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

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. 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 (1Bachère E. Mialhe E. Rodriguez J. Fish Shellfish Immunol. 1995; 5: 597-612Crossref Scopus (111) Google Scholar), 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 (2Söderhäll K. Cerenius L. Johansson M.W. Söderhäll K. Iwanaga S. Vasta G.R. New Directions in Invertebrate Immunology. SOS Publications, Fair Haven, NJ1996: 229-253Google Scholar). 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. 3Iwanaga S. Muta T. Shigenaga T. Miura Y. Seki N. Saito T. Kawabata S. Ann. N. Y. Acad. Sci. 1994; 712: 102-116Crossref PubMed Scopus (45) Google Scholar). 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 (4Hoffmann J.A. Reichhart J.-M. Hetru C. Curr. Opin. Immunol. 1996; 8: 8-13Crossref PubMed Scopus (291) Google Scholar). 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 (5Chisholm J.R.S. Smith V.J. J. Mar. Biol. Assoc. U. K. 1992; 72: 529-542Crossref Scopus (64) Google Scholar). 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 (6Schnapp D. Kemp G.D. Smith V.J. Eur. J. Biochem. 1996; 240: 532-539Crossref PubMed Scopus (171) Google Scholar). 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. (7Hetru C. Bulet P. Cociancich S. Dimarcq J.-L. Hoffmann D. Hoffmann J.A. Hoffmann J.A. Janeway C.A. Natori J.S. Phylogenetic Perspectives in Immunity: The Insect Host Defense. CRC Press, Boca Raton, FL1994: 43-66Google Scholar)) as well as from plants (8Broekaert W.F. Terras F.R.G. Cammue B.P.A. Osborn R.W. Plant Physiol. 1995; 108: 1353-1358Crossref PubMed Scopus (657) Google Scholar). 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. 9Boman H.G. Faye I. Gudmunssson G.H. Lee J.Y. Lidholm D.A. Eur. J. Biochem. 1991; 201: 23-31Crossref PubMed Scopus (236) Google Scholar); (ii) peptides with one to six intramolecular disulfide bridges including the arthropod defensins (10Hoffmann J.A. Hetru C. Immunol. Today. 1992; 13: 411-415Abstract Full Text PDF PubMed Scopus (239) Google Scholar), antifungal peptides from Drosophila, drosomycin (11Fehlbaum P. Bulet P. Michaut L. Lagueux M. Broekaert W.F. Hetru C. Hoffmann J.A. J. Biol. Chem. 1994; 269: 33159-33163Abstract Full Text PDF PubMed Google Scholar) and metchnikowin (12Levashina A. Ohresser S. Bulet P. Reichhart J.-M. Hetru C. Hoffmann J.A. Eur. J. Biochem. 1995; 233: 694-700Crossref PubMed Scopus (165) Google Scholar), thanatin from Podisus (13Fehlbaum P. Bulet P. Chernysh S. Briand J.-P. Roussel J.-P. Letellier L. Hetru C. Hoffmann J.A. Proc. Natl. Acad. Sci., U. S. A. 1996; 93: 1221-1225Crossref PubMed Scopus (243) Google Scholar), tachyplesin, big defensin and tachycitin from limulus (14Nakamura T. Furunaka H. Miyata T. J. Biol. Chem. 1988; 263: 16709-16713Abstract Full Text PDF PubMed Google Scholar, 15Saito T. Kawabata S. Shigenaga T. Takayenoki Y. Cho J. Nakajima H. Hirata M. Iwanaga S. J. Biochem. (Tokyo). 1995; 117: 1131-1137Crossref PubMed Scopus (17) Google Scholar, 16Kawabata S.I. Nagayama R. Hirata M. Shigenaga T. Agarwala K.L. Saito T. Cho J. Nakajima H. Takagi T. Iwanaga S. J. Biochem. (Tokyo). 1996; 120: 1253-1260Crossref PubMed Scopus (125) Google Scholar), and other cysteine-rich antimicrobial peptides isolated from a scorpion (17Ehret-Sabatier L. Loew D. Goyffon M. Fehlbaum P. Hoffmann J.A. van Dorsselaer A. Bulet P. J. Biol. Chem. 1996; 271: 29537-29544Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar) and from a bivalve mollusk (18Charlet M. Chernysh S. Philippe H. Hetru C. Hoffmann J.A. Bulet P. J. Biol. Chem. 1996; 271: 21808-21813Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar, 19Hubert F. Noël T. Roch P. Eur. J. Biochem. 1996; 240: 302-306Crossref PubMed Scopus (169) Google Scholar); (iii) proline-rich peptides, among them the apidaecins and abaecins from Hymenoptera (20Casteels P. Ampe C. Jacobs F. Vaeck M. Tempst P. EMBO J. 1989; 8: 2387-2391Crossref PubMed Scopus (341) Google Scholar, 21Casteels P. Ampe C. Rivière L. Damne J.V. Elicone C. Fleming M. Jacobs F. Tempst P. Eur. J. Biochem. 1990; 187: 381-386Crossref PubMed Scopus (211) Google Scholar) and drosocin from Drosophila hemolymph (22Bulet P. Dimarcq J.-L. Hetru C. Lagueux M. Charlet M. Hegy G. Van Dorsselaer A. Hoffmann J.A. J. Biol. Chem. 1993; 268: 14893-14897Abstract Full Text PDF PubMed Google Scholar); (iv) glycine-rich antimicrobial peptides or polypeptides (9–30 kDa) such as the attacins (23Hultmark D. Engstrom A. Bennich H. Boman H.G. Andersson K. Steiner H. EMBO J. 1983; 2: 571-576Crossref PubMed Scopus (339) Google Scholar), diptericin (24Dimarcq J.-L. Keppi E. Dunbar B. Lambert J. Reichhart J.-M. Hoffmann D. Rankine S.M. Fothergill J.E. Hoffmann J.A. Eur. J. Biochem. 1988; 171: 17-22Crossref PubMed Scopus (106) Google Scholar) and sarcotoxins (25Kanai A. Natori S. Mol. Cell Biol. 1990; 10: 6114-6122Crossref PubMed Scopus (27) Google Scholar). 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 (26Norelli J.L. Aldwinckle H.S. Destefano-Beltran L. Jaynes J.M. Euphytica. 