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Penaeidins, a New Family of Antimicrobial Peptides Isolated from the Shrimp Penaeus vannamei (Decapoda)*

  • Delphine Destoumieux
    Affiliations
    Institut Français de Recherche et d'Exploitation de la Mer/CNRS, Unité Mixte de Recherche 219, “Défense et Résistance chez les Invertébrés Marins,” Université de Montpellier 2, CC 80, 34095 Montpellier, France
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  • Philippe Bulet
    Footnotes
    Affiliations
    Institut de Biologie Moléculaire et Cellulaire, Unité Propre de Recherche 9022, CNRS, “Réponse Immunitaire et Développement chez les Insectes,” 15 rue René Descartes, 67084 Strasbourg Cedex, France
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  • Damarys Loew
    Affiliations
    Laboratoire de Spectrométrie de Masse Bio-Organique, Unité de Recherche Associée 31, CNRS-Université Louis Pasteur, Faculté de Chimie, 1 rue Blaise Pascal, 67008 Strasbourg Cedex, France
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  • Alain Van Dorsselaer
    Affiliations
    Laboratoire de Spectrométrie de Masse Bio-Organique, Unité de Recherche Associée 31, CNRS-Université Louis Pasteur, Faculté de Chimie, 1 rue Blaise Pascal, 67008 Strasbourg Cedex, France
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  • Jenny Rodriguez
    Affiliations
    Centro Nacional de Acuiculture e Investigaciones Marinas-Escuela Superior Politécnica del Litoral, Guayaquil, Ecuador
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  • Evelyne Bachère
    Footnotes
    Affiliations
    Institut Français de Recherche et d'Exploitation de la Mer/CNRS, Unité Mixte de Recherche 219, “Défense et Résistance chez les Invertébrés Marins,” Université de Montpellier 2, CC 80, 34095 Montpellier, France
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  • Author Footnotes
    * This work was supported by the Institut Français de Recherche et d'Exploitation de la Mer and by the Centre National de la Recherche Scientifique.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) Y14925, Y14926, Y14927, and Y14928 and to the Swiss-prot Data Base with accession numbers P81056, P81057, P81058, P81059, and P81060.
    ¶ To whom correspondence regarding isolation and biochemical characterization of antimicrobial peptides should be addressed.
    ‡ To whom all other correspondence should be addressed. Tel.: 33467144710; Fax: 33467144622.
Open AccessPublished:November 07, 1997DOI:https://doi.org/10.1074/jbc.272.45.28398
      We report here the isolation of three members of a new family of antimicrobial peptides from the hemolymph of shrimpsPenaeus vannamei in which immune response has not been experimentally induced. The three molecules display antimicrobial activity against fungi and bacteria with a predominant activity against Gram-positive bacteria. The complete sequences of these peptides were determined by a combination of enzymatic cleavages, Edman degradation, mass spectrometry, and cDNA cloning using a hemocyte cDNA library. The mature molecules (50 and 62 residues) are characterized by an NH2-terminal domain rich in proline residues and a COOH-terminal domain containing three intramolecular disulfide bridges. One of these molecules is post-translationally modified by a pyroglutamic acid at the first position. Comparison of the data obtained from the cDNA clones and mass spectrometry showed that two of these peptides are probably COOH-terminally amidated by elimination of a glycine residue. These molecules with no evident homology to other hitherto described antimicrobial peptides were named penaeidins.
      Living in an aquatic environment rich in microorganisms, crustaceans have developed effective systems for detecting and eliminating noxious microorganisms. The defense mechanisms, largely based on the activity of the blood cells, include encapsulation, phagocytosis and associated oxygen-dependent microbicidal mechanisms (
      • Bachère E.
      • Mialhe E.
      • Rodriguez J.
      ), the prophenoloxidase activating system leading to melanization, and hemolymph coagulation, a rapid and powerful system that prevents blood loss upon wounding and participates in the engulfment of invading microorganisms (
      • Söderhäll K.
      • Cerenius L.
      • Johansson M.W.
      ). In the horseshoe crab (Chelicerata, Merostomata), the oldest existent marine arthropod, the hemocytes respond to a bacterial endotoxin activation by cell adhesion and degranulation. The released granule-specific proteins include clotting factors essential for hemolymph coagulation, lectins, and a large number of antimicrobial substances (for review see Ref.
      • Iwanaga S.
      • Muta T.
      • Shigenaga T.
      • Miura Y.
      • Seki N.
      • Saito T.
      • Kawabata S.
      ). In insects, the synthesis of potent antimicrobial peptides or polypeptides induced upon injury is a major and important component of the humoral innate host defense (
      • Hoffmann J.A.
      • Reichhart J.-M.
      • Hetru C.
      ). Surprisingly, in crustaceans, the role of antimicrobial peptides in the survival against invading microorganisms has hardly been studied. Until now, bactericidal activities have only been demonstrated in the hemocytes of very few crustaceans (
      • Chisholm J.R.S.
      • Smith V.J.
      ). Three constitutive hemocytic proteins have been isolated to date in the shore crab, Carcinus maenas, and one of these, a 6.5-kDa antibacterial peptide, has been partially characterized (
      • Schnapp D.
      • Kemp G.D.
