Molecular characterization and increased expression of the Nile tilapia, Oreochromis niloticus (L.),...

Molecular characterization and increased expression of the

Nile tilapia, Oreochromis niloticus (L.), T-cell receptor beta

chain in response to Streptococcus agalactiae infection

N Nithikulworawong1, A Yakupitiyage1, S K Rakshit2 and P Srisapoome3

1 Aquaculture and Aquatic Resources Management Field of Study, School of Environment, Resource and Development,

Asian Institute of Technology, Pathumthani, Thailand

2 Food Engineering and Bioprocess Technology Program, School of Environment, Resources and Development, Asian

Institute of Technology, Pathumthani, Thailand

3 Department of Aquaculture, Faculty of Fisheries, Kasetsart University, Bangkok, Thailand

Abstract

The complete cDNA sequence of the Nile tilapiaT-cell receptor (TCR) b chain was cloned using 5¢RACE. The full-length, 1263-bp cDNA containeda 942-bp open reading frame (ORF) encoding a314-amino-acid protein. Sequence analyses revealedthat the Nile tilapia TCR b chain contains fourconserved cysteine residues involved in the forma-tion of disulphide bridges and a conserved aminoacid motif believed to be important for assemblyand signalling of the TCR ab/CD3 complex, bothof which are normally found in the TCR b chain ofother vertebrates. As detected using semi-quantita-tive and quantitative RT-PCR, the highest expres-sion level of TCR b was detected in the thymus.Interestingly, Streptococcus agalactiae significantlyinduced the up-regulation of the TCR b chain, andthe strongest up-regulation was detected in thebrain and peripheral blood leucocytes (PBLs). Inin vitro experiments, concanavalin A and Aeromonashydrophila were found to significantly increase theexpression of the TCR b chain in PBLs after 48 h(P < 0.01) and 72 h (P < 0.05), respectively.Furthermore, real-time PCR analysis showed thatintraperitoneal injection (IP) of 107 cfu mL)1 ofS. agalactiae could induce TCR b expression thatwas greater than the expression observed following

administration of 109 cfu mL)1. The presence ofthe TCR b chain in fish detected in this studysuggests the presence of T-cell populations thathave been found in higher vertebrates, which mayplay a crucial functional role in the response to fishpathogens.

Keywords: cDNA cloning, expression analysis,Oreochromis niloticus, T-cell receptor beta.

Introduction

The immune system in fish generally follows thesame model that has been extensively studied inmammals, which can be divided into the innate andadaptive immune systems. The defence againstmicrobes is mediated by the early reactions of theinnate immune system and the later responses of theadaptive immune system (Ellis 1999). The innateimmune system, or non-specific immunity, pro-vides the first line of defence against microbes. Thisimmunity consists of mechanisms that are in placeprior to infection and react essentially the same wayto repeated infections (Ellis 2001). In contrast toinnate immunity, the adaptive or specific immunesystem is stimulated by exposure to infectiousagents, and this response increases in magnitudeand defensive capabilities with each successiveexposure to a particular microbe. The adaptiveimmune system can be further subdivided into cell-mediated immunity and humoral immunity. Cell-mediated immunity is mediated by T lymphocytes,

Journal of Fish Diseases 2012, 35, 343–358 doi:10.1111/j.1365-2761.2012.01353.x

Correspondence P Srisapoome, Faculty of Fisheries, Kasetsart

University, 50 Phahonyothin Road, Chatuchak Bangkok 10900,

Thailand (e-mail: [email protected])

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Blackwell Publishing Ltd

and it functions as the defence mechanism againstmicrobes that survive within phagocytes or infectednon-phagocytic cells.

T lymphocytes specifically recognize a variety ofantigens displayed by major histocompatibilitycomplex (MHC) molecules. On the surface of Tcells, there are receptors that recognize peptide–MHC complexes (T-cell receptors; TCRs). TheTCR is a member of the immunoglobulin super-family, and antigen-binding receptors belonging tothe TCR family are present in all species of jawedvertebrates (Hawke, Rast & Litman 1996). TheTCR is a heterodimer of two disulphide-linkedtransmembrane polypeptide chains, consisting ofeither an a/b or a c/d combination, with twofunctions: ligand binding and signal transduction(Partula 1999). T cells are activated when a TCRheterodimer, in conjunction with the CD3 com-plex, specifically recognizes an external antigen(Ag). TCR ab molecules recognize an Ag-derivedpeptide presented by antigen presenting cells thatexpress MHC class I or class II, whereas the targetsrecognized by cd TCRs are not fully understood.

In fish, the T-cell subpopulations have not beenformally characterized with respect to specificmolecules such as CD4, CD8 or CD3, unlike inmany higher vertebrates. However, the structureand expression of the genes and cDNAs encodingthe TCR b molecules have been identified inteleosts (Partula et al. 1995; Rast et al. 1995;Hordvik et al. 1996; Wilson et al. 1998; Wermen-stam & Pilstrom 2001) and elasmobranchs (Rast &Litman 1994; Rast et al. 1997). Furthermore, TCRb chains have also been successfully cloned andcharacterized from a number of species based onsequence similarity to genes in higher vertebrates(Rast et al. 1995). In addition, Wilson et al. (1998)identified two different cDNAs encoding two TCRb constant regions (Cb) in channel catfish, sug-gesting the existence of two Cb loci or allotypes.Furthermore, genomic organization analysis of theTCR b chain in fish has indicated that the teleostTCR b loci appear to be arranged in a transloconfashion (De Guerra & Charlemagne 1997; Wer-menstam & Pilstrom 2001; Zhou et al. 2003),whereas cartilaginous TCR b genes are arranged inmultiple clusters (Rast & Litman 1994). The T-cellrepertoire of rainbow trout was shown to haveskewed complementarity-determining region 3 sizeprofiles for several VbJb combinations, correspond-ing to T-cell clonal expansions during primaryand secondary responses to viral haemorrhagic

septicaemia virus (VHSV) (Boudinot, Boubekeur& Benmansour 2001).

Nile tilapia, Oreochromis niloticus (L.), is a teleostfish that is considered to be an important commer-cial species for aquaculture both in Thailand andglobally. One of the major problems in Nile tilapiafarming is widespread disease caused by bacterialinfections (e.g. motile aeromonad septicaemiacaused by Aeromonas hydrophila). Additionally,from 2003 to 2006, Streptococcus agalactiae (groupB streptococcus; GBS) were isolated from infectedred tilapia, Oreochromis sp., and Nile tilapiacultured in Thailand (Suanyuk et al. 2008). Strep-tococcus agalactiae is known to severely infect thefarmed fish and can result in major economic losses.Infection with this bacterium has been shown tocause haemorrhagic septicaemia, exophthalmia,meningoencephalitis and multiple necrotic foci invarious tissues (Russo, Mitchell & Yanong 2006;Suanyuk et al. 2008).

An understanding of the adaptive immunesystem of the Nile tilapia is required for theestablishment of effective methods to protectagainst this and other diseases. However, to date,the research regarding the adaptive immune systemof the Nile tilapia has been limited to molecularcloning of immune-related genes involved in theresponse to invading pathogens, such as bacterialinfection. Therefore, this study examined theexpression of TCR b, a gene important in adaptiveimmunity, which plays a crucial role in theelimination of intracellular infections caused byviruses and some bacteria.

Materials and methods

Fish

An adult Nile tilapia weighing approximately 400–500 g was obtained from the Department ofAquaculture, Faculty of Fisheries, Kasetsart Uni-versity. The fish was acclimatized in an 80-L glasstank for 1 week prior to experimental use. Duringthis time, the fish was fed with commercial pelletstwice per day.

Bacteria preparation

Streptococcus agalactiae strain AQSA001 andA. hydrophila strain AQAH001 were obtained frominfected Nile tilapia collected from culture ponds inthe central part of Thailand. The bacteria were

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Journal of Fish Diseases 2012, 35, 343–358 N Nithikulworawong et al. Tilapia T-cell receptor beta chain response inStreptococcus infection

maintained in the Laboratory of Aquatic AnimalHealth Management, Department of Aquaculture,Faculty of Fisheries, Kasetsart University.

