied Biosystem 494). Micro-organisms Bacteria Escherichia coli D31 (streptomycin resistant), Micrococcus luteus (A270), Aerococcus viridans (= Gaffkya homarii) and Bacillus megaterium were a gift from Dr D. Hoffmann (CNRS, Strasbourg, France). The human pathogen strains, Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus faecalis, Enterobacter aerogenes were isolated from patients by H. Darbas (HoÃpital Arnaud De Villeneuve, Montpellier, France), Salmonella thyphimurium (SL 1344), Brucella suis S1 (1330 ATCC) and Listeria monocytogenes (SV1/2a strain EGD) were a gift from Dr J-P. Liautard (INSERM, Montpellier, France). The marine bacteria, Vibrio alginolyticus (DSMZ 2171), Vibrio splendidus (ATCC 33125)

q FEBS 1999

and Vibrio vulnificus (DSMZ 10143) were a gift from J.-L. Nicolas (IFREMER, PlouzaneÂ, France), Salmonella newport was isolated from Thau laguna by P. Monfort (CNRS, Montpellier, France). The marine fungus Fusarium oxysporum (pathogenic for penaeid shrimp) was a gift from A. Vey (INRA, Saint Christolles-AleÁs, France). The protozoan Perkinsus marinus, a parasite affecting the Eastern oyster, Crassostrea virginica, was obtained from M. Faisal (Virginia Institute of Marine Sciences, USA). Antimicrobial assays After each purification step, antibacterial activity was monitored by a liquid growth inhibition assay [9]. Briefly, 10 mL aliquots from each fraction were incubated in microtiter plates with 100 mL of a Micrococcus luteus suspension (starting A600 of 0.001) in Poor-Broth nutrient medium (PB: 1% bactotryptone, 0.5% NaCl w/v, pH 7.5). Bacterial growth was assayed by measurement of A600 after 24 h incubation at 30 8C. To determine the minimal inhibitory concentration (MIC), serial doubling dilutions were carried out following the protocol described above. The MIC values are expressed as an interval of concentration (a±b), where (a) represents the highest peptide concentration at which bacteria are still growing and (b) the lowest concentration that causes 100% growth inhibition. The minimal bactericidal concentration (MBC) was determined according to the method of Hancock [10]. Peptides were dissolved in a solution containing 0.01% acetic acid and 0.2% BSA and then serial doubling dilutions of the stock solution were made in 0.01% acetic acid, 0.2% BSA. Ten-microlitre aliquots from each dilution were incubated in sterile 96-well poly(propylene) microtitre plates with 100 mL of a suspension of bacteria (starting A600 of 0.001) in Mueller Hinton broth medium (MHB, Difco). Bacterial growth was checked after 18 h incubation at 37 8C under agitation, except for the marine bacteria, which were incubated at 25 8C in MHB supplemented with NaCl (15 g´L21). The MBC was determined by plating the contents of the first three wells showing no turbidity onto MH agar plates and incubating at 37 8C for 18 h. The lowest concentration of peptide that prevented any residual colony formation corresponded to the MBC. Antifungal activity was monitored against Fusarium oxysporum as described by Felhbaum et al. [11] by a liquid growth inhibition assay. Briefly, 80 mL of fungal spores (final concentration 104 spores´mL21) suspended in potato dextrose broth (Difco) were added to 10 mL of peptide solution in microtitre plates. Growth inhibition was observed microscopically after 24 h incubation at 30 8C and quantified by measurement of A600 after 48 h. To test antiprotozoan activity, Perkinsus marinus was cultivated in Dubelcco's modified Eagle's medium (DMEM, Gibco) according to the method of Gauthier and Vasta [12]. Pure peptide (10 mm) was added to 4 104 P. marinus in sea water (20 mL final volume) and incubated for 1 h at room temperature. Parasite viability was then estimated using acridine orange and ethidium bromide vital staining technique [13]. Bactericidal assay Ten microlitres of purified peptide, at a concentration 10 times higher than the MIC, was mixed with 90 mL of an exponential phase culture of Micrococcus luteus (starting A600 of 0.01) in PB and incubated at 30 8C. Aliquots of 10 mL were plated after 0, 3, 10 and 30 min, 2, 6 and 24 h of incubation on nutrient agar

