Antibacterial peptides in hemocytes and hematopoietic tissue from freshwater crayfish Pacifastacus leniusculus: Characterization and expression pattern

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Developmental and Comparative Immunology 31 (2007) 441–455

Developmental & Comparative Immunology www.elsevier.com/locate/devcompimm

Antibacterial peptides in hemocytes and hematopoietic tissue from freshwater crayfish Pacifastacus leniusculus: Characterization and expression pattern Pikul Jiravanichpaisala,b, So Young Leea,c, Young-A Kima, Tove Andre´na, Irene So¨derha¨lla, a Department of Comparative Physiology, Uppsala University, Norbyva¨gen 18A, SE-752 36, Sweden National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, 113 Paholyothin Road, Klong 1, Klong Laung, Pathumthani 12120, Thailand c CICBiogune, Scientific Park of Bizkaia 801 A, Derio 48160, Spain

b

Received 17 July 2006; received in revised form 7 August 2006; accepted 10 August 2006 Available online 2 October 2006

Abstract A 14 amino acid residues proline/arginine-rich antibacterial peptide designated as astacidin 2 was purified and characterized from hemocytes of the freshwater crayfish, Pacifastacus leniusculus. Astacidin 2 has a broad range of antibacterial activity against both Gram-positive and Gram-negative bacteria. The primary sequence of astacidin 2 is RPRPNYRPRPIYRP with an amidated C-terminal and the molecular mass is 1838 Da determined by mass spectrometry. Furthermore, the cDNA of three different crustin antibacterial homologs were isolated from a crayfish hemocyte EST library. RT-PCR was used to analyze the expression of the genes coding for astacidin 2 and P. leniusculus crustins (Plcrustin) 1–3 after bacterial challenge. The expression of Plcrustin1 was upregulated in both hemocytes and hematopoietic tissue after challenge with Gram-negative Escherichia coli or Acinetobacter ssp. non pathogenic bacteria as well as by a Gram negative crayfish pathogenic bacterium (Aeromonas hydrophila). The PlCrustin3 transcript was only upregulated after inoculation with the non-pathogenic Acinetobacter ssp. while there was no change in expression of Plcrustin2 or astacidin 2 following a bacterial challenge. r 2006 Elsevier Ltd. All rights reserved. Keywords: Antibacterial protein; Proline-rich peptide; Astacidin 2; Crustin; Carcinin; Crayfish; Pacifastacus leniusculus; Innate immunity

1. Introduction Antimicrobial peptides (AMPs) have become recognized as important components of the nonspecific host defense or innate immune system in a Corresponding author. Tel.: +46 18 4172818; fax: +46 18 4716425. E-mail address: [email protected] (I. So¨derha¨ll).

variety of organisms including bacteria, fungi, plants, insects, birds, crustaceans, amphibians, and mammals [1–6]. The primary structures of these positively charged AMPs are highly diverse, yet their secondary structures share a common feature of amphipathicity [7–9]. Although they exhibit great structural diversity, the peptides are often grouped into three families by certain common structural patterns: (i) linear peptides forming a-helices and deprived of cysteine

0145-305X/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.dci.2006.08.002

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residues (cecropin and magainin family), (ii) peptides containing cysteine residues (defensin family), and (iii) peptides with an overrepresentation of proline and/or glycine residues [10–15]. Antibacterial activity has been demonstrated in the hemolymph of various crustaceans [16] and the first crustacean antibacterial peptide characterized was a proline-rich 6.5 kD peptide from the shore crab Carcinus maenas [17]. Several proline/argininerich AMPs have been characterized from various animals [18–22] and some of these have been shown to exhibit various functions. For example, PR-39 purified from pig intestine [23] has several activities such as inducing the expression of syndecans, the major cell surface heparan sulfate proteoglycans, during wound healing [24] and inhibition of superoxide anion production mediated by neutrophil NADPH oxidase, which influences the oxidative killing mechanisms of neutrophils [25]. Furthermore, PR-39 was shown to act as a chemotactic agent of neutrophils during cell inflammation [26]. Bovine bactenecin 7 also shows dual functions, such as cell permeation ability and antimicrobial activity [27]. Among crustacean AMPs the penaeidin family contains broad-spectrum AMPs with a proline-rich N-terminal domain and a C-terminal domain with six cystein residues. These have been isolated and characterized from the Pacific as well as the Atlantic white shrimp, Litopenaeus vannamei and L. setiferus [28,29]. Previously, we have reported that crayfish hemocyanin can be processed under acidic conditions by a specific cysteine proteinase to produce an antibacterial peptide, referred to as astacidin 1. Hemocyanin is expressed in hepatopancreas of crayfish and its cleavage into astacidin 1 is induced during bacterial infection [30]. Another common group of AMPs within decapod crustaceans is the crustin family. Granular hemocytes of C. maenas were the first source of the crustin family of putative AMPs [31]. The 11.5 kDa crustin peptide (Cm1) is cysteine-rich, hydrophobic and exhibits specific activity towards Gram-positive marine bacteria [31]. Later a group of cDNA sequences encoding putative AMPs from L. vannamei and L. setiferus was added to the crustin family, although these peptides in addition to the cystein-rich C-terminal with strong similarity to that of Cm1, also contain an amino terminal glycine-rich repeat region of 40–50 amino acid residues [16]. Crayfish hemocytes are important in cellular defense mechanisms as well as in release of humoral