Single colonies of these bacteria were separatelygrown in tryptic soy broth (TSB; Merck) andincubated in a shaker water bath at 37 �C for 24 h.After cultivation, the bacteria were harvested bycentrifugation at 600 g for 15 min and washedtwice with sterile 0.85% NaCl. The pellet wasresuspended in sterile 0.85% NaCl. The concen-trations of S. agalactiae and A. hydrophila wereadjusted to 1 · 109 cfu mL)1 (an optical density of0.3 at 540 nm) and 1 · 105 cfu mL)1 (an opticaldensity of 0.11 at 540 nm), respectively, and thesesamples were used as the initial stock. The S. aga-lactiae stock was diluted 10- or 100-fold in 0.85%NaCl to produce bacterial densities of 1 · 108 or1 · 107 cfu mL)1, respectively.

Cloning and characterization of the full-lengthNile tilapia TCR b cDNA

5¢ RACE PCR was used to recover the full-lengthcDNA using a BD Smart RACE cDNA Amplifi-cation Kit (Clontech). Briefly, a healthy fish wasintraperitoneally (IP) injected with 10 lg mL)1

concanavalin A (ConA; Bio Basic Inc.). After3 days, total RNA was extracted from peripheralblood leucocytes (PBLs) using the TRIzol reagent(Gibco BRL) following the manufacturer�s instruc-tions. The mRNA was purified using a QuickPrepMicro mRNA Purification Kit (Amersham Bio-sciences). Approximately 1 lg of mRNA, 1 lL ofBD Smart II A oligo (5¢AAGCAGTGGTAT-CAACGCAGAGTACGCGGG-3¢) and 1 lL of5¢-CDS primer (5¢-(T)25VN-3¢; N = A, C, G or T;V = A, G or C) were used to construct the 5¢RACE-ready cDNA, which was used as a template

to determine the nucleotide sequences in the 5¢direction. Each 5¢ RACE PCR reaction (finalvolume of 25 lL) contained 1.3 lL 5¢-RACE-ready cDNA, 2.5 lL 10· Takara LA Taq buffer,0.5 lL 10 mm dNTP mix (2.5 mm each), 0.5 lLTakara LA Taq Polymerase, 2.5 lL 10· UniversalPrimer Mix, 0.4 lm UPM-long, 2 lm UPM-short(Table 1), 1.0 lL 10 lm gene-specific primer(TCRB-R2; Table 1) and 16.7 lL distilled water.For this study, the gene-specific primer (TCRB-R2)was designed based on the sequence of the constantregion of a partial Nile tilapia TCR b cDNAobtained from a cDNA library (Genbank accessionno. FF28069055139726; Table 1).

The RACE product was purified using a WizardSV Gel and PCR Clean-Up System Kit (Promega)and ligated into the pGEM T-Easy cloning vector(Promega) following the manufacturer�s instruc-tions. The ligation products were transformed intoEscherichia coli strain JM 109 cells, which werespread onto Luria-Bertani agar containing ampicil-lin (0.01 g mL)1), X-gal (50 mg mL)1) and IPTG(100 mm). The plate was incubated overnight at37 �C. The white, positive clones were selected forplasmid extraction using a GeneJET PlasmidMiniprep Kit (Fermentas), as recommended bythe manufacturer. The extracted plasmids weresequenced using a Thermo Sequence FluorescentLabelled Primer Cycle Sequencing Kit (AmershamPharmacia Biotech) with the M13 reverse and M13forward primers (Macrogen).

After sequencing, the vector sequences wereremoved using Vec screen (http://www.ncbi.nlm.-nih.gov/VecScreen/VecScreen.html ). The resultingcDNA sequence was searched for homology withsequences available in GenBank (NCBI, http://www.ncbi.nlm.nih.gov) using BLASTX andBLASTN. The open reading frame (ORF) as well

Table 1 Oligonucleotide primers used for PCR analysis

Primer name Sequence (5¢ to 3¢) Purpose of use

TCRB-F1 GTGAAGCCATTGAACCGCAA RT-PCR

TCRB-R1 TTGGCATAGACTCTCAGCCT RT-PCR

TCRB-F2 GGACCTTCAGAACATGAGTGCAGA Real-time PCR

TCRB-R2 TCTTCACGCGCAGCTTCATCTGTT Real-time PCR and RACE-PCR

BA-F1 GGTCATCACCATTGGCAATG RT-PCR

BA-R1 ACTGAAGCCATGCCAATGAG RT-PCR

BA-F2 ACAGGATGCAGAAGGAGATCACAG Real-time PCR

BA-R2 GTACTCCTGCTTGCTGATCCACAT Real-time PCR

UPM-long CTAATACGACTCACTATAGGGCAAGC RACE-PCR

ATGGTATCAACGCAGAGT

UPM-short AAGCAGTGGTATCAACGCAGAGT RACE-PCR

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as the 5¢ and 3¢ untranslated regions (UTRs) of thecDNA were analysed using ORF Finder (OpenReading Frame Finder, http://www.ncbi.nlm.nih.gov/gorf/gorf.html). The sequence of the full-lengthNile tilapia TCR b cDNA was analysed for thepresence of a signal peptide using SignalP software(http://www.cbs.dtu.dk/services/SignalP/). To iden-tify the two major regions, the variable and constantdomains, the Nile tilapia TCR b cDNA sequencewas compared with gilthead sea bream, Sparusaurata L., TCR b cDNA sequences (Randelli et al.2008). The V domain of the TCR b chain was alsoanalysed using the IMGT (the international ImMu-noGeneTics information system) standardizationnumbering (Lefranc et al. 2003). Multiple aminoacid sequence alignments comparing the TCR b ofNile tilapia; Atlantic cod, Gadus morhua L.(AJ133848); Atlantic salmon, Salmo salar L.(X97435); bicolor damselfish, Stegastes partitus(Poey) (AF324820); channel catfish, Ictaluruspunctatus (Rafinesque) (U39193); Japanese floun-der, Paralichthys olivaceus (Temminck & Schlegel)(AB053427); gilthead seabream, Sparus aurata L.(AM490438); rainbow trout, Oncorhynchus mykiss(Walbaum) (U18122); clearnose skate, Raja eglan-teria (Bosc) (U75769); horn shark, Heterodontusfrancisci (Girard) (U07624); human (M12886);and Norway rat (BC088211) were carried out usingCLUSTALW (Thompson, Higgins & Gibson1994). The identity and similarity scores of theNile tilapia TCR b constant domain comparedwith the TCR b constant domains of otherorganisms were calculated using Matrix GlobalAlignment Tool (MatGAT) version 2.01 (http://bitincka.com/ledion/matgat/).

Evolutionary analysis

We analysed the evolutionary relationship be-tween the Nile tilapia TCR b chain and othervertebrate TCR b chains based on morphologicalcharacteristics and amino acid sequence homol-ogy. ClustalW (Thompson et al. 1994) was usedto perform multiple sequence alignments of theconstant domain of each TCR b chain, excludingthe connecting peptide, transmembrane and cyto-plasmic regions. The alignment was performedto determine the longest possible consensussequence, protein alignment and alignment con-sensus sequence. Twelve amino acid sequences ofTCR b used in the last section for ClustalWanalysis and additional amino acid sequences as

follows: house mouse (M11456); axolotl(X70168); African clawed frog (U60424); dog(D16409); rabbit (M13895); chicken (M37800);and sheep (M94143) were aligned. The phyloge-netic tree was constructed using the neighbour-joining method and was bootstrapped for 1000replicates using MEGA version 4 (Tamura et al.2007).

TCR b tissue distribution

To examine the tissue distribution of TCR b, twoNile tilapia were separately acclimatized in two 80-L glass tanks under ambient laboratory conditionsfor 7 days. The first fish was intraperitoneallyinjected with 0.5 mL of 0.85% NaCl, and thesecond fish was injected with 0.5 mL of1 · 108 cfu mL)1 viable S. agalactiae intraperito-neally. After 3 days, total RNA was isolated fromthe brain, gills, gonads, heart, head kidney, intes-tine, liver, muscle, PBLs, spleen, stomach, thymusand trunk kidney using the TRIzol reagent (Molec-ular Research Center) according to the manufac-turer�s instructions. RNA samples were treated withDNase I (Fermentas) to remove genomic DNA. ARevertAid First Strand cDNA Synthesis Kit (Fer-mentas) was used to synthesize first-strand cDNAusing an oligo (dT)18 primer and 2 lg of total RNAfrom each tissue of each fish.