q FEBS 1999

and number of colony-forming units (c.f.u.) was counted after overnight incubation at 37 8C. Controls consisted in bacterial culture incubated with 10 mL of sterile water. Myticin-A-specific probe, cDNA library screening and 18S ribosomal probe Poly(A)+ RNA from adult mussel haemocytes was used to construct a cDNA library in the ZAP Express vector (Stratagene). Reverse transcription and PCR were used to prepare a DNA probe. From the myticin A amino acid sequence, a degenerate sense oligonucleotide pool corresponding to the residues 8±14 [5 0 -TA(C/T)TGGTG(C/T)GGIAA(A/G)TT(C/T)TG-3 0 ] and a degenerate antisense oligonucleotide pool corresponding to the residues 28±34 [5 0 -(A/G)CAIGC(A/G)CACAT(C/T)TTICCIGG(A/G)TG-3 0 ] were designed by back translation. Three micrograms of total RNA were submitted to reverse transcription using the Ready-to-Go You-primeTM first-strand beads kit (Pharmacia). One-fifth of the reaction was used directly as a template for the PCR with the two degenerate pool primers. PCR was performed with 40 cycles consisting of 1 min at 94 8C, 1 min at 48 8C and 1 min at 72 8C in 1.5 mm MgCl2 and 1 mm primers. The resulting 87-bp fragment (pBSMYTIC.87) was cloned using the PCR script Amp (SK+) cloning kit (Stratagene). The fragment was then [32P]-labelled by random priming using the Ready-to-go DNA labelling kit (Pharmacia) and used to screen 400 000 plaque forming units (p.f.u.) from the cDNA library transferred to Hybond-N filter membranes (Amersham). Highstringency hybridizations were carried out overnight at 65 8C in 5 Denhardt's solution, 5 NaCl/Pi/EDTA (50 mm sodium phosphate, 750 mm NaCl, 5 mm EDTA, pH 7.4), 0.1% SDS, 100 mg´mL21 salmon sperm DNA. The filters were washed in a solution of 0.5 NaCl/Cit (7 mm sodium citrate, 75 mm NaCl, pH 7.0) containing 0.1% SDS at 65 8C, followed by autoradiography. A secondary screening was performed to isolate the positive clones. Phagemids were obtained by in vivo excision according to the manufacturer's instructions and sequenced on both strands. A sense oligonucleotide [5 0 -TGACCTCGCGGAAAGAGCGC-3 0 ] and an antisense oligonucleotide [5 0 -AGGGGACGTAATCAACGCGAGC-3 0 ] were designed from the sequence of the ribosomal RNA 18S small subunit [18] and used in PCR experiments. Five hundred nanograms of mussel genomic DNA were submitted to amplification using 35 cycles consisting of 1 min at 94 8C, 1 min at 60 8C and 1 min at 72 8C in 1.5 mm MgCl2 and 1 mm primers. Northern blot analysis The haemocytes from four mussels (8 106 cells per animal) were centrifuged and suspended in 1 mL of Trizol (Life Technologies). Immediately after haemolymph collection, mantle, foot, labial palps, gills, hepatopancreas and adductor muscle were excised from the same animals and washed extensively in sterile sea water. Tissues (100 mg of each) were homogenized in 1 mL of Trizol using 30 potter strokes and total RNA was extracted using the manufacturer's protocol. Five micrograms of total RNA from each tissue and from each animal were pooled (20 mg total per tissue). Total RNAs and size markers were separated on a 1.2% agarose gel electrophoresis containing 17% formaldehyde, transferred and X-linked to a Hybond N membrane (Amersham) which was then stained with methylene blue. The membrane was hybridized with [32P]-labelled myticin A cDNA

