Experimental immunology
Cloning and analysis of Nile tilapia Toll-like receptors type-3 mRNA

Introduction

Pathogen-associated molecular patterns, or PAMPs, are molecules associated with groups of pathogens that are recognized by cells of the innate immune system. These molecules can be referred to as small molecular motifs conserved within a class of microbes. They are recognized by Toll-like receptors (TLRs) and other pattern recognition receptors (PRRs) in both plants and animals. They activate innate immune responses, protecting the host from infection, by identifying some conserved non-self-molecules. Bacterial lipopolysaccharide (LPS), an endotoxin found on the bacterial cell membrane of a bacterium, is considered to be the prototypical PAMP. Every TLR receptor is mainly specialized in recognizing one or more patterns where LPS is specifically recognized by TLR-4 [1-8], a bacterial flagellin recognized by TLR-5 [3], lipoteichoic acid from Gram-positive bacteria and peptidoglycan recognized by TLR-2 [1, 9-11], and nucleic acid variants normally associated with viruses, such as double-stranded RNA – dsRNA recognized by TLR-3 [12-14] or un-methylated CpG motifs, recognized by TLR-15, TLR-21 [15-20]. Although the term “PAMP” is relatively new, the concept that molecules derived from microbes must be detected by receptors from multicellular organisms has been held for many decades, and references to an “endotoxin receptor” are found in a lot of older literature. Toll-like receptors are the basic components of the vertebrate pathogen recognition system. Despite the uniform general structure, remarkable variability in domain composition can be found in individual TLRs among species. Toll-like receptors are typical type I transmembrane proteins, and contain three major domains: a tandem repeat leucine-rich repeat (LRR) motif, which identifies PAMPs, a transmembrane region and an intracellular Toll/interleukin 1 (IL-1) receptor (TIR) domain, which transmits signals. Knowledge of inter-specific differences is of particular importance to our understanding of selective pressures on TLRs. Toll-like receptors are membrane-bound sensors of the innate immune system, which recognize invariant and distinctive molecular features of invading microbes and are also essential for initiating adaptive immunity in vertebrates. The genetic variation in TLR genes has been directly related to differential pathogen outcomes in humans and livestock. Nonetheless, new insights about the impact of TLR polymorphism on the evolutionary ecology of infectious diseases can be gained through the investigation of additional vertebrate groups not yet investigated in detail. Toll-like receptors are members of the PRRs, which detect PAMPs and have a role in initiating the innate as well as adaptive immune defense [1, 12-15, 18, 21-26]. They play a vital role in host immune responses through the recognition of LPS, lipopeptides, flagellins, dsRNA or CpG DNA motifs [16]. The TLR system is a part of ancient machinery that is evolutionary conserved with homologs present in insects, nematodes, plants, fish, mammals and birds [27]. A range of TLR genes has been identified in non-mammalian vertebrates including birds and fish [12]. The numbers of TLR genes vary among various organisms. Thirteen TLRs (TLR 1-13) have been identified in mammals, and functionally these receptors recognize and respond to a wide range of exogenous as well as endogenous ligands. Of the 13 mammalian TLRs, TLR-11, TLR-12, and TLR-13 were identified only in the murine genome. In teleost fish, orthologs of TLR 1-5, 7-9 have been identified, while various reports indicated that TLR-6 and TLR-10 do not exist in teleost fish [28, 29]. In addition to the orthologs of TLRs in mammals, ‘fish-specific’ TLRs have been reported including TLR-18, TLR-19, TLR-20, TLR-21, TLR-22, and TLR-23 [17, 28, 30, 31]. However, all these fish TLRs and their signaling cascade factors represent high structural similarity to the mammalian TLR system. Currently, most TLRs are characterized only in a limited number of model species, including Salmon fish, according to our knowledge, there is almost no data about the Nile tilapia TLRs, which is considered one of the most common farm fish and has a great economic importance all over the world. Here we want to describe the TLRs in the Nile tilapia. The research dedicated to the description of the enormous diversity of molecules involved in pathogen recognition is of vital importance in human and veterinary medicine. It is equally important to the evolutionary biology of host-parasite interactions. Much effort has been devoted to the characterization of immune system components in human and mouse models, while much less is currently known about the architecture of the immune system in other species [32]. Information concerning any one of the wealth of living species may bring new insights into the principles of the vertebrate immune function. Aiming to describe general patterns of immune system evolution in terrestrial vertebrates, the investigation of the fish clade may be particularly useful. Fish form a well-diversified taxon with origin distinct to mammals but with physiology comparable to them. However, in contrast to mammals, our knowledge of the molecular structure of the fish immune system is limited [8, 31]. Considering these reasons, new models are required to verify the universal validity of the results obtained in human and mice, so we chose Nile tilapia as a representative of the freshwater fish [28, 33-36].

