A peroxiredoxin from kuruma shrimp, Marsupenaeus japonicus, inhibited by peptidoglycan

ARTICLE IN PRESS Developmental and Comparative Immunology (2008) 32, 198–203

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SHORT COMMUNICATION

A peroxiredoxin from kuruma shrimp, Marsupenaeus japonicus, inhibited by peptidoglycan Mary Beth Bacano Maningas, Takashi Koyama, Hidehiro Kondo, Ikuo Hirono, Takashi Aoki Laboratory of Genome Science, Graduate School of Marine Science and Technology, Tokyo University of Marine Science and Technology, Minato, Tokyo 108-8477, Japan Received 4 July 2007; received in revised form 17 July 2007; accepted 17 July 2007 Available online 13 August 2007

KEYWORDS Peroxiredoxin; Crustacean; Kuruma shrimp; Peptidoglycan

Abstract Crustaceans rely on both the cellular and humoral responses of their innate immune system for protection against invading pathogens. Peroxiredoxins (Prx) are a family of anti-oxidant proteins that protect aerobic organisms against oxidative damage by reactive oxygen species (ROS). Although it is ubiquitously found in all organisms, it has not been studied thoroughly in crustaceans. Here, we report a Prx from the crustacean kuruma shrimp, Marsupeneaus japonicus (mjPrx). This crustacean Prx has a full-length cDNA of 659 bp encoding for 198 putative amino acids. It has no signal peptide and is composed of 4 cysteine residues. Based on the conservation of these residues, particularly the N- and C-terminal cysteines, conserved protein domains and on phylogenetic analysis, mjPrx was found to belong to the 2-Cys Prx subgroup. The mjPrx gene is constitutively expressed in heart, hemocyte and lymphoid tissues, and is down-regulated in heart and lymphoid tissues by peptidoglycan (PG) treatment. & 2007 Elsevier Ltd. All rights reserved.

1. Introduction Peroxiredoxins (Prx) (formerly known as 25 kDa thiol-specific oxidant, TSA, and TSA thioredoxin peroxidase, Tpx) is a family of enzymes that protects organisms against oxidation. It is present in all organisms, from yeast to mammals [1]. The first Prx was isolated in the cytosol of yeast, Corresponding author. Tel./fax: +81 03 5463 0689.

E-mail address: [email protected] (I. Hirono). 0145-305X/$ - see front matter & 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.dci.2007.07.004

Sacchromyces cerevisae, then named as TpxI. [2] Since then, Prx has been reported in mammals (grouped in 6 subtypes), in insects, fish, reptiles, plants and other organisms, all containing a conserved cysteine (Cys) residue in the N-terminal region that is the primary target site of oxidative reaction by peroxides such as H2O2 [1,3–6]. The 6 mammalian peroxiredoxins (Prx I–VI) are subdivided into 3 subgroups; 2-Cys type (Prx I–IV), atypical 2-Cys type (Prx V) and the 1-Cys type (Prx VI) [1]. 2-Cys Prx contains conserved N- and C-terminal Cys residues needed for catalytic function. 2-Cys atypical Prx contains a conserved N-terminal Cys residue and