Mollusc antimicrobial peptide (Eur. J. Biochem. 265) 73

in a solution containing formamide (50%), NaCl/Cit (5 ), Denhardt's solution (8 ), sodium phosphate (0.05 m pH 6.5), SDS (0.1%) and salmon sperm (100 mg´mL21) at 55 8C for 12 h then washed in 0.2 NaCl/Cit, 0.1% SDS at 65 8C and autoradiography was carried out. After autoradiography the membrane was stripped by incubating the blot with a boiling solution of 0.1% SDS for 1 h and submitted to a subsequent hybridization with the [32P]-labelled 18S DNA probe. Rapid amplification of 5 0 cDNA end (RACE-PCR) and PCR This was performed using the 5 0 -RACE kit (Boehringer) following the manufacturer's instruction. Briefly, 2 mg of total RNA from haemocytes were subjected to reverse transcription using antisense 21 nucleotides [5 0 -CTGCAATGAACACATGCGCAC-3 0 ] primer deduced from myticin A cDNA sequence. After first-strand cDNA synthesis and addition of a poly(dA) tail at its 5 0 -end, PCR was performed with an oligo(dT)-anchor primer and an internal antisense primer of 21 nucleotides [5 0 -TTACCGGGATGGAGTACTCTG-3 0 ] deduced from myticin A cDNA sequence. Amplification was performed according to the following programme: melting at 94 8C for 1 min, annealing at 50 8C for 1 min, elongation at 72 8C for 1 min (35 cycles). The PCR products were cloned using the PCR Script Amp (SK+) cloning kit (Stratagene) and several different cDNA clones were sequenced. A sense oligonucleotide mytic.1, [5 0 -CAAACGAACAACATGAAGGC-3 0 ] and an antisense oligonucleotide mytic.2 [5 0 -TGTGCAACTAGCAGTTCCACAAAAC-3 0 ] were designed from the sequence of the myticin A cDNA sequence and used in PCR experiments. Five hundred nanograms of mussel genomic DNA were subjected to amplification using 35 cycles consisting of 1 min at 94 8C, 1 min at 60 8C and 1 min at 72 8C in 1.5 mm MgCl2 and 1 mm primers. Southern blot analysis DNA used for the Southern blot analysis was prepared from the sperm of an adult mussel. Sperm cells were homogenized in 1 mL of DNAzolTM Reagent (Life Technologies) using 30 potter strokes and DNA was extracted using the manufacturer's protocol. Twenty micrograms of DNA were digested with various restriction endonucleases, subjected (with size markers) to electrophoresis in 0.7% agarose gel, transferred and X-linked to a Hybond N membrane (Amersham) which was then stained with methylene blue. The membrane was hybridized with the [32P]-labelled myticin A cDNA in a solution containing formamide (50%), NaCl/Cit (5 ), Denhardt's solution (8 ), sodium phosphate (0.05 m pH 6.5), SDS (0.1%) and salmon sperm (100 mg´mL21) at 55 8C for 12 h. The membrane was washed in 0.2 NaCl/Cit, 0.1% SDS at 65 8C and autoradiography was carried out. After autoradiography the membrane was stripped by incubating the blot with a boiling solution of 0.1% SDS for 1 h and subsequently hybridized with the [32P]-labelled myticin B cDNA probe using the same conditions as described above.

R E S U LT S Isolation of antimicrobial peptides from haemocytes and plasma Antimicrobial peptides were purified from 500 mL of haemolymph prepared from 1000 M. galloprovincialis mussels.


Fractions eluted by a linear gradient of 5±50% acetonitrile from the organelle extracted from haemocytes and from the plasma shared a common antimicrobial zone eluted between 33 and 56 min corresponding to 20±33% acetonitrile (Fig. 1). The present study focuses exclusively on the antimicrobial molecules eluted by 25±26% acetonitrile from the haemocyte organelle-rich extracts (Fig. 1A) and by 25% acetonitrile from the plasma acidic supernatant (Fig. 1B). These fractions were submitted to three additional reverse-phase HPLC purification steps. Finally, two peptides were purified from the haemocyte organelle-rich extracts and one from the plasmatic extracts. Partial characterization of isolated antimicrobial peptides from organelle-rich and plasmatic extracts Electrospray mass spectrometry and analysis of the widescan spectrum revealed a deduced molecular mass of 4437.28 ^ 2.46 Da for the molecule isolated from the fraction H5 (460 mg, Fig. 1A). Only one mass was obtained, illustrating the purity of the molecule. This peptide was sequenced by Edman degradation after reduction and alkylation. A 36-residue N-terminal sequence was obtained, including seven alkylated cysteines: HSHACTSYWCGKFCGTASCTHYLCRVLHPGKMCACV. Searches in sequence databases did not yield any homology with known peptides and we propose the name of myticin A for this novel cationic cysteine-rich antibacterial peptide. Using the same strategy, we isolated a peptide of 4563.45 ^ 1.32 Da from the fraction H3 (130 mg, Fig. 1A). According to its partial N-terminal sequence obtained on seven

Fig. 1. Reverse-phase HPLC of two acidic extracts obtained from haemocytes (A) and plasma (B). After pre-purification by solid-phase extraction, the material present in the fraction eluted with 40% acetonitrile was loaded onto a Sephasyl C18 column. Elution was performed with a linear gradient (dotted line) from 5 to 50% actonitrile over 90 min at a flow rate of 0.9 mL´min21. Absorbance peaks were monitored at 225 nm (solid line). Biological activity was detected by rapid antibacterial assay against Micrococcus luteus (white rectangles) and by antifungal assay against F. oxysporum (shaded rectangles). H3, H5 and P3 correspond to the myticins.