defense molecules. Thereby the hemocyte will initiate several immune responses such as activation of the prophenoloxidase activating system, initiation of the coagulation system and production of AMPs [32]. Production of hemocytes in crayfish takes place in the hematopoietic tissue forming a sheet of cell clusters situated in the dorsal side of the stomach. The hematopoietic tissue is actively proliferating, producing hemocyte precursors, which are partly differentiated within this tissue, whereas the final differentiation into functional hemocytes is not completed until the hemocytes are released into the circulation [33]. In this paper, we describe the purification, biological activity, and characterization of one prolinerich antibacterial peptide named astacidin 2, as well as the structure of three different crustins in crayfish hemocytes. We also report the expression pattern of astacidin 2 and the crustins in hemocytes and the hematopoietic tissue. 2. Material and methods 2.1. Animals Freshwater crayfish, Pacifastacus leniusculus, were purchased from Berga Kra¨ftodling, So¨dermanland, Sweden, and from Nils Fors, Torsa˚ng at Lake Va¨ttern, and were maintained in tanks with aerated water at 10 1C. Apparently healthy intermolt crayfish were used in all experiments. 2.2. Purification of a proline-rich antibacterial peptide—astacidin 2 Hemolymph was prepared by collecting blood from 400 crayfish in anticoagulant buffer (0.14 M NaCl, 0.1 M glucose, 30 mM trisodium citrate, 26 mM citric acid, 10 mM EDTA, pH 4.6) [34]. After centrifugation at 4 1C and 800g for 10 min, the plasma was removed and the hemocyte pellet was suspended in crayfish PBS (10 mM Na2HPO4, 10 mM KH2PO4, 0.15 M NaCl, 10 mM CaCl2, 10 mM MnCl2, pH 6.8 ¼ CPBS). The hemocyte pellet was stored at 80 1C until further analysis. For purification of antibacterial proteins, the frozen sample was thawed and homogenized and then trifluoroacetic acid was added to a final concentration of 0.1%. After incubation at 4 1C for 12 h, the sample was centrifuged at 16 000g for 20 min and the supernatant was diluted twice with water containing 0.05% trifluoroacetic acid and directly

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subjected to C-18 reverse-phase chromatography (+ 2.7  9 cm, Waters) equilibrated at room temperature with 0.05% trifluoroacetic acid in water. The sample was eluted with 80% acetonitrile in 0.05% trifluoroacetic acid and the fraction was vacuum dried. The sample was resuspended in 10 mM sodium phosphate buffer, pH 6.0 and loaded to a CM (carboxymethyl) cellulose (Sigma) cationic ion exchange chromatography (+ 2  25 cm) column and eluted with a stepwise gradient of 200, 400 mM, and 1 M NaCl in 10 mM sodium phosphate buffer, pH 6.0. The purity of the fractions was monitored by 20% acid–urea PAGE. The sample was then further purified to homogeneity by RPHPLC (Pharmacia Smart chromatography system) on a C-18 column using acetonitrile/water/0.05% trifluoroacetic acid gradients of 0–60% acetonitrile in 60 min at a flow rate of 100 ml/min. Ultraviolet absorbance was monitored at 280, 254, and 214 nm. The eluted peak fractions were vacuum dried and used for assay of antibacterial activity and determination of amino acid sequence. 2.3. Acid– urea PAGE The purity of the peptide fractions was checked in 20% acetic acid–urea polyacrylamide gel electrophoresis followed by Coomassie staining for peptides as described by Selsted et al. [35]. A low molecular mass calibration kit for electrophoresis (Amersham Biosciences) was used: egg white ovalbumin (43 kDa), bovine erythrocyte carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20.1 kDa), bovine milk a–lactalbumin (14.4 kDa), aprotinin (6.5 kDa). A synthetic peptide of astacidin 1 (1.9 kDa) was also used as a molecular marker. 2.4. Assay of antibacterial activity During the purification procedure, the antibacterial activities of samples were monitored by a radial diffusion assay using Bacillus megaterium BM 11 and Escherichia coli D21 as test organisms as described by Lehrer et al. [36]. Briefly, 10 ml cultures of bacterial cells in mid-logarithmic phase were subjected to centrifugation at 900g for 5 min, washed with 10 mM sodium phosphate buffer, pH 7.4 and then resuspended in 10 ml of the same buffer. One hundred ml of bacterial solution containing 1  106 colony-forming units (CFUs) was added to 10 ml of previously autoclaved agar solution (10 mM sodium phosphate, pH 7.4, 1%

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(v/v) LB (Luria-Bertani) medium, 1% (w/v) agarose, 0.02% (v/v) Tween 20), and the mixture was poured into a Petri dish. Peptide samples were added directly to 3 mm wells made on the solidified agar. After incubation for 3 h at 37 1C, the plates were overlaid with 10 ml of sterile agar containing a double-strength (6% v/v) solution of LB and 1% agarose, and then incubated for 12–24 h at 30–37 1C. The minimal inhibitory concentration was determined using the same method and tested against several species of Gram-negative and Gram-positive bacteria. The lowest concentration of the antibacterial peptide that showed visible suppression of growth was defined as the minimal inhibitory concentration. A liquid growth inhibition assay was performed as described in Lee et al. [37]. Bacteria grown in LB medium (peptone 10 g, yeast extract 5 g, NaCl 5 g, glucose 1 g/distilled water 1 liter) was collected in the exponential phase of growth and resuspended with phosphate buffered saline, pH 6.0 at a density of 1  108 cells/ml. Peptide samples were suspended in 200 ml of 0.2% (w/v) bovine serum albumin and mixed with 190 ml of LB medium and 10 ml of bacterial suspension and then incubated with shaking for 3 h at 37 1C. The optical density at 650 nm was measured on each sample. 2.5. Determination of the amino acid sequence and mass spectrometry analysis The homogenous purified peptide was identified based on Edman sequence analysis using an Applied Biosystem 476A automated amino acid sequencer. For mass analysis and for confirmation of amino acid sequences, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF-MS) was performed in a Q-tof tandem mass spectrometer (Micromass, Manchester UK) equipped with nanospray interphase. Interpretation of mass spectra was done by using MassLynx (Micromass, Manchester, UK) suite of software programs. 2.6. cDNA cloning and nucleotide sequencing of the astacidin 2 A ZAP Express cDNA library constructed from mRNA of crayfish hemocytes was used. For the initial screening approximately 120,000 recombinants of the cDNA library were screened with 50 -[g-32P] ATP-labeled mixed probe (CCI AA(C/T)