      • Smith V.J.
      ).
      Antimicrobial peptides are widespread in the living kingdom, and a large number of these molecules have been isolated from vertebrates and invertebrates (reviewed by Hetru et al. (
      • Hetru C.
      • Bulet P.
      • Cociancich S.
      • Dimarcq J.-L.
      • Hoffmann D.
      • Hoffmann J.A.
      )) as well as from plants (
      • Broekaert W.F.
      • Terras F.R.G.
      • Cammue B.P.A.
      • Osborn R.W.
      ). For the time being and for convenience, these antimicrobial peptides are tentatively classified into four distinct groups based on amino acid sequences, secondary structures, and functional similarities: (i) linear basic peptides forming amphipathic α-helices including the cecropins, the first antimicrobial peptide isolated from insect hemolymph (for review see Ref.
      • Boman H.G.
      • Faye I.
      • Gudmunssson G.H.
      • Lee J.Y.
      • Lidholm D.A.
      ); (ii) peptides with one to six intramolecular disulfide bridges including the arthropod defensins (
      • Hoffmann J.A.
      • Hetru C.
      ), antifungal peptides from Drosophila, drosomycin (
      • Fehlbaum P.
      • Bulet P.
      • Michaut L.
      • Lagueux M.
      • Broekaert W.F.
      • Hetru C.
      • Hoffmann J.A.
      ) and metchnikowin (
      • Levashina A.
      • Ohresser S.
      • Bulet P.
      • Reichhart J.-M.
      • Hetru C.
      • Hoffmann J.A.
      ), thanatin from Podisus (
      • Fehlbaum P.
      • Bulet P.
      • Chernysh S.
      • Briand J.-P.
      • Roussel J.-P.
      • Letellier L.
      • Hetru C.
      • Hoffmann J.A.
      ), tachyplesin, big defensin and tachycitin from limulus (
      • Nakamura T.
      • Furunaka H.
      • Miyata T.
      ,
      • Saito T.
      • Kawabata S.
      • Shigenaga T.
      • Takayenoki Y.
      • Cho J.
      • Nakajima H.
      • Hirata M.
      • Iwanaga S.
      ,
      • Kawabata S.I.
      • Nagayama R.
      • Hirata M.
      • Shigenaga T.
      • Agarwala K.L.
      • Saito T.
      • Cho J.
      • Nakajima H.
      • Takagi T.
      • Iwanaga S.
      ), and other cysteine-rich antimicrobial peptides isolated from a scorpion (
      • Ehret-Sabatier L.
      • Loew D.
      • Goyffon M.
      • Fehlbaum P.
      • Hoffmann J.A.
      • van Dorsselaer A.
      • Bulet P.
      ) and from a bivalve mollusk (
      • Charlet M.
      • Chernysh S.
      • Philippe H.
      • Hetru C.
      • Hoffmann J.A.
      • Bulet P.
      ,
      • Hubert F.
      • Noël T.
      • Roch P.
      ); (iii) proline-rich peptides, among them the apidaecins and abaecins from Hymenoptera (
      • Casteels P.
      • Ampe C.
      • Jacobs F.
      • Vaeck M.
      • Tempst P.
      ,
      • Casteels P.
      • Ampe C.
      • Rivière L.
      • Damne J.V.
      • Elicone C.
      • Fleming M.
      • Jacobs F.
      • Tempst P.
      ) and drosocin from Drosophila hemolymph (
      • Bulet P.
      • Dimarcq J.-L.
      • Hetru C.
      • Lagueux M.
      • Charlet M.
      • Hegy G.
      • Van Dorsselaer A.
      • Hoffmann J.A.
      ); (iv) glycine-rich antimicrobial peptides or polypeptides (9–30 kDa) such as the attacins (
      • Hultmark D.
      • Engstrom A.
      • Bennich H.
      • Boman H.G.
      • Andersson K.
      • Steiner H.
      ), diptericin (
      • Dimarcq J.-L.
      • Keppi E.
      • Dunbar B.
      • Lambert J.
      • Reichhart J.-M.
      • Hoffmann D.
      • Rankine S.M.
      • Fothergill J.E.
      • Hoffmann J.A.
      ) and sarcotoxins (
      • Kanai A.
      • Natori S.
      ). The mode of action, the broad activity, the molecular diversity, and the noncytotoxicity of all these circulating antimicrobial peptides make them very attractive as therapeutic agents for pharmaceutical or agricultural applications (
      • Norelli J.L.
      • Aldwinckle H.S.
      • Destefano-Beltran L.
      • Jaynes J.M.
      ,
      • Jaynes J.M.
      • Nagpala P.
      • Destefano-Beltran L.
      • Huang J.H.
      • Kim J.
      • Denny T.
      • Cetiner S.
      ).
      The cultivation of penaeid shrimp is a worldwide economically important activity especially in intertropical developed and developing countries. However, this industry is now suffering serious problems linked to infectious diseases (
      • Lightner D.V.
      ,
      • Vega-Villasante F.
      • Puente M.E.
      ), which cause a decrease in growth in shrimp production resulting in vast economic losses. In this context, the control of diseases has become a priority in terms of research in immunology and genetics to insure the long term survival of shrimp aquaculture. Therefore, we have undertaken the isolation of antimicrobial peptides in the tropical shrimp Penaeus vannamei.
      We report here, for the first time in a crustacean, the biochemical characterization, the antimicrobial activities, and the cDNA cloning of three antimicrobial peptides purified to homogeneity from the hemolymph of P. vannamei that have not been experimentally infected. These peptides, with molecular masses ranging from 5.5 to 6.6 kDa, are characterized by an over-representation of proline residues in their NH2-terminal domain and by 6 cysteine residues engaged in three intramolecular disulfide bridges concentrated in their COOH-terminal domain. One of these molecules is unusual in that the NH2 and COOH termini are blocked by a pyroglutamic acid residue and an amidation, respectively. These peptides, which cannot be associated to groups hitherto described, were named penaeidins, after the genus Penaeus.