ARTICLE IN PRESS A peroxiredoxin from kuruma shrimp, Marsupenaeus japonicus, inhibited by peptidoglycan a non-conserved one for catalytic activity. 1-Cys only contains and needs the N-terminal Cys residue to function. Prx I was the name given to genes known previously as NKEF A, PAG, MSP33, OSF3 and HBP23, Prx II for NKEF B, Calpromotin and Torin, Prx III for MER5 and SP22, and Prx IV for AOE372 and TRANK [1]. In Drosophila melanogaster, 5 different Prx genes (reported as Tpx) have been studied, 3 of which belong to the 2-Cys subgroup, and 2 to 1-Cys, all observed to be active in the thioredoxin system where they were shown to remove H2O2 in the presence of dithiothreitol [7,8]. In plants, at least 17 Prx isoforms have been determined, classified into 4 classes, 1-Cys, 2-Cys, type II and type Q Prx, and shown to confer protection to the photosynthetic system against stresses that produce reactive oxygen or nitrogen species [3]. Most Prx studies were about its anti-oxidant properties. Recently, however, Prx of the 2-Cys subtypes have been implicated in immune responses against bacterial agents. Mice Prx II was found to play a significant role in the regulation of pro-inflammatory responses to bacterial lipopolysaccharide (LPS), protecting it against lethal shock as a result of endotoxins [9]. Furthermore, LPS was also reported to inhibit Prx I gene expression in mouse RAW264.7 cells [10]. Very recently, in expression profiles and transcriptomic reponses to the white spot syndrome virus (WSSV) infection, some genes involved in the protection of shrimp from oxidative stress were found to be downregulated in Litopenaeus vannamei [11] and Fenneropenaeus indicus [12]. Shrimp is the most important commodity in the aquaculture production market, accounting for almost 20% of the aquaculture commodities in trade. However, the intensification of shrimp farming over the last few decades was also accompanied by the outbreak of infectious diseases of either viral or bacterial origin [13,14]. To address this problem, the use of immunopotentiators like peptidoglycan (PG) has been investigated [15]. It was shown that PG is effective against vibriosis and white spot syndrome in kuruma shrimp and this was suggested to be due to the enhancing effect of PG in the phagocytotic activity of shrimp granulocytes. Knowing the underlying molecular mechanisms of such an effect will be important to the further understanding of shrimp innate immunity as this system is the first line of defense that helps limit infection at an early stage. In spite of the isolation of Prxs in numerous organisms, it is yet to be studied thoroughly in crustaceans, particularly its relation to immunity. In fact, it was only very recently that a crustacean Prx has been reported in Chinese shrimp. In this study, we report the complete cDNA of a Prx from kuruma shrimp, Marsupenaeus japonicus, which we found to be of the 2-Cys Prx type, and an orthologue of the Chinese shrimp Prx. We also show that it is ubiquitously expressed in shrimp tissues but is inhibited in heart and lymphoid tissues following PG treatment.

2. Materials and methods 2.1. Cloning A partial cDNA fragment of kuruma shrimp previously identified by EST analysis to be homologous to a natural

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killer cell-enhancing factor (Genbank accession no. AU176327) [16] was used as a probe to screen a kuruma shrimp cDNA library prepared previously in this laboratory [17]. The cDNA fragment was labeled with P32-dCTP by using a random primer DNA labeling kit (Takara, Japan). Positive plaques were isolated in secondary and tertiary screening. The recombinant plasmids were rescued from the bacteriophage clones by in vivo excision using JM109 (Stratagene, USA) following the manufacturer’s recommendations. The isolated clones were sequenced on an automated DNA sequencer LC4200 (Li-cor, USA) using a ThermoSequenase fluorescent-labeled primer cycle sequencing kit (Amersham Biosciences, USA).

2.2. Sequence data analysis Nucleotide and amino acid sequences were analyzed using GENETYX WIN 7.0.3 (SDC Software development, Japan). Identities were calculated using BLASTp (BLOSUM 62) implemented in BLAST 2 SEQUENCES (http://www.ncbi. nlm.nih.gov/blast/bl2seq/wblast2.cgi), and the complete multiple amino acid alignments were carried out in CLUSTAL X 1.81 using default parameters. SignalP (http://www.cbs. dtu.dk/services/SignalP/) and NetNGlyc 1.0 (http://www. cbs.dtu.dk/services/NetNGlyc/) servers were used to predict signal peptide cleavage and N-glycosylation sites, respectively. The ProDom Server, release 2005.1 (http:// protein.toulouse.inra.fr/prodom/current/html/home.php/), was used to predict protein domains. For phylogenetic analysis, we used the neighbor-joining (NJ) algorithm employing the Poisson correction method with 1000 bootstrap re-sampling and with complete deletion of gap sites implemented in MEGA3 software (http://www.megasoftware. net/index.html/). The bootstrap consensus tree was shown with values in percent.