Amplification was performed in a total volume of30 lL, which contained 1 lL first-strand cDNA,1.5 lL each PCR primer (TCRB-F1 and TCRB-R1; Table 1), 2 lL 10· Taq buffer, 2 lL dNTPmix (2.5 mm each), 21.9 lL sterile distilled waterand 0.1 lL Taq DNA polymerase. The cyclingparameters were an initial denaturation of 5 min at95 �C; 25 cycles of 95 �C for 30 s, 55 �C for 30 sand 72 �C for 60 s; and a final elongation step at72 �C for 5 min. The expression of Nile tilapiab-actin was also analyed as an internal control usingthe primers BA-F1 and BA-R1 (Table 1) under thesame conditions as described above. FollowingPCR, 5 lL of the PCR products was analyed usingelectrophoresis on 1.5% agarose gels in 1· TBEbuffer at 100 V. A 100-bp DNA marker (Fermen-tas) was used to determine the approximate size ofthe PCR fragments.

To more precisely quantify the expression level ofNile tilapia TCR b transcripts in seven selectedtissues (brain, head kidney, intestine, liver, PBLs,spleen and thymus), quantitative RT-PCR wasused. The isolation of total RNA and the

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first-strand cDNA synthesis were carried out asdescribed above.

The expression level of the TCR b transcript ineach tissue of each fish was determined using aMx3000P real-time PCR system (Stratagene)equipped with analytical software version 4.0 andthe Brilliant II SYBR Green qPCR Master Mix(Stratagene), according to the manufacturer�s rec-ommended protocol. Each reaction was performedin a final volume of 25 lL containing 1 lL first-strand cDNA, 12.5 lL 2· SYBR Green qPCRMaster Mix, 9.5 lL dH2O and 1 lL each specificprimer (TCRB-F2 and TCRB-R2; Table 1), whichwere designed based on the sequence of the Niletilapia TCR b constant region. The TCR bexpression level in each sample was normalizedrelative to b-actin using the primers BA-F2 and BA-R2 (Table 1). The PCR conditions were 95 �C for10 min, followed by 40 cycles of 95 �C for 30 s,55 �C for 1 min and 72 �C for 1 min. Followingamplification, DNA melting curve analysis was usedto verify the specificity of the primers. Triplicatereactions were performed for each sample for boththe TCR b and b-actin genes. A standard plasmidcontaining the Nile tilapia TCR b constant regionwas serially diluted in 10-fold increments togenerate the standard curves to assess the PCRefficiency.

The relative TCR b mRNA expression level wascalculated using the 2�DDCT method (Livak &Schmittgen 2001). The difference in the thresholdcycle (DCT) between TCR b and b-actin mRNA ineach sample was used to normalize the level of totalRNA. The tissue that showed the lowest expressionlevel was used as a calibrator.

The effects of Aeromonas hydrophila andconcanavalin A (ConA) on the TCR b mRNAlevel in peripheral blood leucocytes

The change in TCR b mRNA expression duringactivation using different stimulatory conditionswas examined using real-time PCR. A healthy Niletilapia was fed and acclimatized in a glass aquariumtank for 7 days. Blood samples were collected fromthe caudal vein vessel in a heparinized syringe. Theblood was diluted with RPMI 1640 (Gibco,Invitrogen) at a ratio of 2:1, and 3 mL of dilutedblood was put onto a PE tube containing 3 mL ofLymphoprep (Axis-Shield). The tube was centri-fuged at 200 g for 30 min at 25 �C in a swingingbucket centrifuge (Kubota 5800). The opaque

interface (3 mL) was aspirated with a Pasteurpipette and was transferred to a new centrifugetube. The sample was washed with 3 mL ofphosphate-buffered saline (PBS) and centrifugedat 100 g for 10 min three times. The PBLs wereadjusted to a final density of 1 · 108 cells mL)1

and incubated with either 1 · 105 cfu mL)1 A. hy-drophila or 10 lg mL)1 of ConA in a 15 mLconical tube. The control group was treated withPBS only. The tubes were incubated at 25 �C withshaking for 0, 6, 12, 24, 48 or 72 h and thenharvested by centrifugation at 5100 g. Total RNAwas extracted and reverse-transcribed as describedabove. For each reaction, 1 lL of cDNA fromeach sample at the different time points wasused for real-time qPCR. The specific primers, thereal-time PCR conditions and the calculation aredescribed above. The expression of TCR b at the0 h time point was used as the calibrator for thisexperiment.

TCR b mRNA expression in response to viableStreptococcus agalactiae

Eighty-one healthy Nile tilapias were divided intothree groups of nine tanks (three tanks per groupand nine fish per tank). All fish were acclimatizedand fed with commercial feed twice per day for7 days prior to use. Each fish was intraperitoneallyinjected with 0.5 mL 0.85% NaCl (control),0.5 mL of 1 · 107 cfu mL)1 S. agalactiae or1 · 109 cfu mL)1 S. agalactiae. The spleen from afish in each tank was collected 0, 1, 3, 6, 12, 24, 48,72 or 96 h after injection. Total RNA isolation andfirst-strand cDNA synthesis were performed asdescribed above. For each reaction, 1 lL of cDNAwas used for qPCR analysis. The specific primers,real-time PCR conditions, calibrator and thecalculations are described above.

Statistical analysis

The relative expression ratios of each group atdifferent times are presented as the mean � stan-dard deviation (SD). All data obtained from theexperiments were analyed using a one-way analysisof variance (ANOVA), and Duncan�s new multi-ple range test was used to determine the signifi-cance of the differences in the TCR b mRNAexpression levels between groups. P values <0.05and 0.01 were considered statistically significant,respectively.

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Results

Cloning and characterization of a cDNAencoding the full-length Nile tilapia TCR bchain

A partial TCR b cDNA isolated from a Nile tilapiaspleen cDNA library (GenBank accession no.FF28069055139726) was fully sequenced, resultingin an 802-bp nucleotide sequence. 5¢ RACE using aTCR b reverse primer and a universal primer wasused to recover the remaining sequences to producethe full-length cDNA. The amplified product wasapproximately 600 bp long and was cloned into thepGEM T-easy vector. The exact length of the 5¢RACE product, as determined following sequenc-ing, was 574 bp (data not shown), and the twooverlapping products resulted in a 1263-bp full-length cDNA (Fig. 1). The nucleotide sequence ofthis clone has been submitted to GenBank with theaccession number HM162889. The full-length Niletilapia TCR b cDNA contained a 945-bp ORF thatencodes 314 amino acid (aa) residues followed by astop codon (TGA). The cDNA also contains both a35-nucleotide-long 5¢ UTR and a 283-nucleotide-long 3¢ UTR, which contains one polyadenylationsignal (AATAAA) that is 18-bp upstream of the

poly (A) tail. A potential signal peptide cleavage sitewas predicted using SignalP (http://www.cbs.dtu.dk/services/SignalP/), and the signal peptiderepresents 27 amino acid residues of the wholeprotein.