      MATERIALS AND METHODS

      Animals and Hemolymph Collection

      Juvenile white leg shrimp, P. vannamei (Penaeidae, Decapoda) were obtained from an intensive shrimp farm in the province of Guayas, Ecuador. A total of 225 ml of hemolymph from five hundred animals (weight ranging from 10 to 30 g) was collected from the ventral sinus located at the base of the first abdominal segment, under 110 volume of anticoagulant buffer (10% sodium citrate, pH 7) supplemented with 200 μm phenylthiourea as a melanization inhibitor and 40 μg/ml aprotinin as a protease inhibitor. The hemolymph was then centrifuged at 700 × g at 4 °C for 15 min to remove the blood cells. Plasma (cell-free hemolymph) and hemocytes were separately frozen at −70 °C until use.

      Peptide Extraction

      Plasma Sample

      The plasma was first diluted (1:1 v/v) with MilliQ water and further (1:1 v/v) with 0.1% trifluoroacetic acid. The pH was then brought to 3.9 with 1 m HCl in an ice-cold water bath under gentle stirring for 1 h. Two successive centrifugations (8000 × g, 20 min, 4 °C) were performed to clarify the supernatant, which was kept in an ice-cold water bath at 4 °C until use.

      Hemocyte Sample

      After thawing, the hemocytes were homogenized using a Dounce apparatus (maximum, 152 μm; minimum, 76 μm) in 50 mm Tris buffer, pH 8.7, containing 50 mm NaCl. After centrifugation (8000 × g, 20 min, 4 °C), the supernatant (cytosolic fraction) was acidified to pH 3.6 by the addition of 1 m HCl and kept without freezing at 4 °C until further purification. The pellet containing cellular organelles was extracted in 2 m acetic acid by sonication (3 × 30 s) at medium power (Branson Ultrasons, Annemasse, France) in an ice-cold water bath. Debris was eliminated by centrifugation (8000 × g, 20 min, 4 °C), and the organelle acid extract was kept at 4 °C until use.

      Solid Phase Extraction Prepurification

      The plasmatic fraction and the cellular cytosolic and organelle acid extracts were separately loaded onto 35 cc Sep-Pak C18Vac cartridges (10 g, Waters Associates) equilibrated in acidified water (0.05% trifluoroacetic acid). After washing with acidified water, three stepwise elutions were performed with successively 5, 40, and 80% acetonitrile in acidified water. The different fractions obtained were lyophilized and reconstituted with MilliQ water before subjection to reversed-phase HPLC.
      The abbreviations used are: HPLC, high performance liquid chromatography; MALDI-TOF-MS, matrix-assisted laser desorption/ionization-time of flight-mass spectrometry; nanoES-MS-MS, nanoelectrospray ionization tandem mass spectrometry; MIC, minimal inhibitory concentration; PCR, polymerase chain reaction; ORF, open reading frame; MES, 4-morpholinoethanesulfonic acid; ACTH, adrenocorticotropic hormone.

      HPLC Purification

      Step 1: Reversed-phase HPLC

      The 40% Sep-Pak fractions were subjected to reversed-phase chromatography on an Aquapore RP300 C8 column (4.6 × 220 mm, Brownlee™) equilibrated in acidified water (0.05% trifluoroacetic acid). Separation of the 40% Sep-Pak fractions was performed with a linear gradient of 2–60% acetonitrile in acidified water over 80 min (0.72% acetonitrile/min) at a flow rate of 1 ml/min. Fractions were hand collected, dried under vacuum (Speed-Vac, Savant), reconstituted in MilliQ water, and tested for antimicrobial activity as described below.

      Step 2: Size Exclusion Chromatography

      Reversed-phase fractions showing the antimicrobial activity were further purified by size exclusion chromatography using two serially linked HPLC columns (Ultraspherogel SEC 3000 and SEC 2000 columns, 7.5 × 300 mm, Beckman) protected by a precolumn (Ultraspherogel SEC, 7.5 × 40 mm, Beckman). Elution was performed under isocratic conditions with 30% acetonitrile in acidified water (0.05% trifluoroacetic acid) at a flow rate of 0.5 ml/min. Fractions were hand collected and treated as above.

      Step 3: Reversed-phase Chromatography

      Different gradients were used for this third purification step of peptides 1–3. Peptides 1 and 2 were purified on the same reversed-phase column as in Step 1 at a controlled temperature of 35 °C with a linear biphasic gradient of 2–21% acetonitrile in acidified water (0.05% trifluoroacetic acid) over 10 min (1.9% acetonitrile/min) and of 21–35% over 50 min (0.28% acetonitrile/min) at a flow rate of 0.25 ml/min. Peptide 3 was purified with a linear biphasic gradient of 2–23% acetonitrile in acidified water over 10 min (2.1% acetonitrile/min) and of 23–37% over 50 min (0.28% acetonitrile/min) at a flow rate of 0.25 ml/min at 35 °C.

      Step 4: Final Purification Steps

      The last purification steps for peptides 1–3 were performed on a narrow bore C18reversed-phase column (Delta Pak HPI C18, 2 × 150 mm, Waters Associates) at 40 °C at a flow rate of 0.25 ml/min using the biphasic gradients described above in Step 3.
      All HPLC purification steps at room temperature were carried out on a Beckman Gold HPLC system equipped with a Beckman 168 photodiode array detector. For the HPLC purifications under controlled temperature, a Waters HPLC system (Waters 626 pump) attached to a tunable absorbance detector (Waters 486) was used. Column effluent was monitored by its UV absorption at 225 nm. Fractions corresponding to absorbance peaks were hand collected in polypropylene tubes (Microsorb 75 × 12 mm, Nunc immunotubes), concentrated under vacuum (Savant), and reconstituted in MilliQ water (Millipore™) before antimicrobial activity was tested.