2.3. Expression analysis PG was prepared from Bifidobacterium thermophilum as reported previously [15]. Apparently healthy kuruma shrimp, each weighing about 12–15 g, were acclimatized in re-circulating culture tanks containing 30–32 ppt with approximately the same rearing conditions. Each individual shrimp was then fed with 0.2 mg of PG per body weight kilogram for 3 consecutive days and subsequently injected with PG (1 mg/kg body weight) on the 4th day. Heart, hemocyte and lymphoid tissues were dissected from 5 shrimps sampled at 1, 3 and 7 days post-PG treatment. Tissue samples were also collected on day 0 prior to PG feeding to serve as initial and negative controls. After this, total RNA was extracted from the tissues collected and pooled, and then cDNA was synthesized using a cDNA firststrand synthesis kit with AMV reverse transcriptase (Life Sciences, USA). Resulting cDNAs were amplified using the following primers (PrxF, 50 -GATGGCCAGTTCAAGGAGAT-30 and PrxR, 50 -GTGGGATCAGCCTTCATTGT-30 ). PCR conditions were initial denaturation at 95 1C for 5 min, 30 cycles (95 1C—30 s, 55 1C—30 s, 72 1C—1 min), and final elongation at 72 1C for 5 min. Elongation factor 1-a (EF1-a) was used as control; EF1-aF, 50 -TGGTTGTCAACTTTGCCCC-30 and EF1-aR, 50 -TTGACCTCCTTGATCACACC-30 . The PCR products were

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M.B. Bacano Maningas et al. residue, FYPLDFTFVCPTEI, and before the C-terminal Cys, GEVCPA, are also conserved. The position of the mjPrx Cys residues shows that mjPrx belongs to the 2-Cys subgroup. Identity comparison shows that mjPrx, like other Prxs, is also conserved, with homology to human Prxs ranging from 31% to 70%, to Drosophila Prxs from 31% to 74% and to its Chinese shrimp orthologue 96%. It does not possess any signal peptide as well as any glycosylation sites as predicted by signalP and NetNGlyc 1.0 servers, respectively. The putative protein is composed mainly of charged, hydrophilic amino acids—aspartic acid (6.4%), lysine (8.4%) and glutamic acid (7.4%), accounting for its low (acidic) isoelectric point at 5.18. The protein does align with the peroxiredoxin domain as shown by multiple alignments with ProDom protein domain database (Fig. 2).

electrophoresed on a 1.0% agarose gel containing ethidium bromide and visualized using a Densitograph (Atto, Japan). The same set of primers was used to amplify kuruma shrimp genomic DNA to confirm the integrity of the sequence.

3. Results 3.1. Kuruma shrimp peroxiredoxin (mjPrx) An EST fragment was used to screen a kuruma shrimp cDNA library. Kuruma shrimp peroxiredoxin (mjPrx) complete cDNA consists of 659 nucleotides encoding for a 198 amino acid sequence (Genbank accession no. AB302094). It contains 4 Cys residues and a typical polyA signal AATAAA. Clustal alignment of amino acid sequences shows that the 1st Cys residue (Cys52) of mjPrx is conserved all throughout the animal species. The 2nd Cys residue (Cys71) is conserved with human Prx I–IV, the 3rd (Cys76) with human Prx IV and V and the 4th (Cys173) with human Prx I–V (Fig. 1). The motifs important for functional activity before the N-terminal Cys

3.2. Phylogenetic analysis Phylogenetic analysis using the NJ method revealed that mjPrx was very similar to a recently reported shrimp Prx, *

*

*

Kuruma Prx ChiShr Prx Hum PrxIII Dro Prx3 Dro Prx1 Carp Prx Catfish Prx Flounder Prx Pufferfish Prx Trout Prx Hum PrxI Hum PrxII Hum PrxIV Dro Prx2 Dro PrxU Hum PrxVI Dro Prx6a Dro Prx6b Hum PrxV