The full-length TCR b cDNA was furthercompared with other known sequences availablein the GenBank database using BLAST. Thesequences with the highest per cent identities werethe TCR b chains of the gilthead seabream and thebicolor damselfish, with E-value scores of 1 · 10)80

and 1 · 10)79, respectively. These sequences werepreviously characterized by Randelli et al. (2008)and Kamper & McKinney (2002), respectively. TheNile tilapia TCR b chain sequence contains bothvariable (V) and constant (C) domains. The Niletilapia TCR b_V region is 333 bp/111 aa long.Two potential carbohydrate acceptor sites (Asn104-Cys105-Thr106 and Asn115-Ser116-Ser117) were iden-tified in the TCR b_V region. Based on the IMGTunique numbering, variable (V), diversity (D) andjoining (J) regions could be identified in the Vdomain, and these regions comprised frameworkregions 1–4 and complementarity-determiningregions (CDR) 1–3 (Fig. 2a). The Nile tilapiaTCR b_C region is 516 bp/172 aa long and

< Signal peptideM F M N V V M H H N M M I T F F C I S F N I I L V S G S S 29

TACATGGGGACAGTTATGAATGCACAGAAGACATAATGTTCATGAATGTAGTCATGCATCACAACATGATGATCACTTTCTTCTGCATAAGCTTTAACATCATCCTCGTGTCAGGTTCAT 120

>< variable region L S D Q V H Q N P A D M Y K N P G Q T A E I T C S H R I D T Y N Q I L W Y K Q T 69

CACTCAGTGACCAAGTCCACCAGAATCCAGCTGATATGTATAAAAATCCAGGACAAACAGCAGAAATCACCTGTTCACACCGTATAGATACCTACAATCAAATCCTCTGGTACAAGCAAA 240><

K T G Q L Q L L G Y M L G T S K S L E P G V T V A I R G D A N K D K N C T L T I 109CAAAAACTGGCCAACTGCAGCTCCTGGGATACATGTTGGGAACAAGCAAATCTCTAGAGCCTGGAGTGACTGTGGCAATTCGTGGGGACGCAAATAAAGACAAGAACTGCACACTGACAA 360

diversity and joining region >< K E V F M N S S A V Y F C A V G Q G E S E A Y F G A G T K L T V L D R E A I E P 149

TTAAAGAGGTCTTCATGAACAGCAGTGCAGTGTACTTCTGTGCTGTTGGACAGGGGGAGTCAGAAGCTTATTTTGGAGCTGGAACTAAACTCACTGTTTTAGACCGTGAAGCCATTGAAC 480

constant Ig domain Q E L K I L G P S E H E C R N S K D K K R K K T L V C V A S G F Y P D H V K M Y 189

CGCAAGAACTAAAAATACTGGGACCTTCAGAACATGAGTGCAGAAACAGCAAAGACAAAAAAAGGAAGAAGACCCTCGTCTGTGTGGCCTCTGGTTTCTACCCTGACCATGTCAAAATGT 600

W Y L N E K T V T D G V A T D E A A R E E K D E N D N T V Y K I T S R L R V Y A 229ATTGGTATTTGAATGAGAAAACAGTCACTGATGGTGTGGCAACAGATGAAGCTGCGCGTGAAGAAAAAGATGAAAATGACAACACGGTGTACAAAATCACCAGCAGGCTGAGAGTCTATG 720

>< K Q W E N P K N T F K C T V K F F D G K N Y K Y N S T D T K G V T A E T T S M S 269CCAAACAATGGGAAAATCCTAAAAATACCTTTAAATGCACTGTCAAATTCTTCGATGGCAAAAATTATAAATATAATTCAACAGATACTAAAGGAGTTACGGCTGAGACAACATCTATGT 840

CPS >< TM >< CYT R E K Y L T T T Q N F K V V Y S V L T V K S C A Y G A F V G F L V W K L Q R F G 309

CAAGAGAGAAGTATTTGACAACTACACAAAATTTCAAGGTCGTCTACAGCGTGCTTACTGTCAAGAGCTGTGCCTATGGAGCTTTTGTTGGATTTTTGGTGTGGAAACTTCAGCGTTTTG 960

> G K Q K Y * 314GTGGAAAGCAGAAGTACTGAGAGCTGAGCTGAAGCCTCAAAAAATAACTTTGGGACATGAGTTACCTTGCTCTCTGTAAATATCGTTCACAAACATAGTCTATTGTTAAAGTGCACACTG 1080TTAATCGCCTGTTCAAGTTCTGCTGTGGATTTTTATTTGCTTATTTGTCTTTATATAATTAATAACTGCCTTTCAAATAGACTGATGTCAATTTCTTTAGATACAAAGTGATTTCTGTTT 1200GTAACTTGATTTTCTTCTCAATAAAAATATGTTTGAGTATTTTAAAAAAAAAAAAAAAAAAAA 1263

Figure 1 The complete cDNA sequence of the Nile tilapia TCR b chain. The top line is the deduced amino acid sequence, and the

bottom line is the nucleotide sequence. The numbers on the right indicate the positions of the nucleotides and amino acids. The start

(ATG) and stop (TGA) codons are in bold. The putative N-glycosylation sites are underlined. The polyadenylation signal (AATAAA) is

indicated by the box.

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contains one potential site for N-linked glycosyla-tion (Asn254-Ser255-Thr256). The immunoglobulin(Ig), connecting peptide (CPS), transmembrane(TM) and cytoplasmic (CYT) regions could bereadily identified in the Nile tilapia TCR b_Cregion (Fig. 3).

The amino acid sequence of the Nile tilapia TCRb chain was compared with the sequence of otherknown TCR b chains (Figs 2b & 3) to analyse theconservation of key amino acid residues that areimportant for maintaining the structure of thedifferent domains. The Nile tilapia TCR b_Vregion includes two conserved cysteines (Cys53 andCys122), which most likely form an intrachaindisulphide bridge, and one conserved J motif (Phe-Gly-X-Gly) (Fig. 2b). Moreover, the TCR b_Cregion contains two conserved cysteines (Cys176 andCys241), which most likely form another intrachaindisulphide bridge. A glutamic acid residue (Glu161)is well conserved in all species except for rainbowtrout (Fig. 3), and this residue is presumed to forma hydrophilic bond with the TCR a chain (Garciaet al. 1996). We did not observe the cysteineresidue that is involved in forming the interchaindisulphide bond with the TCR a chain in theconnecting region. The conserved antigen receptortransmembrane (CART) motif residues were iden-tified and included tyrosine (Tyr284, 294), leucine

(Leu287), lysine (Lys290), serine (Ser291) and valine(Val298) (Fig. 3).

The per cent identity of the nucleotide sequenceof the Nile tilapia TCR b_C region compared withthat of mammals, cartilaginous fish and teleost fishranged from 45.1% to 51.7%, 49.7% to 51.3% and53.5% to 63.1%, respectively (Table 2). Corre-spondingly, the identity of the predicted amino acidsequence of the Nile tilapia TCR b_C regionranged from 36.8% to 39.3%, 25.0% to 34.4% and37.9% to 46.8% when compared with that ofmammals, cartilaginous fish and teleost fish, respec-tively. Moreover, the nucleotide sequence of theNile tilapia TCR b_C region was 56.8% to 63.5%similar to mammalian TCR b_C regions, 50.0% to54.0% similar to cartilaginous fish TCR b_Cregions and 53.2% to 63.7% similar to teleost fishTCR b_C regions (Table 2).

Evolutionary analysis

The relationship of the Nile tilapia TCR b to othervertebrate TCR b_C regions was examined byphylogenetic analysis of the deduced TCR b_Cregion amino acid sequence. The tree branch lengthswere proportional to the differences in the aminoacid sequences. The phylogenetic tree of the TCR bamino acid sequences confirmed the evolutionary

(a) FR1-IMGT CDR1-IMGT FR2-IMGT CDR2-IMGT FR3-IMGT CDR3-IMGT FR4-IMGT (1-26) (27-38) (39-55) (56-65) (66-104) (105-117) (118-128)

1 10 20 30 40 50 60 70 80 90 100 110 120 130

........ ......... ...... ... ........ . ......... ..... .... ..... .... .... ..... .... ..... ...... ... .... ..... ....... .. ......... Nile tilapia SDQVHQNPADMYKNPGQTAEITCSHR IDT......YNQ ILWYKQTKTGQLQLLGY ML......GT SKSLEPGVT VAIRGDAN.K DKNCTLTIKEVF MNSSAVYF CAVGGQ.GESEAY FGAGTKLTVLEDDR

O. mykiss SNQVHQSPASLYKNQGKSAKMVCSHS ISG......YDR VLWYKQSNYREFVFLGY MI......GT SGFPEAG.F DIEGDAN.AG GTSTLTIKQLTP NSSAVYYC AVD.......... ..............

