J Vet Sci.  2015 Mar;16(1):37-46. 10.4142/jvs.2015.16.1.37.

Cloning, expression and functional analysis of the duck Toll-like receptor 5 (TLR5) gene

• Cheng Y
• Sun Y
• Wang H
• Shi S
• Yan Y
• Li J
• Ding C
• Sun J

• KMID: 2160819
• DOI: http://doi.org/10.4142/jvs.2015.16.1.37

Abstract

Toll-like receptor 5 (TLR5) is responsible for the recognition of bacterial flagellin in vertebrates. In the present study, the first TLR5 gene in duck was cloned. The open reading frame (ORF) of duck TLR5 (dTLR5) cDNA is 2580 bp and encodes a polypeptide of 859 amino acids. We also cloned partial sequences of myeloid differentiation factor 88, 2'-5'-oligoadenylate synthetase (OAS), and myxovirus resistance (Mx) genes from duck. dTLR5 mRNA was highly expressed in the bursa of Fabricius, spleen, trachea, lung, jejunum, rectum, and skin; moderately expressed in the muscular and glandular tissues, duodenum, ileum, caecum, and pancreas; and minimally expressed in the heart, liver, kidney, and muscle. DF-1 or HeLa cells transfected with DNA constructs encoding dTLR5 can activate NF-kappaB leading to the activation of interleukin-6 (IL-6) promoter. When we challenged ducks with a Herts33 Newcastle disease virus (NDV), mRNA transcription of the antiviral molecules Mx, Double stranded RNA activated protein kinase (PKR), and OAS was up-regulated in the liver, lung, and spleen 1 and 2 days post-inoculation.

Keyword

duck; innate immune response; myeloid differentiation factor 88; Newcastle disease virus; Toll-like receptor 5

MeSH Terms

2',5'-Oligoadenylate Synthetase/genetics/metabolism
Animals
Cell Line
*Cloning, Molecular
Ducks
Gene Expression Regulation/*physiology
Humans
Immunity, Innate
Myeloid Differentiation Factor 88/genetics/metabolism
Myxovirus Resistance Proteins/genetics/metabolism
Newcastle Disease/metabolism
Newcastle disease virus/classification
RNA, Messenger/genetics/metabolism
Species Specificity
Toll-Like Receptor 5/genetics/*metabolism
2',5'-Oligoadenylate Synthetase
Myeloid Differentiation Factor 88
Myxovirus Resistance Proteins
RNA, Messenger
Toll-Like Receptor 5

Figure

• 

  Fig. 1 Amino acid alignment of Pekin duck (dTLR5, accession no. KF717594), goose (GoTLR5, accession no. AEP71332.1), chicken (ChTLR5, accession no. ACR26276.1), and human (HuTLR5, accession no. AAI09119.1) TLR5. Alignment was performed using Clustal X software and edited with Boxshade. Black shading indicates amino acid identity and gray shading indicates similarity (50% threshold).

• 

  Fig. 2 Phylogeneticanalysis of TLR5. Aneighbor-joining (NJ) tree was constructed using MegAlign. The sequences were derived from the predicted amino acidsequences of thePekin duck TLR5 (KF717594) and GenBank entries for the domestic goose (Anser anser, accession no. AEP71332.1), chicken (Gallus gallus domesticus, accession number. ACR26276.1), cattle (Bos Taurus, accession no. AFR42399.1), chimpanzee (Pan troglodytes, accession no. NP_001123934.1), cat (Felis catus, accession no. XP_004001379.1), gray wolf (Canis lupus, accession no. NP_001184105.1), human (Homo sapiens, accession no. AAI09119.1), macaque (Maca camulatta, accession no. NP_001123901.1), mouse (Mus musculus, accession no. NP_058624.2), pig (Sus scrofa, accession no. AGT79978.1), rabbit (Oryctolagus cuniculus, accession no. AEA11027.1), rat (Rattus norvegicus, accession no. NP_001139300.1), sheep (Ovis aries, accession no. NP_001129398.1), turkey (Meleagris gallopavo, accession no. ADX33343.1), water buffalo (Bubalus bubalis, accession no. AEY63776.1), zebra finch (Taeniopygia guttata, accession no. XP_002188762.1), carp (Cyprinus carpio, accession no. AGH15501.1), fugu rubripes (Takifugu rubripes, accession no. AAW69374.1), zebrafish (Danio rerio, accession no. NP_001124067.1), rattlesnake (Crotalus adamanteus, accession no.AFJ51724.1), American bison (Bison bison, accession no. AEY63777.1), chamois (Rupicapra rupicapra, accession no. AFR42404.1), and deer (Odocoileus virginianus, accession no. AFR42402.1). The phylogenetic tree was generated with Clustal X software using the NJ method.