-----------------------------------------------------MSNT--------------------VPAIGKRAPVFKGTAVV-DGQFKEISLEDYKG-KYVIFFFYPLDFTFVCP-TEIIAFSDRVEEFKKIGCEVVACS-TDSHFSH -----------------------------------------------------....--------------------......P..........-..............-...........Y.....-.............R..........-....... MAAAVGRLLRASVARHVSAIPWGISATAALRPAACGRTSLTNLLCSGSSQAKLF.TSSS----------------CHA..VTQH..Y.......-N.E..DL..D.F..-..LVL............-...V....KAN..HDVN.....V.-V...... MSFVARSLIRN--------VPL------------MGKAILS---QQKQIAARLLHQ.AP----------------LAAVRVQQP..D...L...-.NS.Q.VK....R.-..LVL............-...V...E.IK..HD.NT..LGV.-V...... -----------------------------------------------------------------------------M.QLQ.P..A.A.....-N.V..D.K.S....-..LVL............-.......ESAA..R..N...IG..-...Q.T. -----------------------------------------------------.AAG--------------------KAH...P..D.TAK..MP.....DL..SE...-...VL............-........A....R..N...IGA.-V....C. -----------------------------------------------------..AG--------------------KAQ...P..D.TAK..MP.....DLK.S..R.-...V.............-........AA......N...IGA.-V....C. -----------------------------------------------------.AAG--------------------SAQ...P..D.TAK..MP.....DLT.SN.R.-...V.............-........AA.Q.R......I.A.-V...... -----------------------------------------------------.AAG--------------------KAQ...L..D.TAK..MP....HDLK.S..R.-...V.............-........AA.D.R......I.A.-V...... -----------------------------------------------------.AAG--------------------KAR..HL..G.TAK..MP.....D..MS..R.-...V.............-........AA...R......IGA.-V....C. -----------------------------------------------------..SG--------------------NAK..HP..N..A...MP.....D...S....-...V.............-.........A.....LN.Q.IGA.-V....C. -----------------------------------------------------.ASG--------------------NAR...P..D..A....-..A...VK.S....-...VL............-.......N.A.D.R.L....LGV.-V..Q.N. MEALPLLAATTPDHGRHRRLLLLPLLLFLLPAGAVQGWETEERPRTREEECHFYAGGQVYPGEASRVSVADHSLHLSKAK.S.P..YWE....I-..E...LK.T..R.-..LV.............-......G..L...RS.NT......-V..Q.T. --------------------MSKYLSVLLLSAALVGAAKPED-----NESCYSFAGGSVYPDQPK----GDHQLQYTKAV.S.P..Q.E.....-NKEIVKL..SQ.L.-...VLL...........-.........IA.....KT..IGV.-V....T. ---------------------------------------------------------------------------MRMLN.NQV..N.TTN...-S.GYRNFA.T.LR.-R..LLV...A..SY...-..LQ.....AP..RNV....L...-.....V. -------------------------------------------------------------------------MPG-GLLL.DV..N.EANTT.-----GR.RFH.FL.DSWG.L.SH.R...P..T-..LGRAAKLAP..A.RNVKLI.L.-I..VED. -------------------------------------------------------------------------MSGKALN..DQF.N.TAETSE-----GR.DFY.WMQDSWA.L.SH.A...P..T-..LSRVAALIP..Q.R.VKPI.L.-C.PVE.. ------------------------------------------------------------------------------MRL.QTV.N.EADTTK-----GP.KFHEWQ.NSW.VL.SH.A...P..T-..LGRIAVHQP..A.RNTKCL.H.-V.ALN.. --------------------------MGLAGVCALRRSAGYILVGGAGGQSAAAAARRCSEGEWASGGVRSFSRAAAAM.PI.VGDAIPAVE.FEGEPGNKVN.AELFKG.KGVL.GV.GA..PG.SK.HLPG.VEQA.AL.AK.VQ....LSVNDA.VT

Kuruma Prx ChiShr Prx Hum PrxIII Dro Prx3 Dro Prx1 Carp Prx Catfish Prx Flounder Prx Pufferfish Prx Trout Prx Hum PrxI Hum PrxII Hum PrxIV Dro Prx2 Dro PrxU Hum PrxVI Dro Prx6a Dro Prx6b Hum PrxV