(b)

Nile tilapia DQVHQNPADMYKNPGQTAEITCSH-RIDTYNQILWYKQTKTGQLQLLGYMLGTSKSLEP--GVTVAIRGDANKDKNCTLTIKEVFMNSSAVYFCAVG--QGE---SEAYFGAGTKLTVLGilthead seabream EQVHQTPADMFKQPGEEAKFNCFH-TISNYDKILWYKQTN-EQLQFLGYMNINNGYPEN--GAGVKVEGGAKKDQNCTLTIERLKLNSSAVYLCAAR--RTG---REAYFGGGTKLTVLBicolor damselfish DKVDQNPPNMYKNPQEKAKLYCSH-SIANWNVILWYKQLKNRQLQFLGFMQAGSGFPESGLDEDVTMGGSADEGRNCTLTINSVSVNSSAVFFCAAS--YRGPGYNPAYFGKGTKLTVLJapanese flounder VLITQWPHYISRFPSGSAEMHCYQ-NDTDYQFMYWYRQQKGKEPQLVVYLVASSAN-FEE-GFKSGFEAEIVQKKKWSLKIPSIQEKDEAVYLCAAR--RQRSG-YEAHFGQGTKLTVLAtlantic cod VIIKQSSAKIVRKGAKGIQIDCSH-DDSSYPLMYWYQRKDESPSLTLIGFGYESSTQNYEDRFEERLNITRESVLQGTLVLTEAAESDSAVYFCAAS--MGEGGSEPAFFGKGTKLTVLChannel catfish ANDVLQPDILWAQFGQSVTINCSHTKGSVYREMYWFRQYQGESMELIVYTTSFGTQDFGK-SDQKKFSAIKTVPENGSFTVKDVDYNDNAVYFCAVR----DRGTQPAYFGQGTKLTVLClearnose skate -SVHQSPGALTRSPGQTVKVKCIQ-QDSS-GYIYWYRQYSGAGAQNLFYSAAANIVVPPP-PVTG-FTAERPNNNEFYLKSSGLEADSSAVYFCAWSVPGTDYNNAEAYFGKGTKLVVLHorn shark RSRYPVGNRLTVAEGKTVEMHCFQ-NDTSDSYMYWYRQQSGAGLLLIVTSIGTSDTSPEE-GFKERFKVTRPDLKTCSLKILRVDQTDRAVYYCAASGHPSDSN-SEAYFGDGTKLVVLNorway rat SGVVQSPRHIIKEKGGRSILKCIP--ISEHNNVAWYQQTHGQELKFLIQHYEKSEREKGN--LPSRFSVQQFDDYHSEMNMSALELEDSAMYFCASS--PGGLVQETQYFGPGTRLLVLHuman AVVSQHPSWVICKSGTSVKIECRS-LDFQATTMFWYRQFPKQSLMLMATSNEGSKATYEQGVEKDKFLINHASLTLSTLTVTSAHPEDSSFYICSAR-ESTSDPKNEQFFGPGTRLTVL : . * : *::: : . . : . :.: *: .** **:* **

53 122

Figure 2 (a) The predicted amino acid sequence of the Nile tilapia and rainbow trout TCR b V domains. The IMGT unique

numbering for the V domain (Lefranc et al. 2003); the FR-IMGT and complementarity-determining regions-IMGT designations are

indicated above the sequence. (b) An alignment of the inferred amino acid sequences of the Nile tilapia TCR b V region with the TCR bV regions of other vertebrates. The amino acid sequence for the Nile tilapia is shown in bold. Asterisks (*) indicate residues conserved in

all sequences, and dots (:) indicate residues that are conserved in most of the sequences. Hyphens (-) have been introduced to optimize

the alignment. The conserved cysteine residues are indicated by grey shading. Positions 53 and 122 are the locations of the cysteine

residues that form an intrachain disulphide bridge. The conserved J motif residues are indicated by a box. The accession numbers for the

sequences are as follows: gilthead seabream, Sparus aurata AM490438; bicolor damselfish, Stegastes partitus AF324820; Japanese

flounder, Paralichthys olivaceus AB053427; horn shark, Heterodontus francisci U07624; clearnose skate, Raja eglanteria U75769; channel

catfish, Ictalurus punctatus U39193; Atlantic cod, Gadus morhua AJ133848; Norway rat BC08821; and human, M12886.

349� 2012



<constant Ig domainNile tilapia -DREAIEPQELKILGPSEHECRNSKDKK-RKKTLVCVASGFYPDHVKMYWYLN-------Gilthead seabream GKDDKITPPTVKVLEPSEKECRNKVEKEKRKKTLLCVISRFYPDHVNVTWKIN-------Bicolor damselfish EKGQPVTSPTVKILPPSANECRNKKDDI-RKKTLVCVASGFYPDHVSVSWEKN-------Japanese flounder EPGQAVKSPKVKVFRPSSKECRNPIDNE-REKTLVCVASDFYPDHVSVYWQIIQLNVTSGAtlantic cod EPGCIVSPPTVVVLPPSEKECRDRKEQL--KKTLVCVASGFYPDHVGVSWTVN-------Channel catfish DPDMKLQAPTVTVLNVSEKEVCTKENVT-----LVCVAKGFYPDHVKVYWTVD-------Atlantic salmon DPNIKVTEPTVEVLAPSAKECKDRNKKK--KKTLVCVATRFYPDHVTVFWQVN-------Rainbow trout DPNIKVTEPTVKVLAPSAKKCEDRNKKK--KKTLVCVATRFYPDHVTVFWQVN-------Clearnose skate DPKFKLRPPQVTILQPSDREIKNKGKAT-----VVCLITDFYPDNIKIRWIFDD------Horn shark GENDTIRPAKVTVFEPSPEEIREKKKAT-----VVCLVSDFYPDNIKIHWLVDG------Norway rat EDLKTVTPPKVSLFEPSEAEIADKQKAT-----LVCLARGFFPDHVELSWWVN-------Human EDLKNVFPPEVAVFEPSEAEISHTQKAT-----LVCLATGFYPDHVELSWWVN------- : : :: * : . ::*: *:**:: : *

Nile tilapia -----EKTVTDGVATDEAAREEKDENDNTVYKITSRLRVYAKQWENPKNTFKCTVKFFDGGilthead seabream -----NEEMSKGVATDNMPAQPND---GKFYKITSRLKVDANKWFDPENEFKCIASFFNGBicolor damselfish -----GNGVKDGVATDSAAKRTP----DKTYRITSRLRVSADDYNKLGNTFKCIVSFYNGJapanese flounder VNVIRGENVTRGVTTDEAALRK-----DKVYTITSRLKVSAEDWYKPEWNFECIVRFFNGAtlantic cod -----GQSVIKGVASDHPALRVDD-----KYQITSRLRVEARKWYTGGNIFTCNVSYFNGChannel catfish -----EVNRTIDVSTDEAAVQGSD----KYYTISSRLNIDYKTEWTRGKTFTCIVNFFNGAtlantic salmon -----NVNRTEGAGTDNKALWDKD----SLYSITSRLRVPAKDWHNPDNKFTCIVSFYNGRainbow trout -----NVNRTEGAGTDNRALWDKD----GLYSITSRLRVPANEWHKPENRFTCIVSFYDGClearnose skate ---VVQDKDSDNIHTDASSQSEDE---GMTFSISSRFRLDARDYAKTEK-IVCEVDHYRNHorn shark ---KEKDANDTNIHTDLNAILSKE---NTSYSISSRLRFDALDWARSKN-VECRVDLYTNNorway rat -----GKEIRNGVSTDPQAYKES---NNITYCLSSRLRVSAPFWHNPRNHFRCQVQFYGLHuman -----GKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGL . :* . : ::**:.. . * . :

>< CPS >< TM Nile tilapia KNYKYNSTDTKG--------VTAETT--SMSREKYLTTTQNFKVVYSVLTVKSCAYGAFVGilthead seabream TGTTYHENGTRG--------IEAPKTGQNITTEAYLKRSQTAKLSYGVLIIKGCVYGAFVBicolor damselfish TENVLRHASIDS--------IRGKSE-GGMTKEKYLKHLQNAKLSYGVLIVKSCIYGAFIJapanese flounder THDTDYNDSISG--------EQGPD---ILTREKYLRITRQAKLSYSVLIIKSSVYGAFVAtlantic cod NDTIYTSAEVYGGG----DVRWIKTEPDGETREEFVKVTQTAKLSYIVMIVKNIVYGVFVChannel_catfish TG-INYKDSITG----------PKLTIDEDNYETYVRSVKTTMLRYGMFVAKSIAYGIFIAtlantic salmon NGDINVNDTISG--------DLQGQSGGEITTDYYVKSTQTAKLAYSIFIAKSTFYGLVVRainbow trout TDNIRVNDTISG--------DLQGQSGGEITTDYYVKSTQTAKLAYSIFIAKSTFYGLVVClearnose skate GSTPQTEQGTHY----------IKKETCGLSKEAKIQTMETAKLTYLILICKSILYGIIVHorn shark ESVPTTSSSTLA----------VKAEMCGISKEAKIQSMATAKLTYLILICKSIFYTIFINorway rat TEEDNWSEDSPKPVTQNISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVHuman SENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLV . : * :: * * .:

>< CYT > Nile tilapia GFLVWKLQRFGGKQKY--Gilthead seabream MFLVWKLPGSSGKRNN-- Bicolor damselfish GFLVWKLQSSARKYS--- Japanese flounder AFLVWRLQSSAEKQNH-- Atlantic cod TILAWKLGLGRSHATAKK Channel catfish MYIVRRQGFMSK------ Atlantic salmon MVMIWKFQGSSEKQI--- Rainbow trout MVMIWKFQGSSEKQI--- Clearnose skate SVLACKAKTSYNKRFV-- Horn shark STIAWKTKTSYSKRFD-- Norway rat SALVLMAMVKKKNS---- Human SALVLMAMVKRKDSRG-- :

241

161 176

Figure 3 An alignment of the inferred amino acid sequence of the Nile tilapia TCR-b_C region with the TCR-b_C region sequences of

other vertebrates. The regions corresponding to the constant Ig domain, connecting peptide (CPS), transmembrane (TM) domain and

cytoplasmic tail (CYT), are shown according to Hein (1994). Asterisks (*) indicate residues conserved in all sequences, and dots (:) mark

residues conserved in most of the sequences. Hyphens (-) have been introduced to optimize the alignment. The conserved cysteine

residues are indicated by grey shading. Positions 176 and 241 indicate the positions of the cysteines that form intrachain disulphide

bridges. The conserved cysteine residue involved in an interchain disulphide bridge is indicated by the arrow. The conserved amino acid

motif proposed to be important for the interaction of the TCR a and b chains and for surface expression is shown in italics (Arnaud

et al. 1997). The residues of the Nile tilapia TCR b sequence that match the conserved antigen receptor transmembrane are underlined

(Campbell et al. 1994).

350� 2012



relationships of the Nile tilapia with related speciesbased on morphological characters. The first clusterwas comprised of teleost fish, including the Niletilapia and elasmobranchs. The tree clearly showsthat the Nile tilapia TCR b_C region is closelyrelated to that of the Japanese flounder, bicolordamselfish, gilthead seabream and Atlantic cod.The second groups consisted of TCR b sequencesfrom six mammalian vertebrates and an amphibian(axolotl). The third major branch included thechicken and the African clawed frog (Fig. 4).

Expression and tissue distribution of TCR b

The expression of the TCR b mRNA in varioustissues was examined using semi-quantitative RT-PCR (Fig. 5). The PCR products in both untreatedand stimulated fish were identical in size (216 bp).In untreated fish, weak TCR b expression wasdetected only in the spleen and thymus. Interest-ingly, after stimulation of fish with S. agalactiae,strong expression was detected in the brain andPBLs, while moderate expression was observed inthe head kidney. Relatively low expression was alsodetected in the gills. TCR b expression was not

detected in the gonads, heart, intestine, liver,muscle, stomach or trunk kidney. The expressionof b-actin, a housekeeping gene, was detected in alltissues examined, with an amplification product sizeof 592 bp.

The expression of TCR b in various tissues ofNile tilapia was also carried out using quantitativereal-time RT-PCR (Fig. 6). In untreated fish, thehighest expression level was observed in the thymus,followed by the intestine and spleen; lower TCR blevels were observed in the brain, PBLs and headkidney. Interestingly, intraperitoneal injection (IP)of S. agalactiae induced a significant increase(P < 0.05) in the expression of TCR b in allexamined tissues except for the liver. Relative to thecontrol, the highest increase in TCR b expressionwas detected in the brain (7.16-fold) followed bythe PBLs (4.78-fold), spleen (3.82-fold), headkidney (2.53-fold), intestine (1.87-fold) and thy-mus (1.65-fold). The lowest level of expression wasdetected in the liver, which was used as thecalibrator in this experiment.

Effects of A. hydrophila and ConA on theexpression of TCR b mRNA in vitro

The relative expression of TCR b in PBLs followingexposure to A. hydrophila or ConA was determinedusing quantitative real-time RT-PCR (Fig. 7).From 0 to 24 h, the expression levels of TCR bof in all treated PBLs were similar and notsignificantly different from that of the controlgroup. Forty-eight hours after stimulation withConA, the expression in PBLs was up-regulatedapproximately 3.4-fold, a statistically significantincrease when compared with the control cells orPBLs incubated with A. hydrophila. At 72 h afterstimulation with A. hydrophila, the expression wassignificantly greater than that of the control(approximately 2.30-fold), but was not differentfrom the expression observed in ConA-stimulatedcells. The expression in PBLs stimulated with ConAwas lower at 72 h than at 48 h, and this expressionlevel was not significantly different from theexpression level in the control group or theA. hydrophila–treated group (P > 0.05).

Effects of S. agalactiae on the expression of TCRb mRNA in vivo

The expression level of the TCR b chain in thespleen of Nile tilapia was determined following

Table 2 Amino acid and nucleotide sequence homology

between the Nile tilapia T-cell receptor (TCR) b and other

known TCR b sequences

Name

Accession

number

Identity (%)

Similarity

(%)

Amino

acid Nucleotide

Teleost fish

Bicolor damselfish AF324820 45.6 63.1 58.9

Gilthead seabream AM490438 46.8 62.0 63.7

Atlantic cod AJ133848 42.6 53.5 60.5

Japanese flounder AB053427 37.9 60.1 58.1

Atlantic salmon X97435 43.7 59.1 60.5

Rainbow trout U18122 42.1 58.4 58.9

Channel catfish U39193 38.9 58.6 53.2

Cartilages fish

Clearnose skate U75769 25.0 49.7 50.0

Horn shark U07624 34.4 51.3 54.0

Mammals

Human M12886 37.6 49.8 61.2

House mouse M11456 36.8 51.7 56.8

Norway rat BC088211 36.8 51.6 63.5

Dog D16409 36.8 48.5 59.7

Rabbit M13895 39.1 51.0 62.8

Sheep M94143 39.3 45.1 62.3

Avain

Chicken M37800 38.1 49.9 57.3

Amphibians

African clawed frog U60424 24.6 50.7 45.2

Reptile

Axolotl X70168 36.0 50.3 54.8

351� 2012



experimental inoculation of fish with S. agalactiaeusing real-time RT-PCR (Fig. 8). In fish injectedwith 107 cfu mL)1 of S. agalactiae, the expressionof TCR b was significantly higher than that ofcontrol fish and fish injected with 109 cfu mL)1 ofS. agalactiae (P < 0.05) 1 and 3 h after injection.Six and 12 h post-injection, the expression ofTCR b among the experimental groups wassimilar and no significant differences were

observed. Surprisingly, 24 and 48 h post-injection,the expression of TCR b in fish inoculated with107 cfu mL)1 of S. agalactiae was 2.65- and 7.83-fold greater than in control fish and 6.45- and5.47-fold greater than in fish injected with109 cfu mL)1 of S. agalactiae, respectively. Incontrast, at 72 and 96 h post-inoculation, nosignificant differences among the experimental fishgroups were observed and TCR b expression in

Bicolor damselfishJapanese flounder

Nile tilapia Gilthead seabream

Atlantic cod Atlantic salmon

Rainbow Trout Channel catfish

Clearnose skate Horn shark

AxolotlRabbitHouse mouseNorway rat

DogHumanSheep

African clawed frogChicken

99

100

94

4632

39

99

56

50

31

90

27

29

221428

0.1

Figure 4 Phylogenetic analysis of the amino

acid sequences of the TCR-b_C regions of

Nile tilapia and other organisms. The

numbers indicate the bootstrap per cent

values from 1000 replicates. The bottom

scale refers to the per cent sequence

divergence.