q FEBS 1999

residues including one alkylated cysteine (HPHVCTS), we propose the name of myticin B. Finally, the same method was used to demonstrate that the antimicrobial peptide present in plasmatic fraction P3 (20 mg, Fig. 1B) was identical to myticin A. Cloning of cDNAs encoding myticin isoform A and B To fully identify the amino acid sequences of the myticins and to obtain information on the precursor of these peptides, cDNA clones were screened. To isolate the myticin A cDNA, degenerate sense oligonucleotides corresponding to the segment composed of residues 8±14 of the mature peptide were designed and used in RT-PCR experiments with degenerate antisense oligonucleotides corresponding to the segment composed of residues 28±34 on haemocyte RNA. A 87-bp PCR fragment was obtained and identified by sequencing as the expected cDNA fragment encoding amino acids 8±34 of myticin A. This cDNA fragment was then cloned and used to screen a haemocyte cDNA library, isolating 168 clones out of 4 105 plaques. Four clones were sequenced, three of which being identical and containing an ORF encoding a 96-amino acid sequence corresponding to myticin A (Fig. 2A). The deduced amino acid sequence begins with a 20-residue signal peptide. The cleavage site for signal peptidase is most likely located after the alanine residue preceding the histidine in position 1 as predicted by Signal P VI. & software [15]. This signal peptide is directly C-terminally flanked by a 76 amino acid sequence, which confirmed the partial sequence of myticin A obtained by the biochemical methods described above and completed the primary sequence by adding the four end amino acids. Adding the mass values corresponding to the four residues HCSR deduced from the cDNA at position 37±40 of the precursor (Fig. 2A) and the 36 residue N-terminal sequence obtained by the biochemical approach, a calculated mass of 4446.21 Da was obtained for this 40-residue peptide. Assuming that the difference in mass was attributable to the arrangement of the eight cysteine residues into four intramolecular disulfide bridges (8 Da), the 40 amino acid sequence possessed a mass of 4438.21 Da, which is in agreement with the mass measured for the native myticin A (4437.28 ^ 2.46 Da). Moreover, the sequence of the mature myticin was followed by a C-terminal extension peptide consisting of 36 residues. The fourth clone obtained had a different sequence and contained also an ORF encoding a 96 amino acid sequence (Fig. 2B). This sequence confirmed the partial sequence of myticin B obtained by biochemical methods and completed the myticin B primary structure. A calculated mass of 4570.34 Da was obtained for the 40 residues starting with a histidine (position 1, Fig. 2B). Assuming that the difference in mass was attributable to the arrangement of the eight cysteine residues into four intramolecular disulfide bridges (8 Da), the mass of this 40 amino acid compound was 4562.34 Da, which is in agreement with the mass measured for the native myticin B (4563.45 ^ 1.32 Da). Consequently, we propose that myticin A and B represent two isoforms of a novel 40 residue cationic (pI 8.74 and 9.10, respectively), cysteine-rich antimicrobial peptide. Analysis of the deduced amino acid sequences showed that myticin A and B shared a common structure (Fig. 3): a 20-residue signal peptide highly conserved between the two isoforms, a 40-residue sequence corresponding to the mature peptide, and a highly conserved C-terminal extension of 36 residues. The differences between the signal sequences were

q FEBS 1999


Fig. 2. Nucleotide sequence of myticin A (A) and B (B) cDNA. The two cDNAs were obtained by screening a haemocyte cDNA library. Deduced amino acid sequences of the ORF are shown under the nucleotide sequences. Three asterisks indicate the stop codons. Polyadenylation signals are doubleunderlined. The double-headed arrows indicate the limits of the mature peptides.

only on two residues: I and L at respective position 4 and 9 on myticin A signal sequence were replaced by M and V in myticin B. In addition, 12 residues are different between the two mature peptide isoforms. Finally, the 36 residue C-terminal extensions of myticin A and B differ by nine residues located at the N-terminal end. Location of the mRNA expression To determine the size of the mRNA and to investigate tissue expression of the myticin precursor, Northern blot analysis was carried out using total RNA from various mussel tissues (Fig. 4). One band hybridized strongly with the myticin A cDNA insert. The length of this RNA was estimated to be < 0.72 kb, thereby indicating that the sequence in Fig. 2A represents the full-length cDNA, when taking the length of the

Fig. 3. Deduced amino acid sequences and alignment of the two isoforms of myticin precursors. Myticin A and B clones were obtained by screening a haemocyte cDNA library. Conserved amino acids are boxed. Sequences of mature peptides are underlined.

poly(A) tail into account. The strong intensity of the hybridization signal and the high frequency of the cDNA encoding this peptide in the haemocyte cDNA library (168 clones out of 4 105 p.f.u.) suggested that the myticin gene is abundantly expressed in haemocytes. Among various tissue RNAs, those from the mantle, labial palps and gills showed a faint band with the same mobility as that of the haemocytes. It was unclear, however, whether these bands were derived from small numbers of contaminating haemocytes in the dissected tissues or whether these tissues contain cells capable of expressing myticin transcripts. Southern blot analysis The gene of the myticin precursor was examined by Southern blot analysis with the same myticin A cDNA insert, which was


q FEBS 1999 Table 1. Activity spectrum of myticin A and B. For antibacterial test, MBC was determined by testing different concentrations of pure myticins in liquid growth inhibition assay according to the Hancock method [10]. For antifungal test, MIC was monitored against F. oxysporum according to Felhbaum et al. [11]. NT, not tested.