LAWTNTPRKEG---GLGTMKIPLLADKSMEVAKAYGVLKEDE------GIAFRGLFVIDGKQNLRQVTINDLPVGRDVDETLRLVQAFQFT-DEHGEVCPAGWKPGA--KTMKADP--TGSKEYFQNEN-----------------...I.......---...................T........------..............D............................-...............--.......--A..........-----------------...I.....N.---...H.N.A..S.LTKQISRD....L.GS------.L.L....I..PNGVIKHLSV.......S.E......K...YV-ET.......N.T.DS--P.I.PS.--AA......KV.Q----------------.T.C.VD..N.---.V.QL.Y...S.LTKKISAD.D..LDK.------..SL..T.I..PNGI...YS........S...V...IK....V-EQ.......N.N.NSNPA.I.P.V--EE..K..SKHG-----------------...I.....Q.---...S.D.........K..RD....D.ET------..P.....I..D......I.V.......S.E..........Y.-.KY......N....Q--...V...--.K.....ETTS-----------------...I.....Q.---...H.NV..V..SLRSISQD........------...Y....I..D.GI...I.........SI.............-.K............K--D.I.P.V--QQ..D..SKQH-----------------...I.....Q.---...H..V..V..TKRSISQD........------...Y....I..D.GI...I.........SI.............-.K............K--D.I.P.V--QK..DF.SKQ.-----------------F........Q.---.........VS.TRRTISTD........------...Y....I..D.GI...I.........S.E............-.K............S--D.I.P.V--QK..DF.SKQ------------------F........Q.---.........VS.TRHTISTD........------...Y....I..P.GI...I.........S.E............-.K............S--D.I.P.V--QK...F.SKH------------------.........H.---...A.....V..TLRSISTD........------...Y....I..D.GV...I.........S..............-.K............S--D.I.P.V--QK..DF.SKQQ-----------------...V...K.Q.---...P.N...VS.PKRTI.QD.....A..------..S.....I..D.GI...I.V..P.CC.S..............-.K............S--D.I.P.V--PKT....SKQK-----------------...I.......---...PLN....G.VTRRLSED.....T..------...Y....I....GV...I.V.......S...A........Y.-..............S--D.I.PNV--DD.....SKH.-----------------...I....RQ.---...PIR....S.LTHQIS.D...YL..S------.HTL....I..D.GI...I.L.......S............Y.-.K............S--E.IIP..--A.KLK..DKL.-----------------...I.......---...DV.....S.LTHKIS.D...YL.SS------.H.L....I..QTGV...I.M.......S....I.......Y.-.T.........R...--D.IVPN.--EEKTK..AKN.-----------------C..M.....N.---...ELD.......N.KI.RD....D..T------.L.L.A..I..REGRI..I.V..MG...S...A.........S-..F.....VN.R...--......A--..KE...KHAI-----------------...SKDINAYNCEEPTEKLPF.IID.RNR.L.ILL.M.DPA.KDEKGMPVTA.VV..FGPDKK.KLSILYPATT..NF..I..V.ISL.L.-A.KRVAT.VD..D.DS-VMVLPTIPEEEA.KL.PKGVFTKELPSGKKYLRYTPQP KG.IEDIKSF.---K.SSFDY.II..DKR.L.LKFNM.DK..INAEGIPLTC.AV..V.D.KK..LSILYPATT..NF..I..VIDSL.L.-QTKSVAT..D..Q.GK-CMVLPTVKAEDVPKL.PDGIETIELPSGKSYLRITPQP VD.V.DIKSYCLD-IP.DFPY.II..PTRDL.VSL.M.D.EQKKDPEV.KTI.A..I.SPDHKV.LSMFYPMST..N...I..TIDSL.L.DRLKVVAT..N.T..TK-VMILPTVTDEEAHKL.PKGFDKVSMPSGVNYVRTTENY GE.GRAHKA..--------.VR....PTGAFG.ETDL.LD.S------------.VS.F.NRR.KRFSMVVQDG---------I.K.LNVEP.GT.LT.SLAP-------NIISQL-------------------------------

*

IDENTITY --96% 62% 58% 74% 71% 70% 70% 73% 72% 69% 70% 67% 64% 62% 31% 32% 31% 32%

Fig. 1 Clustal alignment of kuruma shrimp peroxiredoxin (mjPrx) protein with other members of the peroxiredoxin family in other organisms. Cysteine (Cys) residues are boxed black marked with an asterisk (*), the important FYPLDFTFVCPTEI and GEVCPA motifs are boxed gray; dots (y) represent conserved amino acid residues and gaps in dash () were introduced to maximize alignment. 0

100

PD000498

Amino acid

ProDomID

8 Ð54 55 Ð86 82 Ð161 162 Ð186 158 Ð198

PD000498 PD738734 PD000721 PD586906 PD947500

PD738734

200

PD000721

PD586906 9475

# in Family Domain Name 711 216 623 159 1

Peroxidaseantioxidant peroxiredoxin Peroxidaseantioxidant thioredoxin Peroxidaseantioxidant bacterioferritin Peroxidasethioredoxinantioxidant Peroxidasethioredoxin

E-value 2e-16 6e-09 1e-29 4e-07 1e-06

Fig. 2 Result of the ProDom multiple alignment using the kuruma shrimp perociredoxin (mjPrx) amino acid sequence as the query sequence. Consensus domains pertaining to peroxiredoxin and thioredoxin motifs are indicated accordingly.