• 

  Fig. 3 Quantitative analysis of the tissue distribution of dTLR5 transcripts in healthy Pekin ducks. dTLR5 mRNA levels are expressed as the relative mRNAs relative to those in kidney.

• 

  Fig. 4 Functionality of dTLR5. (A and B) HeLa (A) and DF-1 (B) cells were transfected with 0.1 µg/well of a reporter plasmid (pGL-NF-κB) along with 0.025 µg/well of pRL-TK plasmid and the expression constructs (pCAGGS-dTLR5 or empty vector) using Fugen HD. (C and D) HeLa (C) and DF-1 (D) cells were transfected with 0.1 µg/well of pGL-IL-6-Luc along with 0.025 µg/well of pRL-TK plasmid and the expression constructs (pCAGGS-dTLR5 or empty vector). Twenty-four hour post-transfection, 50 ng/mL of flagellin was added to the transfected cells and luciferase assays were performed after stimulation for 6 h using a Dual-Luciferase Assay Kit. Data are presented as the mean values from three independent experiments. Significance was analyzed with a two-tailed Student's t-test (*p < 0.05).

• 

  Fig. 5 Fold-changes in gene expression of PKR (A), OAS (B), and Mx (C) in different tissues of virus-infected ducks compared to control animals. The controls were inoculated with PBS while the experimental ducks were infected with Herts33 NDV. Each bar represents the level of target gene mRNA relative to that in the control group. *Significant differences (p < 0.05) between the experimental and control groups. Error bars indicate the SD.

Reference

1. Barber MRW, Aldridge JR Jr, Webster RG, Magor KE. Association of RIG-I with innate immunity of ducks to influenza. Proc Natl Acad Sci U S A. 2010; 107:5913–5918.

2. Bernasconi D, Schultz U, Staeheli P. The interferon-induced Mx protein of chickens lacks antiviral activity. J Interferon Cytokine Res. 1995; 15:47–53.

3. Brownlie R, Allan B. Avian toll-like receptors. Cell Tissue Res. 2011; 343:121–130.

4. Chen S, Cheng A, Wang M. Innate sensing of viruses by pattern recognition receptors in birds. Vet Res. 2013; 44:82.

5. Daviet S, Van Borm S, Habyarimana A, Ahanda ML, Morin V, Oudin A, Van Den Berg T, Zoorob R. Induction of Mx and PKR failed to protect chickens from H5N1 infection. Viral Immunol. 2009; 22:467–472.

6. Didierlaurent A, Ferrero I, Otten LA, Dubois B, Reinhardt M, Carlsen H, Blomhoff R, Akira S, Kraehenbuhl JP, Sirard JC. Flagellin promotes myeloid differentiation factor 88-dependent development of Th2-type response. J Immunol. 2004; 172:6922–6930.

7. Fang Q, Pan Z, Geng S, Kang X, Huang J, Sun X, Li Q, Cai Y, Jiao X. Molecular cloning, characterization and expression of goose Toll-like receptor 5. Mol Immunol. 2012; 52:117–124.

8. Gewirtz AT, Navas TA, Lyons S, Godowski PJ, Madara JL. Cutting edge: bacterial flagellin activates basolaterally expressed TLR5 to induce epithelial proinflammatory gene expression. J Immunol. 2001; 167:1882–1885.

9. Hayashi F, Smith KD, Ozinsky A, Hawn TR, Yi EC, Goodlett DR, Eng JK, Akira S, Underhill DM, Aderem A. The innate immune response to bacterial flagellin is mediated by Toll-like receptor 5. Nature. 2001; 410:1099–1103.

10. Huang Z, Chen X, Zhang K, Yu B, Mao X, Zhao Y, Chen D. Molecular cloning and functional characterization of Tibetan Porcine STING. Int J Mol Sci. 2012; 13:506–515.

11. Hug H, Costas M, Staeheli P, Aebi M, Weissmann C. Organization of the murine Mx gene and characterization of its interferon- and virus-inducible promoter. Mol Cell Biol. 1988; 8:3065–3079.