Material and methods

Samples



Kidney, brain, spleen, intestine, muscle, liver, gills and heart and skin samples were collected from the Nile tilapia fish. The Nile tilapia fish was brought as a live mature fish from common farm and kept under inspection for 5 days to be sure that it is free from any clinical infection, then the samples were collected and stored in –80°C.



Primer design



We downloaded the complete TLR-3 mRNA sequences of the rainbow trout (Oncorhynchus mykiss): AAX68425; Takifugu rubripes (Fugu rubripes): AAW69373; Larimichthys crocea (large yellow croaker): ADW79423; Paralichthys olivaceus (Japanese flounder): BAM11216; Epinephelus coioides (orange-spotted grouper): AEX01718 from the GenBank. The sequence was aligned using the ClustalW application (primer premier 5.1 software) where we designed degenerative primers match with the alignment of the sequences to the clone short sequence, then we got the full sequence by RACE system SMARTer RACE cDNA Amplification kit (Clontech, CA, USA) following the manufacturer’s instructions, to get the full length for both 3’end and 5’end direction, all the primers were designed in our lab (unpublished data).



Molecular cloning of Nile tilapia TLR-3



Total RNA from fish kidney, brain, spleen, skin, intestine, muscle, liver, gills, heart and skin were extracted by TRIZOL (Invitrogen, USA) according to the manufacturer’s protocol. Both the quantity and quality of total RNA were assessed at OD260 and OD280 using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, USA). RNA samples were used for synthesis from the cDNA library in 10 µl reaction mixture using BioRT cDNA first strand synthesis kit (Hangzhou Boiler, China) according to the manufacturer’s instructions. Briefly, oligo dT primer (0.5 µg) was used to reverse transcribe 1 µg of respective RNA in the presence of dNTP’s (250 µM), reverse transcriptase buffer (50 mM Tris-HCl, 100 mM KCl, 4.0 mM DTT and 10 mM MgCl2), AMV Reverse Transcriptase (5 units/µg) and RNasin Ribonuclease inhibitor (40 unit/µl) at 42°C for 45 min following inactivation at 95°C for 5 min. The PCR was performed to amplify the target gene using specific primers, where 25 µl PCR mixtures contained 50 pmol for each forward and reverse primer, 2 µl template cDNA, 200 µM each of dNTP mix and 2.5 U Ex Taq polymerase (Takara Bio, Dalian, China) in 1× Ex buffer. Amplification conditions were as follows: initial denaturation at 94°C for 5 min, 35 cycles at 94°C for 35 s, annealing at 56-60°C for 35 s, and extension at 72°C for 2 min, followed by the final extension at 72°C for 10 min. PCR amplicons were verified by 1.2% agarose gel electrophoresis in TBE buffer at 70 mA for 45 min and products visualized by staining with ethidium bromide, we checked the band and got the images with GelDoc™ XR+ system (Bio-RAD, USA). We used SMARTer RACE cDNA amplification kits (Clontech, USA) according to the manufacturer’s instructions to get the full length toward the 3’end and 5’end.



Sequence analysis



The sequence of the Nile tilapia TLR-3 mRNA was blast in the GenBank using nucleotide blast and the translated amino acids were also blast by protein blast to check whether the new sequence is related to any other cloned gene. The sequence of Nile tilapia TLR-3 was compared with the known TLR-3 mRNA sequences from different species which were downloaded from the GenBank and aligned by CUSTALW, MEGA 5 software [37]. A phylogenetic tree was constructed from the amino acid alignments using two methods: (A) the neighbour-joining method with options of pairwise deletion, poisson correction and different evolutionary rates with a gamma parameter of 1 and (B) the maximum parsimony method using the close neighbour-interchange search method with random additive trees (10 replicates), all the phylogenetic analyses were made by MEGA5 software [37] and the reliability of branching was tested by 1000 bootstraps. The extracellular, transmembrane, and cytoplasmic domains of these protein sequences were predicted with the analysis tools provided at the websites (http://smart.embl-heidelberg.de and http://split.pmfst.hr).