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TA(C/T) (A/C)GI CCI (A/C)GI CCI AT(A/C/T) TA; I is inosine), which was designed according to the following amino acid sequence of astacidin 2, PNYRPRPIY. The membranes were prehybridized at 65 1C for 1 h in 5  SSC (750 mM NaCl, 75 mM Na-citrate, pH 7.0), 5  Denhardt’s solution (100  Denhardt’s solution is 2% (w/v) bovine serum albumin, 2% (w/v) Ficoll, and 2% (w/v) polyvinylpyrrolidone), 100 mg/ml salmon sperm DNA, and 0.5% SDS. The membranes were then hybridized at 65 1C for 12 h in the same solution as used for prehybridization. After the second screening, positive clones were amplified with PCR using a pair of specific primers corresponding to T7 and T3 promoters in the ZAP Express vector, pBK-CMV. PCR conditions were 94 1C for 45 s, 55 1C for 30 s, and 72 1C for 2 min carried out for 30 cycles. The largest PCR product was subcloned into TOPO cloning vector (Invitrogen). The insert was digested out with the restriction enzyme EcoRI run on 1% agarose gel to confirm its size and finally sequenced with an Applied Biosystems PRISM dye terminator cycle sequencing ready reaction kit (Perkin-Elmer). To get the complete cDNA sequence two specific oligonucleotide primers were constructed corresponding to residue 526–549 (50 -CCT TCT ACG AGA GAT ATT TAC CGT-30 , in the sense direction; 50 -ACG GTA AAT ATC TCT CGT AGA AGG-30 , in the antisense direction). The PCR was done using the same conditions as above using a combination of these two primers and primers corresponding to the T7 or T3 promoters using the hemocyte cDNA library as template. The obtained cDNA sequences were analyzed with the MacVector 6.5.1. Software (Kodak). The nucleotide and the deduced amino acid sequences were compared with the BLAST program (National Center Biotechnology International, Bethesda, MD). 2.7. Northern blot analysis Total RNA was isolated from hemocytes and hepatopancreas, respectively, by using Trizol LS reagent (Life Technologies) according to the manufacturer’s instructions. Approximately 15 mg of total RNA from hemocytes or 20 mg of total RNA from hepatopancreas were run on 1% agarose gel in the presence of formaldehyde with ethidium bromide and transferred to a nylon membrane (Hybond-N, Amersham Biosciences) following standard procedures. The 0.24–9.5 kilobase RNA ladder (Life Technologies) was electrophoresed

simultaneously. To examine the regulation of mRNA expression of astacidin 2, 100 mg of LPS were injected into crayfish. After this total RNA was extracted from crayfish hemocytes at 6 h and 12 h postinjection. No injection was made in control crayfish. A cDNA probe, corresponding to nucleotides 143–549 was labeled with [a-32P]dCTP using the Megaprime labeling kit (Amersham Biosciences). Northern blot hybridization was performed overnight at 65 1C in a solution composed of 5  SSC, 5  Denhardt’s solution, 0.5% (w/v) SDS, and 100 mg/ml denatured salmon sperm DNA. The membrane was washed once with 2  SSC and 0.1% SDS at 65 1C for 20 min, and three times with 0.2  SSC and 0.1% SDS at 65 1C for 20 min. The membrane was subjected to autoradiography. 2.8. LPS challenge and mass spectrometric analysis of plasma proteins Crayfish were injected at the base of a walking leg with 100 ml of 1 mg/ml crude LPS. The crude LPS solution (Sigma from E. coli 055:B5) was dissolved in CPBS, vortexed until no particles were visible and then sonicated for 30 s and sterile filtered through a 20 mm membrane. After 4 h the hemolymph was collected and centrifuged at 2875g for 5 min. The supernatant plasma was ultracentrifuged at 200,000g for 4 h at 4 1C. Trifluoroacetic acid (TFA) was added to a final concentration of 0.05% to the clear supernatant. The sample was incubated for 12 h at 4 1C and the treated plasma was then centrifuged at 16,000g for 30 min. The supernatant was subjected to WatersTM Sep-Paks C18 cartridge equilibrated with 0.05% TFA water, and eluted with 80% acetonitrile containing 0.05% TFA water. The eluted sample was vacuum-dried and dissolved in water. As a control, CFS-injected plasma prepared in the same way was used. The same amount of protein was loaded on a 15% SDSPAGE. Proteins were excised from the SDS-PAGE and cleaved with trypsin by in-gel digestion. Peptides were analyzed by electrospray ionization mass spectrometry on a Q-tof mass spectrometer using Mass lynx software. 2.9. cDNA cloning and nucleotide sequencing of the crustins A crayfish hemocyte cDNA library was constructed using the ZAP Expresss cDNA synthesis kit (Stratagene). The cDNA library was screened

