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8/14/2019 Molecular Cloning and Characterization of Toll-Like Receptor 9 in Japanese
1/24
Molecular cloning and characterization of Toll-likereceptor 9 in Japanese flounder, Paralichthysolivaceus
Tomokazu Takanoa, Hidehiro Kondoa, Ikuo Hironoa, Makoto Endoa, Tatsuo Saito-
Takib and Takashi Aokia, ,
aLaboratory of Genome Science, Graduate School of Marine Science and Technology,
Tokyo University of Marine Science and Technology, Konan 4-5-7, Minato, Tokyo 108-
8477, Japan
bDepartment of Microbiology, School of Allied Health Science, Kitasato University,
Kitasato 1-15-1, Sagamihara-city, Kanagawa 228-8555, Japan
Received 4 August 2006;
revised 5 October 2006;
accepted 10 October 2006.
Available online 21 November 2006.
Abstract
Toll-like receptor (TLR) 9 cDNA and gene were cloned from Japanese flounder,
Paralichthys olivaceus. The Japanese flounder TLR9 cDNA encodes 1065 amino acids.
The leucine-rich domain (LRD) and the Toll/interleukin-1 receptor (TIR) domain found
in other vertebrate TLR9s were conserved in Japanese flounder TLR9. The gene is
composed of three exons and two introns. The Japanese flounder tumor necrosis factor
(TNF) gene promoter was activated in Japanese flounder TLR9-transformed hirame
natural embryo (HINAE) cells upon stimulation with synthesized CpG
oligodeoxynucleotide (ODN), but not by stimulation with GpC ODN. The Japanese
flounder TLR9 gene was highly expressed in epithelial and lymphoid organs, such as the
gills, intestines, kidney, spleen and stomach in an apparently healthy fish. The mRNA
http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T9R-4MD460X-2&_user=4429&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_searchStrId=1142572870&_rerunOrigin=scholar.google&_acct=C000059602&_version=1&_urlVersion=0&_userid=4429&md5=df6dee87e18d496f5b6e8f1f6c842fa4#aff1%23aff1http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T9R-4MD460X-2&_user=4429&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_searchStrId=1142572870&_rerunOrigin=scholar.google&_acct=C000059602&_version=1&_urlVersion=0&_userid=4429&md5=df6dee87e18d496f5b6e8f1f6c842fa4#aff1%23aff1http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T9R-4MD460X-2&_user=4429&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_searchStrId=1142572870&_rerunOrigin=scholar.google&_acct=C000059602&_version=1&_urlVersion=0&_userid=4429&md5=df6dee87e18d496f5b6e8f1f6c842fa4#aff1%23aff1http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T9R-4MD460X-2&_user=4429&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_searchStrId=1142572870&_rerunOrigin=scholar.google&_acct=C000059602&_version=1&_urlVersion=0&_userid=4429&md5=df6dee87e18d496f5b6e8f1f6c842fa4#aff1%23aff1http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T9R-4MD460X-2&_user=4429&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_searchStrId=1142572870&_rerunOrigin=scholar.google&_acct=C000059602&_version=1&_urlVersion=0&_userid=4429&md5=df6dee87e18d496f5b6e8f1f6c842fa4#aff2%23aff2http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T9R-4MD460X-2&_user=4429&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_searchStrId=1142572870&_rerunOrigin=scholar.google&_acct=C000059602&_version=1&_urlVersion=0&_userid=4429&md5=df6dee87e18d496f5b6e8f1f6c842fa4#aff1%23aff1http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T9R-4MD460X-2&_user=4429&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_searchStrId=1142572870&_rerunOrigin=scholar.google&_acct=C000059602&_version=1&_urlVersion=0&_userid=4429&md5=df6dee87e18d496f5b6e8f1f6c842fa4#aff1%23aff1http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T9R-4MD460X-2&_user=4429&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_searchStrId=1142572870&_rerunOrigin=scholar.google&_acct=C000059602&_version=1&_urlVersion=0&_userid=4429&md5=df6dee87e18d496f5b6e8f1f6c842fa4#cor1%23cor1mailto:[email protected]:[email