molecular cloning and characterization of toll-like receptor 9 in japanese

8/14/2019 Molecular Cloning and Characterization of Toll-Like Receptor 9 in Japanese

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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
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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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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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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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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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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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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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molecular cloning and characterization of toll-like receptor 9 in japanese

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