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with 50 -[g-32P] ATP-labeled mixed probe; 50 -TAYTGYTGYGGICCIGA-30 , which was designed according to the amino acid sequence: YCCGPE, found after sequencing of protein bands appearing in plasma after crude LPS challenging. Recombinant lambda phages (50,000) were transferred to Hybond N+ nylon membrane according to the instruction manual (Amersham). Hybridizations were carried out overnight at 47 1C in 6  SSC, 5  Denhardt’s solution (0.1% bovine serum albumin, 0.1% Ficoll, and 0.1% polyvinylpyrollidone), 0.5% SDS, and 20 mg/ml denatured salmon sperm DNA. The membranes were washed with 6  SSC, 0.5% SDS for 15 min at 47 1C followed by autoradiography. Secondary screenings were performed following the same procedure. After the second screening, positive clones were amplified with PCR using T7 promoter primer and T3 promoter primer. The PCR products were subcloned into TOPO cloning vector (Invitrogen). The plasmids were released using GenElute Plasmid Miniprep kit according to the instructions of the manufacturer (Sigma), and the inserts were digested by the restriction enzyme EcoRI and then run in a 1% agarose gel and sequenced with an Applied Biosystems PRISM dye terminator cycle sequencing ready reaction kit (PerkinElmer Life Sciences). The cDNA sequence was analyzed with MacVectorTM 7.0 software (Oxford Molecular Group plc.). The nucleotide and the deduced amino acid sequences were compared using the BLAST program (National Center for Biotechnology Information, Bethesda, MD). A crayfish hemocyte EST library (So¨derha¨ll I unpublished) was also searched for the presence of similar transcripts and several crustin sequences were found that could be divided into three different groups. 2.10. Bacterial challenge To study the expression of the putative AMPs, infection experiments were performed using nonpathogenic Gram-negative (E. coli D21) and Acinetobacter sp. as well as a crayfish-pathogenic Gram-negative bacterium (Aeromonas hydrophila) (Jiravanichpaisal unpublished). All bacteria were grown in LB broth for 18 h at 22 or 37 1C, then viable cells were obtained by washing the bacterial pellet three times with CPBS and finally suspending these bacteria in CPBS. For heatkilling of bacteria they were harvested by centrifu-

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gation and washed three times in CPBS before resuspending in CPBS. The suspension was then incubated in a water bath at 80 1C for 3 h with occasional shaking. Inactivation was confirmed by incubating the bacterial suspension on LB agar for 7 days. If no growth occurred the bacterial suspension was considered heat-inactivated. For non-pathogenic strains approximately 2.5  107 CFUs ml1 or 5  106 CFUs per crayfish (2572 g) was injected. For pathogenic A. hydrophila, 7.5  105 CFUs ml1 or 1.5  105 CFUs per crayfish was used in case of viable cells and 5.0  105 cells per crayfish was used in case of heat-killed A. hydrophila. The pathogenicity of the A. hydrophila had been previously tested and 1.5  105 CFUs proved to kill the crayfish within 24 h at 2072 1C. At a higher dose than 5  105 CFUs per crayfish mortality occurred within 12 h. Two hundred ml of each bacteria solution in CPBS were injected separately into the hemolymph of crayfish using a 0.8  50 mm needle. Control crayfish were injected with 200 ml CPBS. The concentration of bacteria was estimated based on absorbance at 600 nm and counting with a plate count standard method. 2.11. RNA isolation and RT-PCR Bleeding and isolation of the hematopoietic tissue were made according to So¨derha¨ll et al. [33]. Total RNA was extracted from hemocytes and hematopoietic tissue of unchallenged, and challenged crayfish using Trizol (Gibco BRL) according to the manufacturer’s instructions. The precipitated RNA was resuspended in 44 ml of diethyl pyrocarbonate (DEPC)-treated sterile water and stored at 80 1C until used. These RNA samples were subjected to DNase treatment with RNase-free DNase I (Ambion) at a concentration of 2 unit/ sample for 30 min at 37 1C. Next, the DNase I enzyme activity was terminated and RNA was extracted with phenol plus chloroform once and then followed by chloroform only, and finally the RNA samples were precipitated with isopropanol and washed with 70% ethanol and suspended in 15 ml of DEPC-treated sterile water. These RNA samples were used for conventional PCR and RTPCR to detect the presence of DNA contamination and expression of the genes of interest. First strand cDNA was synthesized from 1 mg of total RNA from each sample using of 50 mM of Oligo (dT)20 primer and ThermoScript reverse transcriptase (Invitrogen) in a 20 ml total volume

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reaction according to the manufacturer’s specifications. PCRs were subsequently performed with the following specific primers: Plcrustin1 forward 52+50 -GGT AAC CAT GGC TCG ATC AC-30 and reverse 41950 -TGT AAT GGT GAG ACC GCT CC-30 ; Plcrustin2 forward 131+50 -CTG CAA GAA GCC TGA AGG TC-30 and reverse 48850 GCA TAA CAA GCA AGT CAG CCA-30 ; Plcrustin3 forward 61+50 -AGC GCC CAG AAC ACT AAC AC-30 and reverse 47750 -GGC AGG TTT GCA GAC GTA GT-30 ; astacidin 2 forward 47+50 -CCT ACA ACA CCA CCA TGC GTC-30 and reverse 18650 -CTT GCC AGG TCG GTA GAT TGG-30 ; ProPO forward 622+50 -AGT GAA CAG GAC TCC ACC TAC TGC-30 and reverse 104550 -ACT GAT GTC TAT GAA ATC CAG CCC-30 (GenBank accession no. X83497), respectively, and a control RT-PCR analysis of expression of the crayfish housekeeping gene, a 40S ribosomal protein was also undertaken; 40S forward 50 -CCA GGA CCC CCA AAC TTC TTA G-30 and reverse 50 -GAA AAC TGC CAC AGC CGT TG-30 respectively. The amplification reaction of all genes was performed for 25 cycles (at which no saturation of the housekeeping gene is obtained) in a 50 ml reaction solution, containing 1 ml of the first strand cDNA reaction solution and 10 mM each of the forward and reverse primers. The PCR conditions were 94 1C for 4 min followed by 25 cycles of 94 1C for 30 s, 60 1C (for all crustins and 58 1C for astacidin 2 and 40S ribosomal protein) for 40 s, 72 1C for 30 s and one cycle of final extension at