A-1

A-2

BR PBLsGI GO HA MU TKLIINHK SP ST TH

216 bp

M

592 bp

B-2 216 bp

BR PBLsGI GO HA MU TKLIINHK SP ST THM

B-1592 bp

Figure 5 The expression of the TCR b chain in various tissues of Nile tilapia injected with 0.85% NaCl (A-2) or Streptococcus agalactiae(B-2) detected using semi-quantitative RT-PCR. The expression in the brain (BR), gills (GI), gonads (GO), heart (HA), head kidney

(HK), intestine (IN), liver (LI), muscle (MU), peripheral blood leucocytes, spleen (SP), stomach (ST), thymus (TH) and trunk kidney

(TK) was examined. The expression of b-actin in each fish (A-1, B-1) was used as an internal control. Lane M is the 100-bp DNA

marker.

352� 2012



fish injected with 107 cfu mL)1 of S. agalactiaedecreased to the normal level.

Discussion

This study aimed to clone and characterize thecomplete cDNA sequence of the Nile tilapia TCR bchain and to analyse the tissue distribution usingsemi-quantitative and quantitative RT-PCR. Inaddition, the changes in the transcription of the

TCR b chain following stimulation with viablebacterial pathogens (A. hydrophila and S. agalactiae)and with the T-cell-specific mitogen ConA weredetermined both in vitro and in vivo. The sequenc-ing of the 5¢ RACE product demonstrated that thenucleotide sequence of the TCR b cDNA was574 bp long. It was very difficult to amplify thePCR fragment that contained the 5¢ portion of theTCR b sequence, suggesting that the Nile tilapiaTCR b_C region may be highly polymorphic, asobserved in bicolor damselfish (Kamper & McKin-ney 2002). Moreover, the resulting 5¢ RACEproduct appeared as a thin band in the gel,suggesting that the expression of TCR b is low inhealthy fish. This hypothesis is supported by theresults of the normal tissue distribution analysisusing semi-quantitative RT-PCR.

The results of the current study show that theNile tilapia TCR b contains V and C domains,similar to the TCR bs of other animals. The Vdomain is composed of four FRs and three CDRs,and the Ig, CPS, CYT and TM regions could beidentified within the C domain. The V domain alsocontains the conserved J motif (Phe-Gly-X-Gly) inFR4, similar to all other reported fish TCR bmolecules. This motif is structurally important forstabilizing the conformation of the V region.CDR3, or the hypervariable loop, is located at theV-(D)-J junction of the TCR b that contains aglycine (G) residue. This residue, a typical feature ofTCR b, TCR d and IgH chain V region sequences,provides polypeptide flexibility (McCormack et al.1991). Generally, the CDR3 region encodes aglycine-containing b-turn, and such a turn mayserve to properly position the amino acid sidechains of the hypervariable TCR b chain loop withrespect to the antigen-binding groove of the MHCmolecules (McCormack et al. 1991). However, theCDR3 of the Nile tilapia TCR b chain does notcontain a proline residue (P), which has beenobserved in Japanese flounder (Nam, Hirono &Aoki 2003). A proline residue in this region hasbeen proposed to have the opposite effect andreduce flexibility. The V and C domains contain atotal of four cysteines (Cys39, Cys107, Cys162 andCys227) that are involved in forming the intrachaindisulphide bridges that have also been found to beconserved in the TCR b chain of other species.However, the Nile tilapia TCR b sequence does notcontain the cysteine residue that is involved in theinterchain disulphide bridge that covalently linksthe TCR a and b chains in mammalian T cells.

0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00

40.00

45.00

BrainkidneyHead Intestine Liver PBLs Spleen Thymus

Tissue

Rel

ativ

e ex

pres

sion

rat

io

NormalStimulated

*

*

*

**

*

Figure 6 The relative expression of TCR b (mean � SD) in

different tissues following the injection of 0.85% NaCl or

108 cfu mL)1 of Streptococcus agalactiae detected using quanti-

tative RT-PCR. The TCR b expression level is expressed as the

ratio relative to the b-actin level in the same sample. The lowest

tissue expression level (liver) was used as the calibrator.

Significant differences (P < 0.05) are indicated with an asterisk.

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

0 6 h 12 h 24 h 48 h 72 hHours after stimulation

Rel

ativ

e ex

pres

sion

rat

io

Control A. hydrophilaConA

b*

b

aaa a

aa

aa a

aaa

ab

Figure 7 The relative expression of the TCR b chain in Nile

tilapia peripheral blood leucocytes stimulated with 105 cfu mL)1

of Aeromonas hydrophila or 10 lg mL)1 of concanavalin A

detected using quantitative RT-PCR. The TCR b expression

level is expressed as the ratio relative to the b-actin level in the

same sample. The data are presented as the mean � SD. The

different letters above each bar indicate a significant difference

between the samples at the same time point (P < 0.05), and the

asterisk indicates P < 0.01.

353� 2012



Although the absence of this cysteine has also beenreported in several teleost species (Partula et al.1995; Hordvik et al. 1996; Wilson et al. 1998;Wermenstam & Pilstrom 2001; Kamper & Mc-Kinney 2002; Nam et al. 2003; Randelli et al.2008), it is present in cartilaginous fish (Rast et al.1995, 1997). Importantly, it has been recentlydemonstrated that the interchain disulphide bondbetween the TCR a and b chains may not berequired for membrane expression or signal trans-duction through TCR ab–CD3 complexes (Arnaudet al. 1997). A free cysteine (Cys162) residue thatmay be used for interchain disulphide bonds in fishwas also detected in the Nile tilapia TCR b chainand this residue is highly conserved in the TCR bsequences of other teleost fish (Partula 1999).Furthermore, it has been proposed that a conservedamino acid motif, YCLSSRLRVSA, is one area thatis important for the interaction between the TCR aand b chains and for surface expression. ReducedTCR ab dimerization was found when the serine(S) residue (underlined in the motif) was mutated(Arnaud et al. 1997). The Ser-Arg-Leu sequence(italicized in the motif) in this motif is strictlyconserved in all vertebrates, including the Niletilapia. This core sequence is probably involved inthe formation of the TCR ab heterodimer. TheNile tilapia TCR b, like that of other vertebrates,contains a similar motif (YKITSRLRVYA), whichshould be able to direct TCR dimerization andsurface expression, as observed in humans (Arnaudet al. 1997). Another conserved motif, known as

the CART, is present in the TCR b transmembranedomain in other vertebrates. This CART motif isthought to assume an alpha helical conformationand function as a structural unit that plays a role inthe assembly and/or the signalling properties oflymphocyte antigen receptors (Campbell et al.1994). Moreover, we also found an importantresidue for TCR ab cell surface expression, Lys276,in the Nile tilapia TCR b. This residue is located inthe transmembrane region and is important in theassembly and/or stabilization of the TCR-CD3complex (Alcover et al. 1990; Manolios, Kemp &Li 1994).

A comparison of sequences of the TCR b_Cregion among vertebrates demonstrated that theNile tilapia TCR b has high amino acid andnucleotide sequence identity with the TCR bs ofteleost fish, especially the gilthead seabream (46.8%and 62.0%) and bicolor damselfish (45.6% and63.1%). A phylogenetic tree illustrated that the Niletilapia TCR b gene is closely related to that of otherteleosts in the same subcluster. Among fish TCR bs,the Nile tilapia TCR b is closely related to that ofthe bicolor damselfish; both of these fish belong tothe order Perciformes (Nelson 1984).

The results from semi-quantitative RT-PCRdemonstrate that the Nile tilapia TCR b isnormally expressed in the spleen and thymus ofhealthy fish. These results have also been observedin Atlantic cod, rainbow trout, sea bass, Dicentra-chus labrax (L.), and gilthead seabream (Wermen-stam & Pilstrom 2001; Bernard et al. 2006;

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

0 1 h 3 h 6 h 12 h 24 h 48 h 72 h 96 hHours after injection

Rel

ativ

e ex

pres

sion

rat

io

Control 107 cfu mL–1

109 cfu mL–1b*

b*

a

aa

aa

a

a aaa

a

a aa a

a

a aa

a

bb

Figure 8 Changes in splenic TCR b expression in Nile tilapia following inoculation with Streptococcus agalactiae. The TCR bexpression level is expressed as the ratio relative to the b-actin level in the same sample. The data are presented as the mean � SD. The

different letters above each bar indicate significant differences between the samples at the same time point (P < 0.05), whereas the

asterisks indicate P < 0.01.