protected]://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T9R-4MD460X-2&_user=4429&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_searchStrId=1142572870&_rerunOrigin=scholar.google&_acct=C000059602&_version=1&_urlVersion=0&_userid=4429&md5=df6dee87e18d496f5b6e8f1f6c842fa4#cor1%23cor1http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T9R-4MD460X-2&_user=4429&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_searchStrId=1142572870&_rerunOrigin=scholar.google&_acct=C000059602&_version=1&_urlVersion=0&_userid=4429&md5=df6dee87e18d496f5b6e8f1f6c842fa4#aff1%23aff1http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T9R-4MD460X-2&_user=4429&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_searchStrId=1142572870&_rerunOrigin=scholar.google&_acct=C000059602&_version=1&_urlVersion=0&_userid=4429&md5=df6dee87e18d496f5b6e8f1f6c842fa4#aff1%23aff1http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T9R-4MD460X-2&_user=4429&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_searchStrId=1142572870&_rerunOrigin=scholar.google&_acct=C000059602&_version=1&_urlVersion=0&_userid=4429&md5=df6dee87e18d496f5b6e8f1f6c842fa4#aff1%23aff1http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T9R-4MD460X-2&_user=4429&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_searchStrId=1142572870&_rerunOrigin=scholar.google&_acct=C000059602&_version=1&_urlVersion=0&_userid=4429&md5=df6dee87e18d496f5b6e8f1f6c842fa4#aff2%23aff2http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T9R-4MD460X-2&_user=4429&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_searchStrId=1142572870&_rerunOrigin=scholar.google&_acct=C000059602&_version=1&_urlVersion=0&_userid=4429&md5=df6dee87e18d496f5b6e8f1f6c842fa4#aff1%23aff1http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T9R-4MD460X-2&_user=4429&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_searchStrId=1142572870&_rerunOrigin=scholar.google&_acct=C000059602&_version=1&_urlVersion=0&_userid=4429&md5=df6dee87e18d496f5b6e8f1f6c842fa4#aff1%23aff18/14/2019 Molecular Cloning and Characterization of Toll-Like Receptor 9 in Japanese
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copy numbers of Japanese flounder TLR9 and its adapter protein, the myeloid
differentiation factor 88 (MYD88) were increased in some organs including blood, gill,
kidney and spleen afterEdwardsiella tarda challenge. Immunohistochemical analysis
revealed that TLR9 and MYD88 were expressed in the same cells of kidney. Few TLR9-
expressing cells were found in gill, kidney and spleen in healthy Japanese flounder, but
many were found in these organs afterE. tarda challenge and were coincident with
lesions that had been colonized by the bacteria.
Keywords: Innate immunity; Japanese flounder; Luciferase; Myeloid differentiation
factor 88; Promoter; Reporter assay; Toll-like receptor; Tumor necrosis factor
Article Outline
1. Introduction
2. Materials and methods
2.1. Cloning of Japanese flounder TLR9 cDNA and gene
2.2. Construction of plasmid vector for reporter assay
2.3. Transfection and luciferase assay
2.4. RT-PCR analysis
2.5. Experimental challenge of E. tarda
2.6. Real-time RT-PCR analysis
2.7. Polyclonal antibody preparation against Japanese flounder TLR9
2.8. Immunohistochemical analysis3. Results
3.1. Japanese flounder TLR9 cDNA and gene
3.2. Activation of Japanese flounder TNF gene promoter by CpG ODN
3.3. Gene expression of Japanese flounder TLR9 and MYD88
3.4. Immunohistochemical analysis of Japanese flounder TLR9
4. Discussion
Acknowledgements
References
1. Introduction
Toll-like receptors (TLRs), which are type I transmembrane receptors, are crucial
molecules for recognizing pathogen-associated molecular patterns (PAMPs) (Takeda and
Akira, 2005). After TLRs recognize the PAMPs, the intracellular domain of the
Toll/interleukin-1 receptor (TIR) domains binds to the TIR domain of adapter molecules
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Molecular Cloning and Characterization of Toll-Like Receptor 9 in Japanese
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including myeloid differentiation factor 88 (MYD88) (Akira and Takeda, 2004). TLR
signalling via MYD88-dependent or -independent pathways results in the activation of
the appropriate immune response (Akira and Takeda, 2004).