Results

Nile tilapia TLR-3



The complete mRNA sequence of Nile tilapia TLR-3 was deposited in the NCBI GenBank database under accession no. JQ809460. Where it consists of 2736 nucleotides and the consensus cDNA sequence showed 79% identity with Larimichthys crocea and 78% identity with Epinephelus coioides, while it showed 72% identity with Takifugu rubripes, which confirmed that the new sequence is probably homolog to fish TLR-3. The predicted protein encoded by Nile tilapia TLR3 mRNA sequence is composed of 912 amino acids where it begins with (ATG) which is similar to the other fish TLR-3 sequence. The Nile tilapia TLR-3 domain structure has been estimated using SMART web tool, where Nile tilapia TLR-3 started by signal peptide (20 amino acids from 1-20), then 16 LRR domains (residues 21-639) and one C-terminal LRR domain (LRR-CT, residues 652-705) in the extracellular region, and a TIR domain (residues 764-907) in the cytoplasmic region as shown in Table 1. The encoded amino acids vary among different available amino acids where it has 20 different amino acids; the highest amino acid encoded is Leucine while the lowest is Tryptophan, we made a chart explaining the amount and ratio of the encoded amino acid in the Nile tilapia TLR-3 as shown in Fig. 1.



Similarity with other TLRs



We used two methods to construct a phylogenetic tree (neighbour-joining and maximum parsimony) based on the amino acid of TLR-3, which was downloaded from the GenBank. The phylogenetic analysis had been performed using the translated Nile tilapia amino acid sequence with almost all the known amino acid sequences found in the GenBank, both phylogenetic methods provided almost the same results, where the phylogenetic analysis showed that Nile tilapia TLR-3 is closely related to Larimichthys crocea TLR-3, Epinephelus coioides TLR-3 and Takifugu rubripes TLR-3. The structure of Nile tilapia TLR-3 amino acids is in general similar to other identified TLR-3 sequence with 36-48% identity to different mammalians, 71% identity to rainbow trout (Oncorhynchus mykiss), Epinephelus coioides (orange-spotted grouper), Paralichthys olivaceus (Japanese flounder) and Larimichthys crocea (large yellow croaker) TLR-3 while 68% to Takifugu rubripes (Fugu rubripes) TLR-3 and 39% with chicken as shown in Fig. 2.



Expression pattern of Nile tilapia TLR-3



The transcription of Nile tilapia TLR-3 was highly expressed in kidney, brain, spleen, intestine, muscle, liver, gills, heart and skin (Fig. 3), semiquantitative PCR following the reverse transcription showed differences in the expression level among the tested tissue, where the expression was the highest in the spleen, muscle and liver, also showed a moderate expression level in the kidney and intestine.

Discussion

This is the first study to characterize the Nile tilapia TLR-3 as most studies focus on non-fish vertebrates. Our results provided Nile tilapia TLR-3, which is considered a homologe for rainbow trout, orange-spotted grouper, zebra fish and other vertebrates TLR-3; the structure analysis showed that the Nile tilapia receptor is very close to rainbow trout, orange-spotted grouper with 71% identity and more closer to rain trout than to zebra fish.

The transmembrane structure analysis for the Nile tilapia receptor showed that rainbow trout and orange-spotted grouper have one motif which is absent in tilapia while tilapia has one motif plus zebra fish, and with rainbow trout, orange-spotted grouper sharing the same signal peptide at the beginning of the gene (Fig. 3). The Nile tilapia TLR domain has 143 amino acids, while rainbow trout has 136 amino acids, and, it may be due to the unequal force distribution during the evolution, also the duck showed a unique LRR domain at position 128-150 which is absent in rainbow trout and orange-spotted grouper as shown in Fig. 4. This may have a role in the difference of the immune response against pathogen between

the Nile tilapia and other fish species. The expression of Nile tilapia TLR-3 varies between different tilapia organs as in Fig. 3 where it is highly expressed in kidney, brain, spleen, intestine, muscle, liver, gills, heart and skin.

Acknowledgements

All the authors show gratitude and appreciation to the Financial Support source where this work was supported by the Program for Changjiang Scholars and Innovative Research Team in University, No. IRT0923 “Supported by PCSIRT, No. IRT0923”.





The authors declare no conflict of interests.



Financial Support: This work was supported by the Program for Changjiang Scholars and Innovative Research Team in University, No. IRT0923 “Supported by PCSIRT, No. IRT0923”.

References

 1. Faure E, Thomas L, Xu H, et al. (2001): Bacterial lipopolysaccharide and IFN-gamma induce Toll-like receptor 2 and Toll-like receptor 4 expression in human endothelial cells: Role of NF-kappa B activation. J Immunol 166: 2018-2024.

 2. Leveque G, Forgetta V, Morroll S, et al. (2003): Allelic variation in TLR4 is linked to susceptibility to Salmonella enterica serovar typhimurium infection in chickens. Infect Immun 71: 1116-1124.

 3. Kogut MH, Iqbal M, He HQ, et al. (2005): Expression and function of Toll-like receptors in chicken heterophils. Dev Comp Immunol 29: 791-807.

 4. Kaiser P (2007): The avian immune genome a glass half-full or half-empty? Cytogenet Genome Res 117: 221-230.

 5. Vinkler M, Bryjova A, Albrecht T, et al. (2009): Identification of the first Toll-like receptor gene in passerine birds: TLR4 orthologue in zebra finch (Taeniopygia guttata). Tissue Antigens 2009; 74: 32-41.