      Capillary Zone Electrophoresis

      Peptide purity was ascertained by capillary zone electrophoresis. Analysis was performed on 2 nl of fractions using a 270A-HT electrophoresis system (Applied Biosystems, Inc.) equipped with a fused silica capillary (length, 72 cm; internal diameter, 50 μm). Electrophoresis was monitored at 30 °C in 20 mm citrate buffer, pH 2.5, at 20 kV. Capillary effluent was detected by its absorbance at 200 nm.

      Reduction and S-Pyridylethylation

      Purified peptides were subjected to reduction and alkylation using the procedures already described (
      • Bulet P.
      • Cociancich S.
      • Reuland M.
      • Sauber F.
      • Bischoff R.
      • Hegy G.
      • Van Dorsselaer A.
      • Hetru C.
      • Hoffmann J.A.
      ). Briefly, the peptide (1–2 nmol) was dissolved in 40 μl of 0.5 m Tris HCl containing 2 mm EDTA and 6 m guanidine hydrochloride, pH 7.5, to which 2 μl of 2.2 m dithiotreitol were added. The samples were incubated under oxygen-free conditions for 1 h at 45 °C. 2 μl of freshly distilled 4-vinylpyridine (Aldrich) were added, and incubation was continued for 10 min at 45 °C under N2 to prevent oxidation. The S-pyridylethylated peptide was desalted on an Aquapore RP300 C8 column (220 × 4.6 mm, Brownlee™) using a linear gradient of 2–60% acetonitrile in acidified water (0.05% trifluoroacetic acid) over 120 min (0.48% acetonitrile/min) at a flow rate of 1 ml/min.

      Enzymatic Cleavage

      Trypsin and α-Chymotrypsin Treatments

      Native andS-pyridylethylated peptides (5 μg) were subjected individually to trypsin and α-chymotrypsin treatments (Boehringer Mannheim). Trypsin and chymotrypsin hydrolysis were performed at an enzyme/substrate ratio of 1:20 (w/w) in a 40-μl reaction containing 0.1 m Tris-HCl at pH 8.5 and in a 50-μl reaction containing 100 mm Tris-HCl at pH 7.5 and 10 mmCaCl2, respectively. Incubations were carried out for 16 h at 37 and 25 °C for trypsin and α-chymotrypsin treatments, respectively. The reactions were stopped by acidification with 0.1% trifluoroacetic acid. Peptidic fragments were separated on a Delta Pak HPI C18 column (2 × 150 mm, Waters Associates) and eluted with a linear gradient of 2–80% acetonitrile in acidified water over 120 min (0.65% acetonitrile/min) at a flow rate of 0.25 ml/min.

      Arginyl Endopeptidase Treatment

      TheS-pyridylethylated peptide (5 μg) was treated with arginyl endopeptidase (Takara, Otsu) at an enzyme/substrate ratio of 1:100 (w/w) in a 20-μl reaction containing 10 mm Tris-HCl at pH 8.0 and 0.01% Tween 20. Incubation was performed for 16 h at 37 °C. Peptidic fragments were separated following the procedure described above.

      Thermolysin Treatment

      10 μg of each native andS-pyridylethylated peptides were separately treated with thermolysin (EC 3.4.24.27) from Bacillus thermoproteolyticus(Boehringer Mannheim) for 1 h at 37 °C at an enzyme/substrate ratio of 1:2 (w/w) in 0.1 m MES at pH 6.5 supplemented with 2 mm CaCl2. The digestion was stopped by adding 50 μl of 1% trifluoroacetic acid. The peptides generated by protease hydrolysis were separated by reversed-phase HPLC for further characterization by MALDI-TOF-MS and microsequencing by Edman degradation.

      Mass Measurement by MALDI-MS

      Instrumentation

      This study was carried out on a Bruker (Bremen) BIFLEX™ matrix-assisted laser desorption time-of-flight mass spectrometer equipped with SCOUT™ High Resolution Optics, an X-Y multisample probe, a gridless reflector, and the HIMAS™ linear detector. This instrument has a maximum acceleration potential of 30 kV and may be operated in either linear or reflector mode. Ionization was accomplished with the 337 nm beam from a nitrogen laser with a repetition rate of 3 Hz. The output signal of the detector was digitized at a sampling rate of 250 MHz in the linear mode using a 1 GHz digital oscilloscope. A camera mounted on a microscope allowed the inspection of the sample crystallization homogeneity before measurement. All spectra were obtained in the linear positive ion mode and externally calibrated with a mixture of three standard peptides (angiotensin II, ACTH 18–39, and bovine insulin with MH+at m/z 1047.2, 2466.1, and 5734.6, respectively).

      Sample Preparation

      Purified peptides or enzymatically derived fragments (1 μl) were deposited on a thin layer of α-cyano-4-hydroxycinnamic acid crystals made by fast evaporation of a saturated solution in acetone (
      • Vorm O.
      • Roepstorff P.
      • Mann M.
      ,
      • Vorm O.
      • Mann M.
      ). The droplets were allowed to dry under gentle vacuum before introduction into the mass spectrometer.