Organism

Fig. 4. Detection of myticin mRNA in various tissues by Northern blotting. Twenty micrograms of total RNA from various tissues were separated by 1.2% agarose±formaldehyde gel electrophoresis, blotted and hybridized with a [32P]-labelled cDNA probe corresponding to myticin A. Relative RNA amounts of the various tissues were controlled by hybridizing the same membrane with a [32P]-labelled DNA probe corresponding to 18S ribosomal RNA. 1, haemocytes; 2, mantle; 3, foot; 4, labial palps; 5, gills; 6, hepatopancreas; 7, adductor muscle.

used for Northern blotting (Fig. 5). The genomic DNA was digested completely by three different restriction enzymes (EcoRV, XhoI and EcoRI, respectively, lane 1, 2 and 3) and by a combination of restriction enzymes, XhoI, EcoRI, EcoRV and XhoI. We controlled that none of these enzymes digested the cDNA. In the EcoRV and XhoI digests, one band of 3.4 and 6.2 kbp, respectively, was observed. Two bands were obtained (5.8 and 4.3 kbp) in the EcoRI digest. In the EcoRI plus XhoI digest, two bands (3.6 and 1.8 kbp) were also observed. The EcoRV and XhoI digest gave only one band at 2.8 kbp. The same bands were obtained with the myticin B cDNA insert. These results suggest that the myticin gene is probably present as a single copy in the genome. One explanation of the results obtained with EcoRI could be the presence of an intron containing an EcoRI restriction site. PCR experiments performed on genomic DNA with oligonucleotides designed from the cDNA sequence gave evidence of an intron in the gene sequence and the sequence of the PCR product obtained revealed an EcoRI restriction site (Fig. 6).

Bacteria: gram-positive Micrococcus luteus Bacillus megaterium Staphylococcus aureus Listeria monocytogenes Enterococcus viridans Enterococcus faecalis Bacteria: gram-negative Escherichia coli D31 Salmonella newport Salmonella typhimurium Brucella suis Pseudomonas aeruginosa Enteromonas aerogenes Vibrio alginolyticus Vibrio vulnificus Vibrio splendidus Fungus Fusarium oxysporum Protozoan Perkinsus marinus

Myticin A MBC (mM)

Myticin B

2.25±4.5 2.25±4.5 . 20 . 20 9±4.5 . 20

1±2 1±2 . 20 . 20 2±4 NT

. . . . . . . . .

. 20

120±210 120±210 . 20 . 20 . 20 NT NT . 20 . 20 . 20 5±10

. 20

. 20

20 20 20 20 20 20 20 20 20

Antimicrobial activity spectrum of myticin A and B and bactericidal assay of myticin A The amounts of myticin A (104 nmol) and B (28 nmol) extracted in this study were sufficient to investigate the activity spectrum against a variety of bacterial strains (Table 1). In liquid growth inhibition, the purified myticin A and B had marked activity against the gram-positive strains, Micrococcus luteus, B. megaterium and A. viridans (MBC: 2.25±4.5 mm and 1±2 mm for myticin A and B, respectively). The other grampositive strains, Staph. aureus, L. monocytogenes and Enterococcus faecalis, were not affected, even at a concentration of 20 mm. Myticin B was also found to be moderately active against the gram-negative, E. coli D31 (MBC: 20±10 mm), whereas myticin A was not active even at a concentration of 20 mm. No activity was found towards the other gram-negative Table 2. Bacteriolytic activity of 20 mm myticin A on Micrococcus luteus.

Fig. 5. Southern blot analysis of genomic DNA. Twenty micrograms of genomic DNA were digested with EcoRV (lane 1), XhoI (lane 2), EcoRI (lane 3), XhoI plus EcoRI (lane 4) and EcoRV plus XhoI (lane5) and separated by 0.7% agarose gel electrophoresis, blotted and hybridized with a [32P]-labelled cDNA probe corresponding to myticin A. The numbers on the left indicate the size of the DNA markers in kbp.