12. Ishibashi M, Wakita T, Esumi M. 2',5'-Oligoadenylate synthetase-like gene highly induced by hepatitis C virus infection in human liver is inhibitory to viral replication in vitro. Biochem Biophys Res Commun. 2010; 392:397–402.

13. Kawai T, Akira S. The roles of TLRs, RLRs and NLRs in pathogen recognition. Int Immunol. 2009; 21:317–337.

14. Kawai T, Akira S. The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors. Nat Immunol. 2010; 11:373–384.

15. Keestra AM, de Zoete MR, Bouwman LI, Vaezirad MM, van Putten JPM. Unique features of chicken Toll-like receptors. Dev Comp Immunol. 2013; 41:316–323.

16. Keestra AM, de Zoete MR, van Aubel RA, van Putten JPM. Functional characterization of chicken TLR5 reveals species-specific recognition of flagellin. Mol Immunol. 2008; 45:1298–1307.

17. Keestra AM, de Zoete MR, van Aubel RAMH, van Putten JP. Functional characterization of chicken TLR5 reveals species-specific recognition of flagellin. Mol Immunol. 2008; 45:1298–1307.

18. Khaperskyy DA, Hatchette TF, McCormick C. Influenza A virus inhibits cytoplasmic stress granule formation. FASEB J. 2012; 26:1629–1639.

19. Ko JH, Asano A, Kon Y, Watanabe T, Agui T. Characterization of the chicken PKR: polymorphism of the gene and antiviral activity against vesicular stomatitis virus. Jpn J Vet Res. 2004; 51:123–133.

20. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG. Clustal W and Clustal X version 2.0. Bioinformatics. 2007; 23:2947–2948.

21. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-ΔΔCT Method. Methods. 2001; 25:402–408.

22. Marques J, Anwar J, Eskildsen-Larsen S, Rebouillat D, Paludan SR, Sen G, Williams BRG, Hartmann R. The p59 oligoadenylate synthetase-like protein possesses antiviral activity that requires the C-terminal ubiquitin-like domain. J Gen Virol. 2008; 89:2767–2772.

23. Medzhitov R. Toll-like receptors and innate immunity. Nat Rev Immunol. 2001; 1:135–145.

24. Melchjorsen J, Kristiansen H, Christiansen R, Rintahaka J, Matikainen S, Paludan SR, Hartmann R. Differential regulation of the OASL and OAS1 genes in response to viral infections. J Interferon Cytokine Res. 2009; 29:199–207.

25. Palm NW, Medzhitov R. Pattern recognition receptors and control of adaptive immunity. Immunol Rev. 2009; 227:221–233.

26. Poltorak A, He X, Smirnova I, Liu MY, Van Huffel C, Du X, Birdwell D, Alejos E, Silva M, Galanos C, Freudenberg M, Ricciardi-Castagnoli P, Layton B, Beutler B. Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science. 1998; 282:2085–2088.

27. Roach JC, Glusman G, Rowen L, Kaur A, Purcell MK, Smith KD, Hood LE, Aderem A. The evolution of vertebrate Toll-like receptors. Proc Natl Acad Sci U S A. 2005; 102:9577–9582.

28. Takeuchi O, Akira S. Innate immunity to virus infection. Immunol Rev. 2009; 227:75–86.

29. Tatsumi R, Sekiya S, Nakanishi R, Mizutani M, Kojima S, Sokawa Y. Function of ubiquitin-like domain of chicken 2'-5'-oligoadenylate synthetase in conformational stability. J Interferon Cytokine Res. 2003; 23:667–676.

30. Underhill DM, Ozinsky A, Hajjar AM, Stevens A, Wilson CB, Bassetti M, Aderem A. The Toll-like receptor 2 is recruited to macrophage phagosomes and discriminates between pathogens. Nature. 1999; 401:811–815.

31. Vaheri A, Ruoslahti E, Hovi T, Nordling S. Stimulation of density-inhibited cell cultures by insulin. J Cell Physiol. 1973; 81:355–364.

32. Webster RG, Bean WJ, Gorman OT, Chambers TM, Kawaoka Y. Evolution and ecology of influenza A viruses. Microbiol Rev. 1992; 56:152–179.

33. Xu Y, Tao X, Shen B, Horng T, Medzhitov R, Manley JL, Tong L. Structural basis for signal transduction by the Toll/interleukin-1 receptor domains. Nature. 2000; 408:111–115.