      Sequencing by Nanoelectrospray Tandem Mass Spectrometry

      Instrumentation

      The nanoelectrospray (nanoES) experiments were done on a triple quadrupole Bio-Q mass spectrometer, upgraded by the manufacturer so that the source and the quadrupoles had Quattro II performances (Micromass Ltd. UK, Altrincham). The conventional electrospray probe was modified so that a glass capillary similar to that described by Wilm and Mann (
      • Wilm M.
      • Mann M.
      ) could be positioned at about 2 mm from the first cone of the electrospray source. The source was used without counter electrode, and the drying gas heated at 50 °C was nitrogen. The glass capillary and extracting cone voltages were 900 and 50 V, respectively. Electrical contact between the probe tip and the metallized glass capillary (long needle type glass capillaries purchased from the Protein Analysis Company, Odense M) was made by using a graphite cone inside the Swagelok union instead of the customary brushing of an organic solution of graphite (
      • Wilm M.
      • Mann M.
      ) giving interference ions in the low m/z range. Before connecting the glass capillary into the mass spectrometer, it was opened by briefly touching a metal capillary (0.5-mm inner diameter × 150 mm) connected to a vacuum source. The opened glass capillary was then washed by applying a N2 pressure to reduce contamination by impurities in the metal layer. After loading the sample solution at a concentration of 1 pmol/μl in acetonitrile/water containing 1% formic acid, the glass capillary was inserted in the MS source, and static air pressure was applied to give a flow rate of approximately 20 nl/min, which allowed a stable signal recording for up to 3 h.

      Nanoelectrospray Tandem Mass Spectrometry

      In a first approach, the parent ion (m/z = 520.1) produced at a low cone voltage (V c = 30 V) was selected in the first quadrupole mass analyzer and fragmented by collision-induced dissociation with argon gas at 4.5 × 10−2 Pa at 40 V. To gain additional structural information, MS-MS experiments on fragments generated by source collision of the parent ion at m/z 520 with an extracting cone voltage of 100 V were performed using 60 and 80 V in the collision cell. The resulting fragment ions were named according to Roepstorff and Fohlman's nomenclature (
      • Roepstorff P.
      • Fohlman M.
      ). The quadrupole analyzers were calibrated by using the multiply charged ions from a separate acquisition of horse heart myoglobin (16951.5 Da).

      Microsequence Analysis

      Native,S-pyridylethylated peptides and peptidic fragments were subjected to Edman degradation on a pulse liquid automatic sequenator (Applied Biosystems, model 473A).

      Antimicrobial Assays

      Microorganisms

      The microbial strains used to determine antimicrobial activities during the purification steps were those used in previous studies (
      • Fehlbaum P.
      • Bulet P.
      • Michaut L.
      • Lagueux M.
      • Broekaert W.F.
      • Hetru C.
      • Hoffmann J.A.
      ,
      • Bulet P.
      • Cociancich S.
      • Dimarcq J.-L.
      • Lambert J.
      • Reichhart J.-M.
      • Hoffmann D.
      • Hetru C.
      • Hoffmann J.A.
      ): Micrococcus luteus(Gram-positive strain), Escherichia coli D31 (Gram-negative strain), and Neurospora crassa as a filamentous fungus. The marine fungus Fusarium oxysporum, pathogenic for penaeid shrimp (gift from Dr. Alain Vey, INRA, St. Christol-les-Alès, France) was used to complete the activity spectrum.

      Antibacterial Assay

      After each step of purification, an aliquot of each eluted fraction reconstituted in MilliQ water was tested by the liquid growth inhibition assay already described (
      • Bulet P.
      • Dimarcq J.-L.
      • Hetru C.
      • Lagueux M.
      • Charlet M.
      • Hegy G.
      • Van Dorsselaer A.
      • Hoffmann J.A.
      ). Briefly, 10-μl aliquots from each test fraction were incubated in microtiter plates with 100 μl of a suspension of a midlogarithmic phase culture of bacteria (E. coli D31 or M. luteus) at a starting optical density ofA 600 = 0.001 in Poor-Broth nutrient medium (1% bactotryptone, 0.5% NaCl, w/v). Bacterial growth was assayed by measurement of the optical density at A 600 after a 24-h incubation at 30 °C.
      An identical procedure was used to determine the minimal inhibitory concentration (MIC) of the molecules on the previously described bacterial strains. The MIC values are expressed as intervals of concentration (a–b), where a is the highest concentration at which bacteria are growing and b is the lowest concentration that causes 100% of growth inhibition (
      • Casteels P.
      • Ampe C.
      • Jacobs F.
      • Tempst P.
      ).

      Bacteriostatic Assay

      A midlogarithmic phase culture ofM. luteus in Poor-Broth nutrient medium was incubated at 30 °C in the presence of the antimicrobial peptides of interest or water (control). The final concentration of the molecules to be tested was eight times over the MIC value. 20-μl aliquots were removed at different time intervals and plated on nutrient agar. The number of colony-forming units was determined after 24 h at 37 °C.

      Antifungal Assay

      Antifungal activity was monitored againstN. crassa and F. oxysporum as described previously (
      • Fehlbaum P.
      • Bulet P.
      • Michaut L.
      • Lagueux M.
      • Broekaert W.F.
      • Hetru C.
      • Hoffmann J.A.
      ,
      • Fehlbaum P.
      • Bulet P.
      • Chernysh S.
      • Briand J.-P.
      • Roussel J.-P.
      • Letellier L.
      • Hetru C.
      • Hoffmann J.A.
      ) by a liquid growth inhibition assay. Briefly, 80 μl of fungal spores (final concentration, 104 spores/ml) suspended in potato dextrose broth (Difco) at half-strength supplemented with tetracycline (10 μg/ml) and cefotaxim (100 μg/ml) were added to 10 μl of fractions in microtitration plates. The final volume was brought to 100 μl by the addition of 10 μl of water. Growth inhibition can be observed microscopically after a 24-h incubation at 25 °C in the dark and measured by the increase in optical density (at 600 nm) after 48 h.