72 1C for 7 min.The products were then analyzed on a 1.5% agarose gel, stained with ethidium bromide, and visualized by ultraviolet light. The expected size of Plcrustin1, 2, 3, astacidin 2 and 40S were 368, 358, 417, 140 and 359 bp, respectively. 3. Results 3.1. Purification and antibacterial activity of astacidin 2 An antibacterial peptide was purified from the hemolymph of a total of 400 crayfish. The acid extract fractions were directly applied to C-18 reverse-phase chromatography. The antibacterial activities were assayed against two bacterial strains, B. megaterium BM 11 and E. coli D21. The fraction eluted with 80% acetonitrile in 0.05% TFA exhibited strong antibacterial activity. The fractions with antibacterial activity were collected, vacuum dried and then subjected to CM cellulose chromatography. The bound proteins were eluted with a stepwise gradient of 200, 400 mM, and 1 M NaCl in 10 mM sodium phosphate buffer. The elution profile of the CM cellulose column and acid–urea PAGE patterns are shown in Figs. 1A and B. Each eluted fraction was applied to C-18 SEP-PAK cartridges for removing sodium chloride and for assay of antibacterial activity. All eluted fractions had antibacterial activity against B. megaterium BM 11 and E. coli D21. The eluted fraction 4 with 1 M NaCl in sodium phosphate buffer was subjected to

Fig. 1. Purification of astacidin 2. (A) Elution pattern of cationic exchange column chromatography. The cationic exchange column, CM cellulose had been equilibrated with 10 mM sodium phosphate buffer, pH 6.0, and samPles were eluted with a step gradient of 200, 400 mM, and 1 M NaCl in the same buffer. The eluted fractions were designated I–V. Fraction IV was used for further purification of astacidin 2. (B) 20% acid–urea PAGE of the eluted fractions from the CM cellulose column. Lane 1, size marker; lane 2, fraction I; lane 3, fraction II; lane 4, fraction III; lane 5, fraction IV; lane 6, fraction V. (C) 20% acid–urea PAGE profile of the purified astacidin 2. A low molecular mass was used: rabbit muscle phosphrylase b (94 kDa), bovine serum albumin (67 kDa), egg white ovalbumin (43 kDa), bovine erythrocyte carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20.1 kDa), bovine milk a-lactalbumin (14.4 kDa), aprotinin (6.5 kDa) and synthetic peptide of 16 amino acid residue peptide, astacidin 1 (1.9 kDa).

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C-18 reverse-phase column chromatography for further purification. Finally, astacidin 2 was purified to homogeneity by reverse-phase HPLC. The purity of the sample was monitored by 20% acid–urea PAGE (Fig. 1C). Antibacterial activities were measured towards eight different microorganisms including Gramnegative and Gram-positive bacteria. As shown in Table 1, astacidin 2 has a strong and broadspectrum antibacterial activity against both Gramnegative and Gram-positive bacteria. 3.2. Determination of the primary structure To determine the amino acid sequence of astacidin 2 the purified sample was subjected to Edman degradation. Astacidin 2 consists of 14 amino acid residues with the sequence RPRPNYRPRPIYRP that was confirmed by mass spectrometry and the C-terminus is amidated. There are no cysteine residues, but the peptide has strong

Table 1 Minimal inhibitory concentration of Astacidin 2 Organism

Astacidin 2 (mg/ml)

Shigella flexneri ATCC 203 Proteus vulgaris OX19 ATCC 6380 Escherichia coli D21 Psedomonas aeruginosa OT 97 Bacillus megateriun B11 Bacillus subtilis ATCC 6633 Staphylococcus aureus JC-1 Micrococcus luteus Ml 11

0.98 (0.5 mM) 7.8 (4.24 mM) 3.9 (2.12 mM) 7.8 (4.24 mM) 1.9 (1.03 mM) 1.95 (1.06 mM) 7.8 (4.24 mM) 10 (5.44 mM)

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cationic property like other known AMPs. This proline/arginine-rich antibacterial peptide has 57% amino acid sequence identity with metalnikowin 1 from the hemipteran insect, Palomena prasina (16) and belongs to the proline/arginine-rich family of antibacterial peptides. The mass of astacidin 2 determined with MALDI–TOF-MS was 1838 Da. 3.3. cDNA cloning and Northern blot The amino acid sequences of astacidin 2 was used to design and synthesize degenerate primers. Using 50 -[g-32P] ATP-labelling and PCR method, specific DNA fragments representing astacidin 2 was amplified from a crayfish hemocyte cDNA library. The nucleotide sequence and deduced amino acid sequence are shown in Fig. 2. The underlined amino acid sequences of the cDNA perfectly match the amino acid sequences of astacidin 2. The first 22 amino acid residues form a signal sequence. A long 30 -untranslated flanking region in the mRNA of astacidin 2 follows the termination codon. Northern blot was performed using a cDNA probe spanning an astacidin 2 sequence to the 30 -untranslated region (143–549 nucleotides in Fig. 2). Northern blot analysis shows that astacidin 2 is synthesized by the blood cells and expressed as a single band of 0.9 kilobases, which corresponds to the cDNA including a 30 -untranslated region and a poly (A) tail region. Expression of astacidin 2 could not be detected in the hepatopancreas (Fig. 3A) and it is not up-regulated after injection of crude LPS and hence the astacidin 2 transcript is expressed constitutively (Fig. 3B).