Incubation time

Control water

Myticin A ( 1024 c.f.u.´mL21)

0 3 10 30 2 6 24

2.19 2.07 2.11 2.27 3.11 4.20 854.40

2.19 1.50 0.38 0.08 0.01 0 0

min min min min h h h

q FEBS 1999


Fig. 6. Sequence of the PCR product obtained with the oligonucleotides mytic.1 and 2. The two primers (bold and double underlined) were designed from the mytilin A cDNA sequence. The sequence represents the fragment of PCR amplification product obtained from genomic DNA. Fragments of cDNA were in bold. Splice site junctions follow the GT±AG rule (bold and underlined). An intron sequence of 776 nucleotides interrupts the myticin A cDNA sequence. The EcoRI restriction site (GAATTC) present in the intron sequence is in italic underlined.

strains tested, S. typhimurium, Br. suis, Ps. aeruginosa, Enterobacter aerogenes, V. alginolyticus, V. splendidus and V. vulnificus. In addition, myticin B was active against the filamentous fungus, F. oxysporum, pathogenic for shrimp (MIC: 5±10 mm) and no activity was found for myticin A, even at 20 mm. The two isoforms were tested against the protozoan parasite affecting the Eastern oyster, C. virginica, and the viability of the parasite was not affected, even at a concentration of 20 mm. When myticin A was incubated with Micrococcus luteus at 20 mm, a concentration 10 times higher than the MIC value, all the bacteria were killed in 2 h (Table 2). Mytilin B was not purified in quantity enough to perform the same test. Consequently, at least myticin A appears to exert a bactericidal activity.

DISCUSSION Here, we report here the full characterization of a new type of cysteine-rich antimicrobial peptide named myticin. Two isoforms (myticins A and B) were purified from the haemocytes and one (isoform A) also from the plasma of experimentally unchallenged mussels M. galloprovincialis. The two peptides were purified to homogeneity and fully characterized at the level of their amino acid sequences, using a combination of reverse-phase chromatography, Edman degradation, mass spectrometry and cDNA cloning. Because the cDNA encoding the two isoforms were obtained from a cDNA library prepared with the haemocytes of 1000 animals, it is not clear whether the two isoforms are expressed in the same mussel. However, Southern blot analysis using genomic DNA suggests that the myticin gene is present as a single copy in the genome and contains at least one intron with splice site junctions following the GT±AG rule with homology to the canonical exon±intron junction consensus sequence [16,17]. The amino acid sequence deduced from the cloned cDNA sequence revealed that the myticins are processed from a precursor molecule consisting of 96 residues. The myticin precursor consists of a signal peptide of 20 amino acid residues, a mature peptide of 40 amino acid residues and an additional C-terminal sequence of 36 residues. Evidence of these structural segments suggests that mature myticin may be generated through conventional processing mechanisms. Firstly, release of the pre-segment, then cleavage between N±V at positions 40 and 41 by an endoproteinase R. Another possibility is that the N-terminal signal sequence of the myticin precursor is processed in a stepwise fashion by a signal peptidase followed by a dipeptidyl aminopeptidase [18], because the E±A sequence preceding the mature myticin peptide is present in the C-terminal portion of the signal peptide. While the 20-residue N-terminal segment is presumed to be a signal sequence for translocation to the lumen of the rough endoplasmic reticulum,

the functional significance of the C-terminal portion is unknown. This portion is highly conserved between the two isoforms (particularly the last 18 residues which are identical) and contains several acidic amino acids, five and six for isoforms A and B, respectively. This acidic region may interact with the cationic part of the peptide to stabilize precursor conformation for proteolytic processing or prevent the membrane interaction of the basic peptide part. The C-terminal extension of the myticin may function as a signal to address the peptide to a particular haemocyte compartment. Such a location of the protein part after the mature peptide is unusual for invertebrate antimicrobial peptide precursors, although it has been observed in the tachyplesin precursor of the horseshoe crab, Tachypleus tridentatus [19]. In this arthropod, the mature peptide is also stored in haemocytes, particularly in the small granules. Our data revealed that mussel haemocytes are a site of production and storage of the myticins, which were found in abundance in the acid extract of haemocyte organelle-rich fraction. Furthermore, myticin cDNAs were isolated from a cDNA library constructed with haemocyte mRNA and the analysis by Northern blot confirmed that haemocytes are the site of myticin precursor production. This implies that the myticins are processed from mRNA to active peptides within the haemocytes, something also observed in other invertebrates, namely the shrimp, Penaeus vannamei [20], and the horseshoe crab, T. tridentatus [19]. In the latter species, the haemocytes are extremely sensitive to microbial substances such as lipopolysaccharides and b glucans. Upon stimulation, the haemocytes degranulate and release into the extracellular fluid a series of substances involved in immune defence [21], including several antimicrobial peptides such as tachyplesins [19], big defensin [22] or tachycitin [23]. A similar mechanism may occur in mussels as the antimicrobial peptides were also present in the plasma of the animals used for this study. Although the animals were not challenged experimentally with bacteria, it is possible that some could already be in a stimulated immune state leading to haemocyte activation and partial release of the peptide in the plasma. Relative to their size, myticins are remarkably rich in cysteine residues (eight cysteines out of 40 residues), which leads to the assumption that their three-dimensional structure is highly compact. The three-dimensional structure has yet to be elucidated, but the position of the cysteines in the primary structure is different from those of the previously characterized cysteine-rich antimicrobial peptides in invertebrates (insect defensins, big defensin, tachyplesine, drosomycine, thanatine, buthinine, mytilin, MGD1, penaedin; [4,5,11,19,20,22,24,25]), vertebrates (mammalian defensins and protegrins, brevinins; [26]) and plants (plant defensins, g-thionins, Ib-AMP1-4; [27,28]). Myticins are essentially active against gram-positive