The mammalian TLR9 recognizes unmethylated CpG DNA of bacterial genomic DNA
and activates the nuclear factor (NF)-B via a MYD88-dependent pathway (Akira and
Takeda, 2004). Synthetic CpG oligodeoxynucleotides (ODNs) mimic the bacterial CpG
DNA and induce the production of cytokines (Hemmi et al., 2000). The TLR9 gene is
expressed primarily on antigen-presenting cells (APCs), such as dendritic cells (DCs)
(Hornung et al., 2002). In human, TLR9 is retained in the endoplasmic reticulum and is
trafficked to endocytic vesicles for sensing bacterial DNA (Leifer et al., 2004).
Synthetic CpG ODNs also activate innate immune responses of fish. For example,
cytotoxicity of non-specific cytotoxic cells in channel catfish (Ictalurus punctatus)
(Oumouna et al., 2002) and respiratory burst in leukocytes of Japanese flounder
(Paralichthys olivaceus) were modulated by stimulation with CpG ODN (Lee et al.,
2003). The expression of cytokine genes in common carp (Cyprinus carpio L.)
leukocytes (Tassakka et al., 2006), and production of type I IFN-like antiviral activity
from Atlantic salmon (Salmo salarL.) head kidney (HK) leukocytes (Jrgensen et al.,
2003) were also induced by CpG ODNs. TLR genes have been found in several fish
species from genomic DNA and EST analysis (Oshiumi et al., 2003,Stafford et al., 2003,
Jault et al., 2004, Meijer et al., 2004,Hirono et al., 2004,Tsujita et al., 2004,Bilodeau
and Waldbieser, 2005 and Tsoi et al., 2006). Fish TLR9 genes have also been identified
in puffer fish (Takifugu rubripes) and zebrafish (Danio rerio) by in silico screening
(Oshiumi et al., 2003, Jault et al., 2004 and Meijer et al., 2004). Further, we identified
Japanese flounder MYD88 gene and demonstrated the assemblage of MYD88-expressing
cells at the lesion that was formed byEdwardsiella tarda infection (Takano et al., 2006).
However, the role of TLR9 and the functional involvement of TLR9 and MYD88 in fish
are unclear.
In this study, a cDNA and gene encoding the Japanese flounder TLR9 were cloned. We
found that activation of the Japanese flounder tumor necrosis factor (TNF) gene promoter
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Molecular Cloning and Characterization of Toll-Like Receptor 9 in Japanese
4/24
in Japanese flounder TLR9 gene-expressing cells was ligand-dependent. We also
investigated the dynamics of TLR9-expressing cells in several tissues after experimental
challenge withE. tarda.
2. Materials and methods
2.1. Cloning of Japanese flounder TLR9 cDNA and gene
Total RNA was isolated from kidney of Japanese flounder using TRIzol (Invitrogen,
USA). cDNA was synthesized from 1 g of total RNA using SuperScript II reverse
transcriptase (Invitrogen, USA) according to the manufacturer's protocol. Degenerate
primers for Japanese flounder TLR9 designated as TLR9-f and TLR9-r (Table 1) were
designed from conserved regions of published nucleotide sequences of TLR9 genes from
human,Homo sapiens (BC032713), mouse, Mus musculus (AF314224) and puffer fish,
Takifugu rubripes (AC156439). PCR was performed using 1 l of the synthesized cDNA
as template. The PCR conditions were: an initial denaturation step of 2 min at 95 C,
followed by 40 cycles of denaturation at 95 C for 30 s, annealing at 48 C for 30 s and
1 min of extension at 72 C and a final elongation step at 72 C for 5 min to complete the
reaction. The nucleotide sequences of the amplified DNA fragments were determined by
an automated DNA sequencer LC4200 (Li-Cor, USA). The full-length TLR9 cDNA wasdetermined by 3- and 5-RACE using the SMART RACE cDNA amplification kit (BD
Biosciences, USA) according to the manufacturer's protocol with gene-specific primers
(Table 1), and the nucleotide sequence of the full-length cDNA was determined as
described. The structure of the deduced amino acid sequence of the TLR9 was
characterized using the SMART program (http://smart.embl-heidelberg.de/).
Table 1.