Fig. 2. Nucleotide sequence and deduced amino acid sequence of the astacidin 2. The mature astacidin peptide is underlined and two polyadenylation sites are indicated in bold. The first 22 amino acids are the putative signal peptide.

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3.4. Analysis of plasma proteins after crude LPS-injection When cell-free hemolymph was analyzed by SDSPAGE after crude LPS injection three protein bands

Fig. 3. (A) Tissue specific expression of astacidin 2. Lane 1, 15 mg total RNA of hemocytes; lane 2, 20 mg total RNA of hepatopancreas; lane 3, RNA size marker. The blot was hybridized with a 32P-labeled astacidin 2 cDNA probe. (B) Expression of astacidin 2 mRNA after LPS injection to crayfish. Lane 1, 10 mg total RNA of hemocytes for control crayfish (without any injection); Lane 2, 10 mg total RNA of hemocytes after 6 h injection of LPS to crayfish; Lane 3, 10 mg total RNA of hemocytes after 12 h injection of LPS to crayfish. The rRNA of crayfish was used for control probe.

were found to increase in strength as shown in Fig. 4A. These bands were further analyzed by MALDI–TOF-MS, and the amino acid sequences obtained for two of the bands were as shown in Fig. 4B. The amino acid sequence YCCGPE was used to design and synthesize degenerate primers, and one full-length crustin-like cDNA-sequence was isolated from hemocyte total RNA. Later cDNAclones corresponding to the partial amino acid sequences shown in Fig. 4B were also found in our P. leniusculus hemocyte EST library (I. So¨derha¨ll, unpublished), and they were all different crustinlike sequences. These cDNA clones were named Plcrustin1, Plcrustin2 and Plcrustin3, respectively. As shown in Fig. 5A–C all are different, but share the common conserved cysteine pattern in the carboxyl terminus. All three peptides are secreted molecules with a signal peptide with predicted cleavage sites between Ala16 and Arg17 for Plcrustin1, between Glu16 and His17 for Plcrustin2, and between Ala19 and Gln20 for Plcrustin3. Aligment using CLUSTAL W indicates that Plcrustin1 shares most similarities with the Homarus gammarus (AJ86653) crustin-like peptide (24% identity, 38% similarity), whereas Plcrustin2 is more similar to the 11.5 kDa peptide (AJ427538) from C. maenas (33% identity, 43% similarity, Fig. 6) [38]. The third clone, Plcrustin3 is different from Plcrustin1 and 2 in its amino terminal part. At this end the crayfish crustin 3 has a glycin-rich domain showing some similarities to the kuruna prawn, Marsupenaeus japonicus crustin as well as to the L. vannamei, and

Fig. 4. (A) SDS-PAGE of plasma from CFS (C) injected or crude LPS-injected crayfish. (B) Internal amino acid sequences of bands 1 and 2. The bands were excised from SDS-PAGE, treated with trypsin and analyzed by MALDI–TOF-MS. The amino acid sequence used for designing degenerated primers is underlined.

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Fig. 5. (A) Nucleotide sequence and deduced amino acid sequence of the Plcrustin1. The putative signal peptide is shown in italics and the mature peptide consists of 102 amino acids. The 14 amino acid ‘‘block 2-WAP’’ motif is underlined. (B) Nucleotide sequence and deduced amino acid sequence of Plcrustin2. The putative signal peptide is shown in italics and the mature peptide consists of 93 amino acids. The 14 amino acid ‘‘block 2-WAP’’ motif is underlined. Bold sequences match with mass spectrometer sequences used for designing degenerated primers. (C) Nucleotide sequence and deduced amino acid sequence of the Plcrustin3. The putative signal peptide is shown in italics and the mature peptide consists of 138 amino acids. The 14 amino acid ‘‘block 2-WAP’’ motif is underlined.

L. setiferus sequences [16,39] although this part in crayfish is much shorter and does not contain as high number of repeats as is the case for the shrimp clones (Fig. 6). 3.5. Expression of Astacidin 2 and Plcrustin1–3 after bacterial challenge The expression in hematopoietic tissue (hpt) cells and hemocytes of the crayfish AMPs was semiquantified by RT-PCR in unchallenged chrayfish and in crayfish after bacterial challenge. The results show that all AMPs are expressed in hemocytes, while in hpt the expression is more variable. Plcrustin2 and astacidin 2 were constitutively expressed in hemocytes and hpt. However, using

primers for Plcrustin2 gave as a result two bands in some animals and one band in some (Fig. 7, lane 2). After sequencing of these two bands, both were confirmed to encode for Plcrustin2, but the longer sequence was found to contain at least one 50 nucleotide repeat in its 30 UTR. Plcrustin1 and 3 expression were not detected in the hpt from unchallenged animals (Fig. 7). The expression of these AMPs, was further studied after bacterial challenge using different Gram-negative bacteria. Fig. 8 shows expression analyzed by RT-PCR, relative to the 40S ribosomal protein transcripts that were not affected by any bacterial injection and served as an internal control together with ProPO, whose transcript is exclusively expressed in mature hemocytes. The response to

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Fig. 6. (A) Alignment of Plcrustin1 and 2 with Carcinus maenas crustin (AJ427538) and Homarus gammarus (AJ86653) crustin-like peptide. (B) Alignment of Plcrustin3 with Marsupenaeus japonicus crustin (BAD15066) as well as to the L. vannamei, (AAL36890) and L. setiferus (AAL36896) sequences. Red letters indicate highly conserved residues, blue color indicate weakly consensus and black color indicate a neutral not conserved residue.