      Penaeidin-specific DNA Probe and Screening of cDNA Library

      Poly(A)+ RNA from juvenile shrimp hemocytes harvested 6 and 12 h after a bacterial challenge were used to construct a cDNA library in the ZAP Express vector (Stratagene, La Jolla, CA) following the manufacturer's instructions.
      Reverse transcription and polymerase chain reaction (PCR) were used to prepare a DNA probe corresponding to the P3 peptide (see “Results”) isolated from P. vannamei. From the peptide sequence obtained by Edman degradation, a degenerate oligonucleotide probe pool corresponding to the residues 38–44 of the mature molecule was designed by back translation: 5′-GGIAT(A/T/C)(A/T)(G/C)ITT(C/T)(A/T)(G/C)ICA(A/G)GC-3′ (see Fig.4 A). 3 μg of total hemocyte RNA were submitted to reverse transcription using the Ready-to-Go You-prime first-strand beads kit (Pharmacia Biotech Inc., Uppsala, Sweden) with a 18-base poly(dT) oligonucleotide as primer. One-fifth of the reaction was directly used as a template for polymerase chain reaction with the degenerate pool primers and the poly(dT) oligonucleotide. PCR was performed with five cycles consisting of 1 min at 94 °C, 1 min at 37 °C, and 1 min at 72 °C and 35 cycles consisting of 1 min at 94 °C, 1 min at 50 °C, and 1 min at 72 °C in 1.5 mm MgCl2and 1 μm primers.
      Figure thumbnail gr4
      Figure 4A, nucleotide sequence of a penaeidin-3 cDNA clone from the shrimp P. vannamei. The deduced amino acid sequence of the ORF is shown above the nucleotide sequence. An asterisk indicates the stop codon. A polyadenylation signal is double-underlined. Thedouble-headed arrow indicates the putative cleavage site by a signal peptidase. A dotted arrow indicates the position of the degenerate probe pool used in reverse transcription-PCR experiments to generate a probe for penaeidin-3 cDNA isolation. ABsaAI restriction site used in cloning of a probe for penaeidin-3 cDNA isolation is in boldface type. Theunderlined sequences correspond to the primers used to generate a probe for penaeidin-2 cDNA isolation. TheEcoRI linker used in construction of the cDNA library is indicated by a dotted line. B, alignment of the deduced amino acid sequences of the three penaeidin-3 cDNA clones isolated. Sequence differences are in boxes. Thedash represents a gap at the indicated position.
      The resulting 497-base pair fragment corresponding to a fragment of P3 cDNA was sequenced and a 440-base pair subfragment, for the most part consisting of the 3′-untranslated region, was generated byBsaAI enzymatic hydrolysis (see Fig. 4 A). This fragment was cloned into a pBluescript vector (Stratagene). It was labeled by random priming using the Ready-to-Go DNA labeling kit (Pharmacia) and used to screen 500,000 plaques from the cDNA library transferred to Hybond-N filter membranes (Amersham Corp.). High stringency hybridizations were carried out overnight at 65 °C in 5 × Denhardt's solution, 5 × SSPE (1 × SSPE = 150 mm NaCl, 1.25 mm EDTA, 10 mm sodium phosphate, pH 7.4), 0.1% SDS, 100 μg/ml salmon sperm DNA. The filters were washed in a solution of 0.5 × SSPE containing 0.1% SDS at 65 °C followed by autoradiography. A secondary screening was performed to purify the positive plaques. Phagemids were obtained byin vivo excision according to the manufacturer's instructions and sequenced on both strands.
      A screening at low stringency was performed to isolate other members of the family. A probe was generated by PCR on a P3 cDNA clone with 5′-GTGTACAAGGGCGGTTACACG-3′ as the upstream primer and 5′-CAACAGGTTGTCAAGCGAGGT-3′ as the downstream primer. The amplified fragment consisted mainly of the P3 open reading frame (ORF). Radiolabeling and hybridization were identical to those previously described with the exception of a reduced hybridization temperature (50 °C).