bacteria, including some pathogens for marine invertebrates [29,30] and are much less active against gram-negative bacteria and fungi. Myticins were found to have a bacteriolytic effect at the concentration tested, but this effect was much less rapid than that of mytilin, which killed bacteria within seconds [4]. In conclusion, myticins constitute a new type of cysteine-rich peptides. Their antimicrobial activity, together with their location in haemocytes, argue in favour of their participation in immune defence. However, they constitute only one partner of the mussel immune system as at least several other antimicrobial peptides and a cytolytic complex are also present in the haemocytes and/or plasma. Further studies will investigate the subcellular location of these peptides together with the influence of microbial substances on their release.

ACKNOWLEDGEMENTS We are grateful to Dr Bernard Calas (Centre de Biochimie Structurale, UniversiteÂ de Montpellier 1) for performing mass spectrometry. We are indebted to Dr Delphine Destoumieux (DRIM) for advices during biochemical purification. We thank Dr Elisabeth Dyrynda (School of Biological Sciences, University of Wales, Swansea, UK) for correcting the manuscript and Judith Atlan (DRIM) for technical assistance.

REFERENCES 1. Renwrantz, L. (1990) Internal defense system of Mytilus edulis. In Neurobiology of Mytilus edulis (Stephano, G., ed.), pp. 256±275. Manchester University Press, Manchester, UK. 2. BacheÁre, E., Hervio, D. & Mialhe, E. (1991) Luminol-dependent chemiluminescence by hemocytes of two marine bivalves, Ostrea edulis and Crassostrea gigas. Dis. Aquat. Org. 11, 173±180. 3. Hubert, F., Cooper, E.L. & Roch, Ph. (1997) Structure and differential target sensitivity of the stimulable cytotoxic complex from hemolymph of the Mediterranean mussel Mytilus galloprovincialis. Biochim. Biophys. Acta 1361, 29±41. 4. Charlet, M., Chernysh, S., Philippe, H., HeÂtru, C., Hoffmann, J. & Bulet, P. (1996) Innate immunity. Isolation of several cysteine-rich antimicrobial peptides from the blood of a mollusc, Mytilus edulis. J. Biol. Chem. 271, 21808±21813. 5. Hubert, F., NoeÈl, T. & Roch, Ph. (1996) A member of the arthropod defensin family from edible Mediterranean mussels (Mytilus galloprovincialis). Eur. J. Biochem. 240, 302±306. 6. Hoffmann, J.A., Reichart, J.M. & Hetru, C. (1996) Innate immunity in higher insects. Curr. Opin. Immunol. 8, 8±13. 7. Iwanaga, S., Kawabata, S.I. & Muta, T. (1998) New types of clotting factors and defense molecules found in horseshoe crab hemolymph: their structure and functions. J. Biochem. 123, 1±15. 8. BacheÁre, E., Chagot, D. & Grizel, H. (1988) Separation of Crassostrea gigas hemocytes by density gradient centrifugation and counterflow centrifugal elutriation. Dev. Comp. Immunol. 12, 549±559. 9. Bulet, P., Cociancich, S., Dimarcq, J.L., Lambert, J., Reichhart, J.M., Hoffmann, D., HeÂtru, C. & Hoffmann, J.A. (1991) Insect immunity. Isolation from a coleopteran insect of a novel inducible antibacterial peptide and of new members of the insect defensin family. J. Biol. Chem. 266, 24520±24525. 10. Hancock, B. (1997) Recently modified methods used by the Hancock Laboratory. http://www.interchg.ubc.ca/bobh/methods.htm. 11. Fehlbaum, P., Balet, Ph., Michaut, L., Lagueux, M., Broekaert, W.F. & Hoffman, J.A. (1994) Insect immunity. Septic injury of Drosophila induces the synthesis of a potent antifungal peptide with sequence homology to plant antifungal peptides. J. Biol. Chem. 269, 33159±33163. 12. Gauthier, J.D. & Vasta, G.R. (1995) In vitro culture of the eastern

q FEBS 1999

13. 14.

15. 16. 17. 18. 19.

20.

21. 22.

23.

24.

25.

26. 27. 28.

29.