PCR primers used in this study
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Primer nameSequence of oligonucleotides (references or accession numbers
and position of the primer in the sequence)
Degenerate primer
TLR9-f 5-CTCTNNGGCTGGGANNTNTGGTA-3
TLR9-r 5-TGTCGAANACCACNAANGCAT-3
TLR9 gene-specific primers for RACE-PCR
jfTLR9(3-RACE) 5-TTTGGGCAGGGCATAAAGGA-3 (AB234023, 27102729)
jfTLR9(5-RACE) 5-TCCTTTATGCCCTGCCCAAA-3 (AB234023, 27102729)
TLR9 gene-specific primers for RT-PCR
jfTLR9RT-f 5-GCTCCTGGATGAAAAGGTGGA-3 (AB234023, 30383058)
jfTLR9RT-r5-TAATTTGAGGTTATCTGACGACAATGC-3 (AB234023,
32013227)
Jf-actinRT-f 5-TTTCCCTCCATTGTTGGTCG-3 (Takano et al., 2004)
Jf-actinRT-r 5-GCGACTCTCAGCTCGTTGTA-3 (Takano et al., 2004)
Primers for construction of TLR9 gene expression vector
jfTLR9(HindIII)-f5-TTTAAGCTTTCATCTGCCTCCTGATTAGAATTAT-3
(AB234023, 3358)
jfTLR9(BamHI)-r5-TTTGGATCCTCAGACAAAACTCTCACTCATGTTGTTGTC-
3 (AB234023, 32313261)
Primers for TNT ptomoter
jfTNF-LUC(KpnI)-f5-GGTACCCGACCTGAAAGAGGCAGTAA-3 (Yazawa et al.,
2005)
jfTNF-LUC(Bg/II)-r5-AGATCTCCAGTCGGTGGACGACTTCT-3 (Yazawa et al.,
2005)
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Molecular Cloning and Characterization of Toll-Like Receptor 9 in Japanese
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Primer nameSequence of oligonucleotides (references or accession numbers
and position of the primer in the sequence)
Primers for real time PCR (for construction of standard curve)
TLR91ong-f 5-GCTCCTGGATGAAAAGGTGGA-3 (AB234023, 30383058)
TLR91ong-r5-TAATTTGAGGTTATCTGACGACAATGC-3 (AB234023,32013227)
MyD881ong-f 5-GTCCTGGAGCCCAACAGAAA-3 (AB241074, 751770)
MyD881ong-r 5-GACGGTATATGAAGCCACAGTTAG-3 (AB241074, 927950)
EF (elongation factor
1 ) long-f
5-ACGTTCTTGATGTTGAAGCCGA-3 (AU090803, 760781)
EFlong-r 5-CCCTGCAGGACGTCTACAAA-3 (AU090803, 930959)
RP (ribosomal
protein L10) long-f
5-GGAATTCAACCCAACTTTCTGACGT-3 (AU050650, 196
230)
RPlong-r 5-TGGTGCGATCAGGTAGCTAC-3 (AU050650, 376395)
For detection of mRNAs
TLR9short-f 5-CGTGCTGGTTCTGTTGGATGAG-3 (AB234023, 30653086)
TLR9short-r 5-AACCTCTTCCTCAGCTGGAGG-3 (AB234023, 31073127)
MyD88short-f 5-CCAGTGGTGTACAAGCCGATG-3 (AB241074, 780800)
MyD88short-r 5-ACACGGTGAGGAAGCGCAAG-3 (AB241074, 821840)
EFshort-f 5-CTCGGGCATAGACTCGTGGT-3 (AU090803, 801820)
EFshort-r 5-CATGGTCGTGACCTTCGCTC-3 (AU090803, 841860)
RPshort-f 5-GCTCCTCTGGTGCAGTTTGTGA-3 (AU050650, 236257)
RPshort-r 5-TGGTGTTTGCTGGCGTCACTCT-3 (AU050650, 324345)
Primers for recombinant TLR9 protein production
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Molecular Cloning and Characterization of Toll-Like Receptor 9 in Japanese
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Primer nameSequence of oligonucleotides (references or accession numbers
and position of the primer in the sequence)
jfTLR9recombinant-f 5-CCCATATGCACCACCACCACCACCACGAGATG
GTTTTCAGAGAGCT-3 (AB234023, 612631)
jfTLR9recombinant-r5-GGGGAATTCTTAGTCAGAGAGGTCCAAACTCT-3
(AB234023, 679698)
The DNA fragment of Japanese flounder TLR9 was used as a DNA probe for screening
of BAC clones from a Japanese flounder genomic BAC library (Katagiri et al., 2000 and
Hirono et al., 2000).