Fig. 7. Expression profiles determined by RT-PCR of AMPs in hemocytes (lane 1) and hematopoietic tissue (lane 2) of unchallenged crayfish. Expression levels of the specific sequences are detected relative to the control ribosomal protein S40 transcripts and also proPO transcripts. The expected size of the PCR products is indicated.

Fig. 8. RT-PCR analysis of AMPs in hemocytes (lanes 1–4) and hematopoietic tissue (lanes 5–8) after challenge with Gramnegative E. coli (lanes 2,6), Acinetobacter sp. (lanes 3,7) or Gramnegative A. hydrophila (lanes 4,8) compared to controls (lanes 1,5). Changes in expression levels of the specific sequences are detected relative to the control ribosomal protein S40 transcripts and also proPO transcripts.

Gram-negative (E. coli and A. hydrophila) and Acinetobacter varied in hemocytes and hpt in as much as that Plcrustin1 was induced effectively by both pathogenic and non-pathogenic bacteria in hemocytes, whereas Acinetobacter sp. was most efficient to induce expression of this transcript in hpt cells. In contrast Plcrustin3, was induced to a very low extent by Acinetobacter sp. in hemocytes, and hardly induced in hpt. The expression of

astacidin 2 and Plcrustin2 was not obviously different in control and challenged animals in either hemocytes or hpt (Fig. 8). Moreover, the expressions of AMPs were investigated after inoculation with live or heat-killed bacteria at a higher dose, 5  105 cells per crayfish. As shown in Fig. 9 Plcrustin1 was clearly upregulated in hpt after inoculation with live as well as with heat-inactivated Gram-negative A. hydrophila

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Fig. 9. Expression profiles, determined by RT-PCR, in hematopoietic tissue (lanes 1–3), hemocytes (lanes 4–5) and hepatopancreas (lanes 6–8) after challenge with live A. hydrophila (3,8), heat killed A. hydrophila (lanes 2,5,7) compared to controls (1,4,6) with CPBS injection. Twenty-five PCR cycles were performed in all reactions. Changes in expression levels of the specific sequences are detected relative to the control ribosomal protein S40 transcripts and also proPO transcripts.

and to some extent in hemocytes by heat-killed bacteria. This dose of live bacteria caused death of the crayfish within 6 h and thus, the detection of AMPs transcripts in hemocytes could not be done since the hemocytes were too few. Interestingly live bacteria were more efficient in inducing Plcrustin1 and 2 transcript in hepatopancreas as compared to that of heat-inactivated bacteria. 4. Discussion This is the first report of crustin-like putative AMPs in a freshwater crustacean, P. leniusculus and of a 14 amino acid proline-rich AMP (astacidin 2) from hemocytes of this animal. Astacidin 2, is isolated from crayfish hemocytes and it shows a very strong antibacterial activity to both Gram-positive and Gram-negative bacteria. Astacidin 2 was synthesized as a prepropetide and is apparently processed at both the N- and C-terminus by two proteinases. In hemocytes, the mRNA of astacidin 2 is expressed constitutively and is not up-regulated by crude LPS infection. Many AMPs are derived from larger precursors and the processing and generation of antibacterial peptides have been reported from several species. For example, in amphibians, buforin I from the stomach gland cells of the Asian toad Bufo bufo is generated by a pepsin-mediated processing of the cytoplasmic histone H2A [40]. In mice, the precusor a-defensin is cleaved by a metalloproteinase, a matrilysin to produce a-defensin [41], and human defensin-5 is also processed by paneth cell trypsin [42]. The crayfish antibacterial peptide named astacidin 1 is

cleaved from the C-terminus of hemocyanin by a cysteine proteinase [30]. A proline-rich bombesinrelated antibacterial peptide isolated from the toad Bombina maxima skin is also processed by cleavage at both the N- and C-terminus [18]. Moreover, the processing sites of the N- and C-terminus are conserved in bombesin-related antibacterial proteins, suggesting that they share the same processing pathways [43–45]. However, the cleavage sites and primary sequence of astacidin 2 are not similar to that of bombesin-related antibacterial peptides, although they belong to the same group of proline-rich antibacterial peptides. Interestingly, astacidin 2 also shows high homology to the C-terminal part of the proline-rich AMP (PR-39) isolated from pig bone marrow cells [46]. In addition to astacidin 2 three different crustinlike proteins were detected in crayfish hemolymph, and the corresponding cDNA sequences were found in the hemocyte EST library. The crustins all share the carboxy-terminal cysteine rich domain, whereas Plcrustin3 in addition has a glycine rich amino terminal domain, but no repeated short sequence was observed as in shrimp crustins. In all known crustins a specific motif with the consensus sequence CXXDXXCXXXXKCC is present, that is homologous to the block 2 of conserved residues of the whey acidic protein (WAP) domain [47]. WAP proteins are major whey acidic proteins of milk isolated from a wide source of mammals containing a four-disulfide core (4-DSC). This 4-DSC is also found in other small secreted proteins with various functions such as proteinase inhibitors, antibiotics, and growth regulators [47,48]. The presence of the block 2 part of the WAP domain in the crustins suggests a conserved function for this motif. The crustins have been described as very abundant transcripts in L. vannamei, and L. setiferus hemocyte cDNA libraries [29] and in our crayfish hemocyte EST library they constitute 2–3% of all clones (I. So¨derha¨ll, unpublished). Thus these small cysteine rich secreted peptides are likely to serve important functions within the hemolymph of these animals. Crustins are usually found to be constitutively expressed in hemocytes, although the expression may be increased 2–3 times by bacterial challenge [39,49]. The results in this study show that the expression of Plcrustin1 in crayfish is affected by challenging crayfish with bacteria and the response is similar after challenge with different kinds of bacteria. Inoculation of different Gramnegative bacteria caused an upregulation in the