30.

oyster parasite Perkinsus marinus: optimization of the methodology. J. Invertebr. Pathol. 66, 156±168. Morvan, A., Iwanaga, S., Comps, M. & BacheÁre, E. (1997) In vitro activity of the Limulus antimicrobial peptide tachyplesin I on marine bivalve pathogens. J. Invertebr. Pathol. 69, 177±182. Kenchington, E.L.R., Landry, D. & Bird, C.J. (1995) Comparison of taxa of the mussel Mytilus (Bivalvia) by analysis of the nuclear small-subunit rRNA gene sequence. Can. J. Fish. Aquat. Sci. 52, 2613±2620. Nielsen, H., Engelbrecht, J., Brunak, S. & von Heijne, G. (1997) Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng. 10, 1±6. Breathnach, R. & Chambon, P. (1981) Organization and expression of eucaryotic split genes coding for proteins. Annu. Rev. Biochem. 50, 349±383. Mount, S.M. (1982) A catalogue of splice junction sequences. Nucleic Acids Res. 10, 459±472. Kreil, G. (1990) Processing of precursors by dipeptiylaminopeptidases: a case of molecular ticketing. Trends Biochem. Sci. 15, 23±26. Shigenaga, T., Muta, T., Toh, Y., Tokunaga, F. & Iwanaga, S. (1990) Antimicrobial tachyplesin peptide precursor cDNA cloning and cellular localization in the horseshoe crab (Tachypleus tridentatus). J. Biol. Chem. 265, 21350±21354. Destoumieux, D., Bulet, P., Loew, D., Van Dorsselaer, A., Rodriguez, J. & BacheÁre, E. (1997) Penaeidines, a new family of antimicrobial peptides isolated from the shrimp Peanaeus vannamei (Decapoda). J. Biol. Chem. 272, 28398±28406. Muta, T. & Iwanaga, S. (1996) The role of hemoplymph coagulation in innate immunity. Curr. Opin. Immunol. 8, 41±47. Saito, T., Kawabata, S.I., Shigenaga, T., Takayenoki, Y., Cho, J., Nakajima, H., Hirata, M. & Iwanaga, S. (1995) A novel big defensin identified in horseshoe crab hemocytes: isolation, amino acid sequence, and antibacterial activity. J. Biochem. 117, 1131±1137. Kawabata, S.I., Nagayama, R., Hirata, M., Shigenaga, T., Agarwala, K.L., Saito, T., Cho, J., Nakajima, H., Takagi, T. & Iwanaga, S. (1996) Tachycitin, a small granular component in horseshoe crab hemocytes, is an antimicrobial protein with chitin-binding activity. J. Biochem. 120, 1253±1260. Fehlbaum, P., Bulet, P., Chernysh, S., Briand, J.-P., Roussel, J.-P., Letellier, L., HeÂtru, C. & Hoffmann, J.A. (1996) Structure±activity analysis of thanatin, a 21-residue inducible insect defense peptide with sequence homology to frog skin antimicrobial peptides. Proc. Natl Acad. Sci. USA 93, 1221±1225. Ehret-Sabatier, L., Loew, D., Goyffon, M., Fehbaum, P., Hoffmann, J.A., Van Dorsselaer, A. & Bulet, P. (1996) Characterization of novel cysteine-rich antimicrobial peptides from scorpion blood. J. Biol. Chem. 271, 293537±229544. Simmaco, M., Mignogna, G., Barra, D. & Bossa, J.M. (1993) Novel antimicrobial peptides from skin secretion of the European frog Rana esculenta. FEBS Lett. 324, 159±161. Broekaert, W.F., Terras, F.R.G., Cammue, B.P.A. & Osborn, R.W. (1995) Plant defensins: novel antimicrobial peptides as components of the host defense system. Plant Physiol. 108, 1353±1358. Tailor, R.H., Acland, D.P., Attenborough, S., Cammue, B.P., Evans, I.J., Osborn, R.W., Ray, J.A., Rees, S.B. & Broekaert, W.F. (1997) A novel family of small cysteine-rich antimiccrobial peptides from seed of Impatiens balsima is derived from a single precursor protein. J. Biol. Chem. 272, 24480±24487. Johnson, P.T., Stewart, J.E. & Arie, B. (1981) Histopathology of Aerococcus viridans var. homari infection (gaffkemia) in the lobster, Homarus americanus, and a comparison with histological reactions to a Gram-negative species, Pseudomonas perolens. J. Invertebr. Pathol. 38, 127±148. Friedman, C.S., Beattie, J.H., Elston, R.A. & Hedrick, R.P. (1991) Investigation of the relationship between the presence of a Grampositive bacterial infection and summer mortality of the Pacific oyster, Crassostrea gigas Thunberg. Aquaculture 94, 1±15.