354� 2012



Picchietti et al. 2008; Randelli et al. 2008). Theseresults confirm that the thymus is the primarylymphoid organ in fish and is responsible for theproduction of T-cell populations (Rombout et al.2005). Additionally, the spleen of teleosts seems torepresent a major secondary lymphoid organ in fishand acts in the initiation of the adaptive immuneresponse (Chaves-Pozo et al. 2005). Importantly,the IP of fish with S. agalactiae resulted in the up-regulation of TCR b very efficiently in the brain,gills, PBLs, spleen and thymus. Most of theseorgans are known to be involved in the immuno-logical responses against invading bacteria in tele-osts. Likewise, quantitative RT-PCR analysisdemonstrated a high expression level in the thymus,similar to that reported for the gilthead seabream(Randelli et al. 2008), European sea bass (Romanoet al. 2007), Atlantic cod (Wermenstam & Pilstrom2001), rainbow trout (Partula et al. 1995) andclearnose skate (Miracle et al. 2001). Because thethymus is the primary lymphoid organ responsiblefor T-cell maturation and the major site of T-celldevelopment (Bowden, Cook & Rombout 2005),high expression in this tissue is expected. In thecyprinid and sea bass, T cells appear to be selectedin the thymus much earlier than the first detectionof T-cell-dependent antibody responses (Romboutet al. 2005). In addition, high expression of TCR bwas detected in intestine of the Nile tilapia. Thischaracteristic has also been observed in the giltheadseabream (Randelli et al. 2008), European sea bass(Romano et al. 2007) and rainbow trout (Bernardet al. 2006). However, no expression of TCR b inthe intestine was detected in the Atlantic cod(Wermenstam & Pilstrom 2001), which was similarto that of RT-PCR analysis in this study, suggestingthat TCR b may be inconsistently expressed inteleost intestine. Previous studies have shown that Tcells predominantly reside in the gut-associatedlymphoid tissue of the sea bass (Picchietti et al.1997; Scapigliati et al. 2000). Romano et al. (2007)demonstrated that TCR b-positive cells mainlyaccumulate in the mucosa of the midgut and that allT cells resident there express TCR b. It has alsobeen suggested that the level of TCR b-positive cellsmay play a special role in antigen-specific cellularimmunity in this intestinal segment (Romano et al.2007).

It is interesting in that both the semi-quantitativeand quantitative RT-PCR techniques revealed thesignificantly increased expression level of TCR bbecause it was detected in the brain and other

examined organs of S. agalactiae-infected fish com-pared with the control. This finding could bepossibly associated with infection with Streptococcus,including S. agalactiae (also known as group Bstreptococcus; GBS), which is a major cause ofbacterial sepsis and meningitis. In addition, Evans,Shoemaker & Klesius (2001) stated that thepresence of S. iniae in the cerebellum may be animportant component responsible for behaviouralabnormalities and disease initiation and thatS. iniae is able to penetrate the blood–brain barrier.Moreover, the recovery of S. agalactiae from brainand eye tissue of mullet and seabream reared in netpens without disease signs suggests that these fishcould be carriers (Evans et al. 2002). Accordingly,in mammals, T lymphocytes can enter the centralnervous system to search for danger signals (Pede-monte et al. 2006), and a similar phenomenon mayoccur in teleosts.

We further conducted an analysis of the regula-tion of the Nile tilapia TCR b chain in response toA. hydrophila (a Gram-negative, pathogenic bacte-ria) and ConA, a T-cell mitogen. Gene expressionwas analyed in Nile tilapia PBLs following stimu-lation with either viable A. hydrophila or ConA.Stimulation with A. hydrophila had only a minimaleffect on the expression of TCR b relative to thecontrol. Previous studies have shown that Salmo-nella enterica serovar typhimurium, and S. typhimu-rium, Gram-negative bacteria, showed an inhibitoryeffect on mice T cells, blocking their proliferationand development of T-cell-mediated adaptiveimmunity to this bacteria (van der Velden, Copass& Starnbach 2005). Additionally, T cells culturedin the presence of S. typhimurium down-modulateexpression of the T-cell b chain, thus targeting thefirst step in T-cell clonal expansion (van der Velden,Dougherty & Starnbach 2008). These results maysupport the low expression of TCR b responses toA. hydrophila observed in this study. Furthermore,the outer layer of the Gram-negative bacterial cellwall is composed of lipopolysaccharide (LPS).Because LPS is well known to be involved in theimmune response by triggering macrophage and Blymphocyte activation (Kai 1998), it is not surpris-ing that, following stimulation with A. hydrophila, alow TCR b expression level was clearly exhibited.

Based on the current study, ConA can beconsidered a stimulator of fish T cells, as in highervertebrates. In this study, there was significantlyhigher expression of the TCR b chain (P < 0.01)48 h post-stimulation. This result strongly suggests

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that Nile tilapia lymphocytes, like lymphocytesfrom other teleosts, are capable of responding toConA. This finding supports the data of Agbede,Adedeji & Adeyemo (2005), who found that Niletilapia T-cell-like lymphocytes proliferated stronglyin response to ConA. This result is similar to thoseof previous reports that were conducted in trout(Estepa & Coll 1992), red drum, Sciaenops ocellatus(L.) (Lopresto, Schwarz & Burnett 1995), sea bass(Galeotti, Volpatti & Rusvai 1999) and spottedwolfish, Anarhichas minor (Olafsen) (Espelid, Steiro& Johansen 2003). Previous studies have demon-strated that ConA is capable of triggering positiveselection in mature T cells by cross-linking the TCRwith high avidity (Lovatt et al. 2000). Moreover,the activation and proliferation of mature T cells byConA are known to mimic the effects of highavidity TCR ligation by activated peripheral T cells,and these processes trigger distinctively differentphysiological changes in developing thymocytes(Pongracz et al. 2003). This finding suggests thatfish lymphocyte subpopulations (T cells) can pro-liferate upon stimulation with specific mitogen,similar to cells isolated from mammals or otherhigher vertebrates.

To date, the relationships between the Niletilapia immune response and S. agalactiae infectionhave not been fully elucidated. In this study, theimmune response against extracellular bacteria wasinvestigated by measuring the change in theexpression of TCR b at the mRNA level. Thespleen was sampled because it is considered to be animportant organ for antigen presentation and seemsto represent a major secondary lymphoid organ infish during bacterial infections. In this study,increased expression of TCR b mRNA was detectedafter injecting fish with viable S. agalactiae. Thisresult proves that stimulation causes increasedexpression of the TCR b chain. The results fromthis study demonstrate that the TCR b transcriptwas slightly up-regulated 1 h after injecting fishwith S. agalactiae. This phenomenon most likelyinvolves the ability of the bacteria to cause acuteinfection, evade innate immunity and engage theadaptive arm of the immune response (Neely,Pfeifer & Caparon 2002). Furthermore, 24 and48 h after the fish were injected with 107 cfu mL)1

of viable S. agalactiae, a dramatic up-regulation inthe expression of TCR b was observed (P < 0.01),suggesting that this dose may be suitable tostimulate TCR b expression or T-cell activity. Incontrast, fish injected with 109 cfu mL)1 of viable

S. agalactiae demonstrated no statistically signifi-cant increase in the expression of TCR b. Thisresult may be the result of the high concentrationof viable S. agalactiae used for stimulation, asAmal et al. (2008) demonstrated that the LD50

of S. agalactiae in Nile tilapia was 3.0 ·107 cfu mL)1.

Acknowledgements

This study is part of a doctoral thesis carried out atthe Asian Institute of Technology (AIT), Thailand.This work was supported by Kasetsart UniversityResearch and Development Institute (KURDI). Wethank Assoc. Prof. Dr Nontawith Areechon foraccess to laboratory facilities.

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Received: 23 October 2010Revision received: 30 May 2011Accepted: 3 June 2011

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