ARTICLE IN PRESS A peroxiredoxin from kuruma shrimp, Marsupenaeus japonicus, inhibited by peptidoglycan most closely related to Drosophila Prx 1 and significantly clusters with Prxs of the 2-Cys subtype, confirming our data on multiple alignment (Fig. 3). It is interesting to note that the Prx identified from different fish species so far appears to be the orthologue of human Prx I as they belong to the same clade.

3.3. Tissue distribution and down-expression of mjPrx gene following PG treatment A set of primers from the mjPrx ORF was used to amplify shrimp hemocyte cDNA as well as genomic DNA to check the integrity of the sequence data. We obtained the expected cDNA size, and a higher single band was also observed in the genomic DNA (Fig. 4a). mjPrx was shown to be constitutively and highly expressed in heart, hemocyte and lymphoid tissues. Its expression remains the same in hemocytes after the kuruma shrimps are treated with peptidoglycan at 1, 3 and 7 days, but interestingly declines in the heart and lymphoid tissue (Fig. 4B).

4. Discussion Peroxiredoxins (Prx) are multifunctional antioxidant thioredoxin peroxidases already identified as a conserved

99 61 85

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molecule in many organisms, and hence are believed to occur in organisms in all kingdoms [1]. In spite of this, however, a complete Prx cDNA has only been reported in the crustacean taxa just very recently in Chinese shrimp [18]. The kuruma shrimp Prx (mjPrx) that we have reported in this study was reported in 2002 [16]. mjPrx showed high similarity and is an orthologue of the Chinese shrimp Prx. Crustacean molecular physiological processes, including its immune system, have been increasingly studied, and the identification of shrimp Prxs would significantly contribute to these efforts. mjPrx is highly homologous to its mammalian, teleost and insect counterparts. This, together with the identification of mjPrx as a 2-Cys Prx, which is composed of the signature Prx motifs (FYPLDFTFVCPTEI and GEVCPA) [19], suggests that this shrimp molecule is capable of the same anti-oxidant attributes as the members of the 2-Cys Prxs. The 2 conserved Cys residues (as was first demonstrated in yeast) are essential for catalytic activity. The N-terminal Cys is the primary site for H2O2 oxidation, while the C-terminal Cys forms an intermolecular disulfide binding to other subunits when the N-terminal Cys is oxidized [20]. More recently, this reaction mechanism has been elucidated further. The redox-sensitive cysteine residue of each of the Prx homodimer subunits is oxidized by hydrogen peroxide (H2O2) to Cys-SOH, which then forms an intermolecular disulfide bond with the neighboring Cys-SH [1].

Flounder Prx Pufferfish Prx Trout Prx Carp Prx

95 61 69

Catfish Prx Human Prx I 2-Cys Prx

Human Prx II

25 91

Human Prx III Drosophila Prx 3

52 35

Human Prx IV 85

Drosophila Prx 2 Drosophila Prx 1

93

Kurumashrimp Prx

39 99

Chinese shrimp Prx 1 Drosophila Prx unknown Drosophila Prx 6b Human Prx VI

100 91

1-Cys Prx

Drosophila Prx 6a Human Prx V

2-Cys atypical Prx

0.1

Fig. 3 Neighbor-joining tree of the peroxiredoxin family including kuruma shrimp peroxiredoxin (mjPrx). Bootstrap values are shown in percent. Accession numbers include Kuruma shrimp Prx (AB302094), Chinese shrimp Prx I (DQ205423), human Prx I (CAG28580), human Prx II (CAG46588), human Prx III (CAG29340), human Prx IV (CAG46469), human Prx V (CAG33484), human Prx VI (CAI20936), carp Prx (BAB39202), trout Prx (AAF71327), Japanese flounder Prx (AAY25400), green spotted pufferfish Prx (AAY21814), catfish Prx (AAU29515), Drosophila Prx 1(Q9V3P0), Drosophila Prx 2 (AAF42986), Drosophila Prx 3 (AAG41976), Drosophila Prx U (N_P648759), Drosophila Prx 6a (AAG47822) and Drosophila Prx 6b (AAG47823).