1994; 77: 123-128Crossref Scopus (86) Google Scholar,27Jaynes J.M. Nagpala P. Destefano-Beltran L. Huang J.H. Kim J. Denny T. Cetiner S. Plant Sci. (Limerick). 1993; 89: 43-53Crossref Scopus (133) Google Scholar). 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 (28Lightner D.V. McVey J.P. Crustacean aquaculture. CRC Press, Boca Raton, FL1983: 289-320Google Scholar, 29Vega-Villasante F. Puente M.E. Prev. Vet. Med. 1993; 17: 271-282Crossref Scopus (6) Google Scholar), 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. 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. 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. 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. 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. 1The 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. 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. 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. 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. 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. 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. Purified peptides were subjected to reduction and alkylation using the procedures already described (30Bulet P. Cociancich S. Reuland M. Sauber F. Bischoff R. Hegy G. Van Dorsselaer A. Hetru C. Hoffmann J.A. Eur. J. Biochem. 1992; 209: 977-984Crossref PubMed Scopus (107) Google Scholar). 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. 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. 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. 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. 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). 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 (31Vorm O. Roepstorff P. Mann M. Anal. Chem. 1994; 66: 3281-3287Crossref Scopus (644) Google Scholar, 32Vorm O. Mann M. J. Am. Soc. Mass Spectrom. 1996; 5: 955-958Crossref Scopus (142) Google Scholar). The droplets were allowed to dry under gentle vacuum before introduction into the mass spectrometer. 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 (33Wilm M. Mann M. Anal. Chem. 1996; 68: 1-8Crossref PubMed Scopus (1696) Google Scholar) 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 (33Wilm M. Mann M. Anal. Chem. 1996; 68: 1-8Crossref PubMed Scopus (1696) Google Scholar) 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. 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 (34Roepstorff P. Fohlman M. J. Biomed. Mass. Spectrom. 1984; 11: 601-606Crossref PubMed Scopus (2382) Google Scholar). The quadrupole analyzers were calibrated by using the multiply charged ions from a separate acquisition of horse heart myoglobin (16951.5 Da). Native,S-pyridylethylated peptides and peptidic fragments were subjected to Edman degradation on a pulse liquid automatic sequenator (Applied Biosystems, model 473A). The microbial strains used to determine antimicrobial activities during the purification steps were those used in previous studies (11Fehlbaum P. Bulet P. Michaut L. Lagueux M. Broekaert W.F. Hetru C. Hoffmann J.A. J. Biol. Chem. 1994; 269: 33159-33163Abstract Full Text PDF PubMed Google Scholar, 35Bulet P. Cociancich S. Dimarcq J.-L. Lambert J. Reichhart J.-M. Hoffmann D. Hetru C. Hoffmann J.A. J. Biol. Chem. 1991; 266: 24520-24525Abstract Full Text PDF PubMed Google Scholar): 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. 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 (22Bulet P. Dimarcq J.-L. Hetru C. Lagueux M. Charlet M. Hegy G. Van Dorsselaer A. Hoffmann J.A. J. Biol. Chem. 1993; 268: 14893-14897Abstract Full Text PDF PubMed Google Scholar). 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 (36Casteels P. Ampe C. Jacobs F. Tempst P. J. Biol. Chem. 1993; 268: 7044-7054Abstract Full Text PDF PubMed Google Scholar). 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 activity was monitored againstN. crassa and F. oxysporum as described previously (11Fehlbaum P. Bulet P. Michaut L. Lagueux M. Broekaert W.F. Hetru C. Hoffmann J.A. J. Biol. Chem. 1994; 269: 33159-33163Abstract Full Text PDF PubMed Google Scholar, 13Fehlbaum P. Bulet P. Chernysh S. Briand J.-P. Roussel J.-P. Letellier L. Hetru C. Hoffmann J.A. Proc. Natl. Acad. Sci., U. S. A. 1996; 93: 1221-1225Crossref PubMed Scopus (243) Google Scholar) 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. 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. 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). Antimicrobial peptides were purified from about 225 ml of hemolymph prepared from P. vannamei shrimps collected in an intensive culture farm and that had not been experimentally bacteria-challenged. Three different acid extracts were prepared, one from plasma (cell free hemolymph; P) and two from hemocytes: cytosolic (HC) and organelle-rich (cellular debris; HO) fractions (see "Materials and Methods"). The three extracts were directly applied to Sep-Pak C18 cartridges. Elutions were successively performed with 5, 40, and 80% solutions of acetonitrile in acidified water. We focused our attention mainly on the 40% Sep-Pak fraction i