      DISCUSSION

      We report here the first isolation and full characterization of antimicrobial peptides from a crustacean (Decapoda). These peptides (penaeidin-1, -2, and -3) were purified from the plasma and hemocytes of experimentally uninfected shrimp P. vannamei (Penaeidae), which were obtained from an intensive Ecuadorian shrimp farm. Among the isolated peptides, three were purified to homogeneity and fully characterized at the level of their amino acid sequences, using a combination of reversed-phase chromatography, Edman degradation, and mass spectrometry (MALDI and nanoES). In addition, cDNA clones encoding two of the three antimicrobial peptides were isolated by screening a cDNA library prepared from hemocytes collected from bacteria challenged P. vannamei. Analysis of the deduced amino acid sequences revealed that the mature peptides are processed from precursor molecules, which have highly conserved signal peptides at their NH2 termini. From the cDNA sequences, we were able to demonstrate that penaeidin-2 and -3 are extended by a glycine residue, which is probably eliminated by COOH-terminal amidation of these two molecules.
      In shrimp, hemocytes have been demonstrated to be a key element of the defense system. As with other crustacean in the Decapoda order, hemocytes are involved in different immune responses such as the prophenoloxidase-activating system or the clotting reaction, which is mediated by the release of a hemocytic transglutaminase (
      • Söderhäll K.
      • Cerenius L.
      • Johansson M.W.
      ,
      • Kopacek P.
      • Hall M.
      • Söderhäll K.
      ,
      • Rodriguez J.
      • Boulo V.
      • Mialhe E.
      • Bachère E.
      ). From our studies and the preliminary results obtained from the shore crab, C. maenas (
      • Schnapp D.
      • Kemp G.D.
      • Smith V.J.
      ), it can be inferred that hemocytes in Decapoda also participate in the production and storage of antimicrobial peptides. Our data show that the blood cells of the shrimp P. vannamei are a site of production and storage of the antimicrobial peptides isolated because they were found in the acid extract of a hemocytic organelle-rich fraction. Moreover, the cDNAs for these peptides were isolated from a library constructed with hemocyte mRNA. This implies that these antimicrobial molecules are processed from mRNA to active compounds within the hemocytes. This is also observed in the horseshoe crab Tachypleus tridentatus (Chelicerata). In this arthropod, the hemocytes are extremely sensitive to microbial substances such as lipopolysaccharides and β-glucans. Upon stimulation, the hemocytes degranulate and release into the extracellular fluid a series of substances involved in immune defense, including several antimicrobial peptides such as tachyplesins (
      • Nakamura T.
      • Furunaka H.
      • Miyata T.
      ), big defensin (
      • Saito T.
      • Kawabata S.
      • Shigenaga T.
      • Takayenoki Y.
      • Cho J.
      • Nakajima H.
      • Hirata M.
      • Iwanaga S.
      ), or tachycitin (
      • Kawabata S.I.
      • Nagayama R.
      • Hirata M.
      • Shigenaga T.
      • Agarwala K.L.
      • Saito T.
      • Cho J.
      • Nakajima H.
      • Takagi T.
      • Iwanaga S.
      ). A similar mechanism is likely to occur in shrimp as suggested by the presence of antimicrobial peptides in the plasma of the animals used for this study. Indeed, although the animals were not infected experimentally, we can assume that they were subjected during their capture and intense manipulation to stress conditions leading to a hemocytic activation and partial degranulation. We do not know whether production of these molecules is induced upon infection or whether, as for the horseshoe crab, the peptides are stored in the hemocytes and released upon infection by hemocytic activation and partial degranulation. Further studies investigating the transcription profiles of penaeidins following microbial infection will address this question.
      Penaeidin-1,-2 and -3 share many general characteristics with other antimicrobial peptides. They are cationic peptides with positive net charges of 7 for penaeidin-1 and -2 and 8 for penaeidin-3, containing 50 (penaeidin-1 and -2) and 62 residues (penaeidin-3). Their calculated isoelectric points vary from 9.34 for penaeidin-1 and -2 to 9.84 for penaeidin-3. In contrast to penaeidin-1 and -2, penaeidin-3 is NH2-terminally blocked by a pyroglutamic acid. Identical NH2-terminal blocking amino acids have already been observed in other antimicrobial peptides such as hymenoptaecin (
      • Casteels P.
      • Ampe C.
      • Jacobs F.
      • Tempst P.
      ) or some bovine β-defensins (
      • Selsted M.E.
      • Tang Y.Q.
      • Morris W.L.A.M.P.
      • Novotny M.J.
      • Smith W.
      • Henschen A.H.
      • Cullor J.S.
      ). The analysis of penaeidin-2 and -3 cDNAs showed the presence of a glycine codon at final position in the ORF. However, the experimentally determined masses clearly indicate that the glycine residue is eliminated in the mature peptide. Therefore, we can assume that the two peptides are COOH-terminally amidated. Because no cDNA clone has been sequenced for penaeidin-1, we do not yet have any conclusive evidence about the possible amidation of the COOH terminus of the molecule. Such a COOH-terminal amidation has also been observed in other marine invertebrate antimicrobial peptides such as the tachyplesins from T. tridentatus (
      • Nakamura T.
      • Furunaka H.
      • Miyata T.
      ) and their Limulus polyphemus analogues, the polyphemusins (
      • Miyata T.
      • Tokunaga F.
      • Yoneya T.
      • Yoshikawa K.
      • Iwanaga S.
      • Niwa M.
      • Takao T.
      • Shimonishi Y.
      ). This modification has also been described in the insect cecropins (
      • Li Z.Q.
      • Merrifield R.B.
      • Boman I.A.
      • Boman H.G.
      ), and in vertebrate antimicrobial peptides (e.g.magainins) (
      • Chen H.C.
      • Brown J.H.
      • Morell J.L.
      • Huang C.M.
      ), where it was shown to be functionally important by increasing antimicrobial activity compared with the same peptides, which have a free carboxyl group.
      The overall structure of the three peptides isolated in P. vannamei, is unique in that it consists of a NH2-terminal domain rich in proline residues and a cysteine-rich COOH-terminal region. The three penaeidins are composed of a proline-rich NH2-terminal domain and a COOH-terminal domain containing 6 cysteines engaged in the formation of three intramolecular disulfide bridges. 4 of the 6 cysteines are organized in two doublets separated by 5 residues. The central-most cysteines are separated by 1, 2, or 3 residues in penaeidin-1, -2, and -3, respectively (Fig. 6). Penaeidins contain the same number of cysteines as the arthropod and mammalian defensins, the β-defensins (for review see Ref.
      • Hetru C.
      • Bulet P.
      • Cociancich S.
      • Dimarcq J.-L.
      • Hoffmann D.
      • Hoffmann J.A.
      ), or the T. tridentatus big defensin (
      • Saito T.
      • Kawabata S.
      • Shigenaga T.
      • Takayenoki Y.
      • Cho J.
      • Nakajima H.
      • Hirata M.
      • Iwanaga S.
      ). However, the cysteine motif in penaeidins has no significant homology with those found in any of the molecules mentioned above. For example, the cysteine stabilized αβ motif characteristic of insect and plant defensins (
      • Bonmatin J.-M.
      • Bonnat J.-L.
      • Gallet X.
      • Vovelle F.
      • Ptak M.
      • Reichhart J.-M.
      • Hoffmann J.A.
      • Keppi E.
      • Legrain M.
      • Achstetter T.
      ,
      • Cornet B.
      • Bonmatin J.-M.
      • Hetru C.
      • Hoffmann J.A.
      • Ptak M.
      • Vovelle F.
      ,