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Plcrustin1 at 6 h of challenging, while the expression levels of Plcrustin2 and astacidin 2, which are highly expressed in hemocytes of unchallenged crayfish relative to ProPO expression, did not change after bacterial challenge. The pattern of these responses are in disagreement with the expression of crustin I in L. vannamei after challenge with heat-killed V. alginolyticus [50], with crustin-like peptides from P. monodon after challenge with pathogenic V. harveyi [51] and also with crustin-like peptides from H. gammarus after challenge with pathogenic L. anguillarum [49]. However, the expression of crustin in H. gammarus is increased at 6 h after injection with live Gram-positive A. viridans var. homari [49] and this response is consistent with the expression of Plcrustin1 in this study, which was highly upregulated in hpt after challenge with the non pathogenic Gram-positive bacterium. Noteworthy is that Plcrustin1 is most closely related to the crustin of H. gammarus. Our studies suggest that Plcrustin1 expression in hemocytes has a broad response to bacterial challenge regardless whether they are non pathogenic or pathogenic and live or killed bacteria. However, in immature cells from the hpt expression of Plcrustin1 is very sensitive to Acinetobacter ssp., whereas Plcrustin3 is hardly affected by challenging. This result may indicate that the different crustin transcripts may be used as markers for steps in the differentiation process in such a way that Plcrustin2 is expressed in immature hemocyte precursors and Plcrustin3 only in mature hemocytes. Another possibility is that the different crustin genes are expressed in different subsets of cells within the hemocyte population and these questions remains to be answered. Intriguingly, as shown in Fig. 7–9 two PCR bands were sometimes found for Plcrustin2, which indicates that these two forms may be differentially regulated. These bands were found to be two different Plcrustin2 variants differing in their 30 UTR region. Whether this repeated sequence has any regulatory function is not yet known, but this detail deserve a further study. Plcrustin1 contains a WAP-conserved domain protein, it lacks one Cys residue after the WAP domain, and Cys is normally present in other crustins [47]. This may indicate that the sequence diversity between the signal peptide and the WAP domain and/or the different conserved Cys residue after the WAP domain may play a specific role for the activity towards various microorganisms. Even though lobster crustin was increased in expression

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after bacterial challenge the animals still died after challenge with A. viridans var. homari, which may suggest that this AMP is not efficient in defense against this pathogen in vivo [49]. A similar phenomenon was observed in this study and crayfish died within 12 h after inoculation with A. hydrophila at doses of 5.0  105 CFUs per crayfish. In this situation we could not harvest hemocytes from the crayfish, because the drop in hemocyte number was dramatic after infection. However, if inoculations were made with heat killed A. hydrophila then Plcrustin1 was upregulated in hpt and hepatopancreas as well as slightly in hemocytes, which may be due to release of new hemocytes from the hpt. When studying the effects of bacterial injections in an animal using pathogens it is important to keep in mind that these bacteria cause significant decreases in hemocyte numbers. If the host can overcome the initial phase of the bacterial infection, new hemocytes are differentiated and released from the hpt to replace destroyed circulating hemocytes and this may affect the expression pattern of the genes of interest. Due to the variation of developmental stages of the cells in hpt, thus, the expression of AMPs in hpt is hardly consistent between different hosts. Therefore, differential and total hemocyte number might be an important parameter to support the study of AMP response in expression to bacterial challenge. Plcrustin2 is most likely to be constitutively and highly expressed in both unchallenged and challenged animals and stored in secretory granules of the granular hemocyte and released to the hemolymph after exocytosis [52]. Therefore, VargasAlbores et al. [50] suggest that its prompt synthesis is not required during bacterial invasion. One interesting observation is that live bacteria was much more effective in inducing Plcrustin1 and 2 transcript in hepatopancreas as compared to heatkilled bacteria, whereas this was not the case in hpt cells or hemocytes. This result may indicate a specific interaction between the live bacteria and hepatopancreas tissue. Interestingly Plcrustin2 as well as astacidin 2 were highly expressed also in hpt cells (immature hemocytes) indicating a role for these molecules in the hematopoietic tissue. We have not isolated or expressed the different crustin proteins and therefore it is impossible to judge whether these molecules have antimicrobial activity or if they play a role in some other process. However, astacidin 2 was proven to be a potent AMP (about 10 times as

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active as C. maenas crustin on a molarity basis) and was also present in hpt cells and thus it seems that this antimicrobial activity is needed early during hemocyte maturation.

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Acknowledgements

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This research has been financed by the Swedish Science Research Council (VR-N) and FORMAS to Kenneth So¨derha¨ll.

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