ARTICLE IN PRESS

cD

M

A

NA

M.B. Bacano Maningas et al.

(kb ) gD NA

202

2.0

0.5

B

Heart M(kb)

0

1d

3d

Hemocyte 7d

0

1d

3d

Lymphoid 7d

0

1d

3d

7d

2.0 0.5

mjPrx

2.0 0.5

EF1-α Peptidoglycan-treated kuruma shrimp

Fig. 4 Amplification of kuruma shrimp peroxiredoxin (mjPrx) cDNA and genomic DNA (A), and RT-PCR expression in the heart, hemocyte and lymphoid of shrimp at 0, 1, 3, 7 days post-peptidoglycan feeding (B). Samples at 0 day were considered as constitutive expression as well as negative controls.

The ubiquitous expression of mjPrx in shrimp tissues indicates that it is a critical molecule that could potentially be involved in numerous physiological functions. It would be interesting to know what types of shrimp cells produce mjPrx and where in the cell is it found. Such information would allow for a greater understanding of this molecule since its location would more or less define its function. For example, mammalian Prx I and II are found in the cytosol, Prx III in mitochondria, Prx IV in the extracellualar space, Prx V in mitochondria and peroxisomes, and Prx in the cytosol. Itami et al. [15] showed that oral feeding of shrimp with peptidoglycan (PG) from Bifidobactrium thermophilum protected the organism against WSSV infection. Recently, it has been reported that LPS markedly inhibits the expression of Prx I and II in mice. Such LPS-dependent regulation of Prx I and II is thought to be very important in the prevention of too many host responses following bacterial infection [9,10]. Very recently, in another report on the expression profiles and transcriptomic responses of Litopenaeus vannamei, it was observed that genes involved in oxidative stress were down-regulated against WSV infection [11]; the same phenomenon was recorded in Fenneropenaeus indicus [12]. The down-regulation of mjPrx by bacterial PG in heart and lymphoid organs is therefore interesting, as this would suggest that this shrimp enzyme is involved in bacterial responses. In the black tiger shrimp, the host response to bacterial infection via oral intubation was traced using immuno-peroxidase and histochemical assays [21]. The authors found that antigens were initially observed in the hemolymph sinuses and subsequently accumulated in the heart and lymphoid organ [21]. In another report on the black tiger shrimp, it was found that bacterial density was highest in the heart and lymphoid organ 48 h post-injection [22]. The accumulation of bacterial components in the heart and lymphoid organ might explain the down-regulation of mjPrx in the heart and lymphoid organ following PG treatment. The exact mechanism, however, of this phenomenon is not clear at the moment and will be worth pursuing in the future.

Interestingly, PG treatment did not induce or inhibit mjPrx expression in the hemocyte, which is a well-known site of shrimp immune responses. Alday-Sanz et al. [21] reported that bacteria entering the digestive tract through the mouth during immersion or intubation were disrupted in the stomach so that only soluble material and fine particles could be observed in the hepatopancreatic tubules. This positively reacting material moved to the hemolymph, where it did not provoke any visible host response such as hemocytic aggregation, although it may have produced other humoral responses. Very recently, Prx expression in the Chinese shrimp hemocyte was reported to change rapidly in response to bacterial injection [18]. Such transient expression and rapid movement might explain the steadiness of mjPrx in the hemocyte. In addition, the Prx transcript in the hemocyte was almost stable 8 h postinjection and this is believed to be related to its phagocytic activity wherein hemocyte needs a relatively stable Prx to eliminate the ROS produced during phagocytosis [18]. It should be noted that samples in this study were collected on days 1, 3 and 7 post-PG treatment in which hemocyte response might have been back to its normal level. The down-regulation of Prx by immune challenges (such as PG and WSSV) suggests their involvement in shrimp responses against viral and bacterial infection and further highlights their functional importance in the shrimp immune system.

Acknowledgment This study was supported in part by the Grants-in-Aid for Scientific Research (S) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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