2.2. Construction of plasmid vector for reporter assay
The ORF of Japanese flounder TLR9 was amplified from the head kidney cDNA by the
PCR primer set jfTLR9(HindIII)-f and jfTLR9(BamHI)-r (Table 1). The amplified TLR9
DNA fragments were cloned into pcDNA3.1(+) (Invitrogen, USA), which was
designated as pcDNA-ParoliTLR9.
The 5-flanking region of the Japanese flounder TNF gene containing 1984 bp (1782 to
+202 from the transcription initiation site) (Yazawa et al., 2005) was also amplified using
a set of PCR primers (Table 1) from a Japanese flounder BAC clone. The fragment was
inserted betweenKpnI andBglII site of pGL3-Basic (Promega, USA), and designated
pGL-ParoliTNF. This plasmid was used as a reporter for TLR9 signal activation. The
phRL-SV40 vector (Promega, USA) was used as an internal control.
2.3. Transfection and luciferase assay
Hirame natural embryo (HINAE) cells (Kasai and Yoshimizu, 2001) were cultured at
20 C in Leibovitz's L-15 medium (Gibco-BRL, USA), supplemented with 20% fetal
bovine serum (FBS) (JRH biosciences, USA), 100 IU/ml penicillin G and 100 g/ml
streptomycin (Gibco-BRL, USA).
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md5=87fd24f9d537fdfc50ae838eaacd166d#tbl1http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T9R-4MD460X-2&_user=4429&_coverDate=03%2F31%2F2007&_rdoc=1&_fmt=full&_orig=search&_cdi=5121&_sort=d&_docanchor=&view=c&_searchStrId=1142572870&_rerunOrigin=scholar.google&_acct=C000059602&_version=1&_urlVersion=0&_userid=4429&md5=87fd24f9d537fdfc50ae838eaacd166d#bib138/14/2019 Molecular Cloning and Characterization of Toll-Like Receptor 9 in Japanese
8/24
HINAE cells (1 105 cell) seeded onto a 24-well cell culture microplate (Corning, USA)
were transiently transfected with 1 g of pGL-ParoliTNF, 0.02 g of phRL-SV40 and
1 g of pGL3-Basic or pcDNA-ParoliTLR9 with Lipofectamine 2000 (Invitrogen,
USA) according to the manufacturer's protocol.
The transformants were washed with PBS at 36 h after transfection, then added 0.2 ml of
culture medium containing synthesized phosphodiesters (Hokkaido System Science,
Japan) of CpG ODN-1668: 5-TCCATGACGTTCCTGATGCT-3 (1, 10 g/ml) or GpC
ODN-1720; 5-TCCATGAGCTTCCTGATGCT-3 (1, 10 g/ml) and cultured for another
36 h.
Luciferase luminescence assays were performed using the Dual-Luciferase reporter
assay system (Promega, USA) with a LUMI-COUNTER 2500 (Microtec Nition, Japan).
Relative light units were calculated using phRL-SV40 as standard. The test was done in
triplicate and relative light units were expressed with standards error of the mean.
Significant differences between relative light units were analyzed by Student's t-test.
2.4. RT-PCR analysis
Peripheral blood, brain, gill, heart, intestine, kidney, liver, muscle, skin, spleen and
stomach were extirpated from a Japanese flounder juvenile (body weight of 8.4 g), and
total RNAs were extracted using TRIzol (Invitrogen, USA). One micrograms of total
RNAs were used for cDNA synthesis in a 20 l reaction mixture as described above.
One microlitre of the reverse-transcribed product was used in a 50 l PCR reaction
mixture. PCR was performed with gene-specific primers for TLR9 and beta-actin
(ACTB) (Takano et al., 2004) (Table 1). The PCR conditions were: an initial denaturation
step of 2 min at 95 C, followed by 30 cycles of denaturation at 95 C for 30 s, annealing
at 58 C for 30 s and 1 min of extension at 72 C, a final elongation step at 72 C for
5 min completed the reaction. The amplified PCR product (5 l) was electrophoresed on
a 0.8% agarose gel.
2.5. Experimental challenge of E. tarda
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