      Fant, F., Santos, L., Vranken, W., Boulez, K., Martin, J.-C., and Borremans, F. A. M. (1994) Proceedings of the 12th European Experimental NMR Conference, Oulo, Finland, 5th-10th June, 1994.

      ) as well as some scorpion toxins (
      • Landon C.
      • Cornet B.
      • Bonmatin J.-M.
      • Kopeyan C.
      • Rochat H.
      • Vovelle F.
      • Ptak M.
      ), which stabilizes an α-helix on a β-sheet through two disulfide bridges (
      • Cornet B.
      • Bonmatin J.-M.
      • Hetru C.
      • Hoffmann J.A.
      • Ptak M.
      • Vovelle F.
      ), is not found in the penaeidins. Moreover, as the penaeidin disulfide bridge positions are still unknown, we cannot predict the three-dimensional structure by homology with any of the antimicrobial peptides whose structures have been characterized to date. These striking features led to the creation of a novel family, the penaeidins, after the genus Penaeus, in which the molecules are characterized by post-translational modifications (COOH-terminal amidation and/or NH2-terminal cyclization by a pyroglutamic acid) and the presence of three intramolecular disulfide bridges, features that confer to the peptides a high stability as observed by their high resistance to proteolysis.
      Figure thumbnail gr6
      Figure 6Sequence comparison of penaeidin-1, -2, and -3 from P. vannamei. The full penaeidin sequences obtained by a biochemical approach and completed by the cDNA cloning data were aligned. Gaps were introduced to optimize the alignment. Cysteines are in boldface type. Identical residues and conservative replacements are in boxes.Asterisks indicate COOH-terminal amidation, andpE stands for pyroglutamic acid.
      During preliminary HPLC purification steps, shrimp hemolymph was shown to display various zones with bactericidal or bacteriostatic activities (data not shown) corresponding to cationic molecules (peptidic or nonpeptidic substances), which will be the subject of further studies. Among these, the penaeidins alone were isolated and characterized. They are essentially active against Gram-positive bacteria and are much less active against the Gram-negative bacteria tested. Like apidaecins (
      • Casteels P.
      • Ampe C.
      • Jacobs F.
      • Vaeck M.
      • Tempst P.
      ), penaeidins were found to have a bacteriostatic effect at the concentrations tested. This suggests that penaeidins do not display a lytic activity on bacteria but rather interfere with cell propagation. Penaeidins were also shown to display activity against the fungal test strain N. crassa and against a fungus F. oxysporum shown to be pathogenic for shrimp as other members of the genus Fusarium (
      • Burns C.D.
      • Berrigan M.E.
      • Henderson G.E.
      ,
      • Rhoobunjongde W.
      • Hatai K.
      • Wada S.
      • Kubota S.S.
      ). Unfortunately, the quantities of all the peptides obtained in the present study were insufficient to conduct a more exhaustive antimicrobial activity spectrum. In this respect the production of recombinant penaeidins will be of special interest. In addition, it will be very important to analyze the activity of the peptides under conditions that more closely resemble the physiological parameters of shrimp, i.e. pH and salt, because several antimicrobial peptides have been shown to be salt-sensitive (
      • Goldman M.J.
      • Anderson G.M.
      • Stolzenberg E.D.
      • Kari U.P.
      • Zasloff M.
      • Wilson J.M.
      ). Our tests were performed under standard conditions that do not correspond to the osmolarity of shrimp extracellular fluid or intracellular pH ranges. It will also be of great interest to check the activity of the penaeidins against a variety of pathogens such as the fungi Lagenidium sp. (
      • Crisp L.M.
      • Bland C.E.
      ), which infects the larvae, and different bacterial species such as V. harveyi (
      • Song Y.L.
      • Lee S.P.
      ) orV. penaeicidae (
      • Ishimaru K.
      • Akagawa-Matsushita M.
      • Muroga K.
      ), which are associated with shrimp diseases, and against viruses that represent the most serious pathogens for shrimp (
      • Vega-Villasante F.
      • Puente M.E.
      ).

      Acknowledgments

      We are grateful to Sandrine Uttenweiler-Joseph for performing mass spectrometry and Emeric Motte for assistance with cDNA library construction. We are indebted to Prof. J. A. Hoffmann and Prof. J.-M. Reichhart for expert advice. We thank Dr. S. Ades and Dr. W. van der Knaap for critical reading of the manuscript and Dr. A. Vey for the gift of F. oxysporumfungal strain.

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