 6. Zhang C, Wu XL, Zhao YF, et al. (2011): SIGIRR inhibits toll-like receptor 4, 5, 9-mediated immune responses in human airway epithelial cells. Mol Biol Rep 38: 601-609.

 7. Pan Z, Fang Q, Geng S, et al. (2012): Analysis of immune-related gene expression in chicken peripheral blood mononuclear cells following Salmonella enterica serovar Enteritidis infection in vitro. Res Vet Sci 93: 716-720.

 8. Elfeil WK, Abouelmaatti RR, Sun CJ, et al. (2012): Identification, cloning, expression of a novel functional anas platyrhynchos mRNA TLR4. J Anim Vet Adv 11: 1727-1733.

 9. Fukui A, Inoue N, Matsumoto M, et al. (2001): Molecular cloning and functional characterization of chicken toll-like receptors. A single chicken toll covers multiple molecular patterns. J Biol Chem 276: 47143-47149.

10. Boyd Y, Goodchild M, Morroll S, et al. (2001): Mapping of the chicken and mouse genes for toll-like receptor 2 (TLR2) to an evolutionarily conserved chromosomal segment. Immunogenetics 52: 294-298.

11. Elfeil W, Soliman E, Sobeih M (2011): Epidemiological studies on environmental pollution in poultry farms. GRIN Publishing GmbH, Munich.

12. Roach JC, Glusman G, Rowen L, et al. (2005): The evolution of vertebrate Toll-like receptors. P Natl Acad Sci U S A 102: 9577-9582.

13. Miggin SM, O'Neill LA (2006): New insights into the regulation of TLR signaling. J Leukocyte Biol 80: 220-226.

14. Brownlie R, Allan B (2011): Avian Toll-like receptors. Cell Tissue Res 343: 121-130.

15. Alcaide M, Edwards SV (2011): Molecular evolution of the Toll-like receptor multigene family in birds. Mol Biol Evol 28: 1703-1715.

16. Werling D, Jann OC, Offord V, et al. (2009): Variation matters: TLR structure and species-specific pathogen recognition. Trends Immunol 30: 124-130.

17. Li YW, Luo XC, Dan XM, et al. (2012): Molecular cloning of orange-spotted grouper (Epinephelus coioides) TLR21 and expression analysis post Cryptocaryon irritans infection. Fish Shellfish Immunol 32: 476-481.

18. He H, Genovese KJ, Swaggerty CL, et al. (2012): Co-stimulation with TLR3 and TLR21 ligands synergistically up-regulates Th1-cytokine IFN-gamma and regulatory cytokine IL-10 expression in chicken monocytes. Dev Comp Immunol 36: 756-760.

19. Ramasamy KT, Verma P, Reddy MR, et al. (2011): Molecular characterization of coding sequence and mRNA expression pattern of Toll-like receptor 15 in Japanese Quail (Coturnix japonica) and indigenous chicken breeds (Aseel and Kadaknath). J Poultry Sci 48: 168-175.

20. Abouelmaatti RR, Elfeil W, Wang Y, et al. (2013): Pattern recognition receptors mini review. Global Anim Sci J 1: 1118-1127.

21. Jin MS, Lee JO (2008): Structures of the toll-like receptor family and its ligand complexes. Immunity 29: 182-191.

22. Temperley ND, Berlin S, Paton IR, et al. (2008): Evolution of the chicken Toll-like receptor gene family: a story of gene gain and gene loss. BMC Genomics 2008; 9: 62.

23. Chaussé AM, Grepinet O, Bottreau E, et al. (2011): Expression of Toll-like receptor 4 and downstream effectors in selected cecal cell subpopulations of chicks resistant or susceptible to Salmonella carrier state. Infect Immun 79: 3445-3454.

24. Yilmaz A, Shen SX, Adelson DL, et al. (2005): Identification and sequence analysis of chicken Toll-like receptors. Immunogenetics 56: 743-753.

25. Ruan WK, Wu YH, An J, et al. (2012): Toll-like receptor 2 type 1 and type 2 polymorphisms in different chicken breeds. Poultry Sci 91: 101-106.

26. Perez de la Lastra JM, de la Fuente J (2007): Molecular cloning and characterisation of the griffon vulture (Gyps fulvus) toll-like receptor 1. Dev Comp Immunol 31: 511-519.

27. Beutler B, Rehli M (2002): Evolution of the TIR, tolls and TLRs: functional inferences from computational biology. Curr Top Microbiol Immunol 270: 1-21.

28. Rebl A, Goldammer T, Seyfert HM. Toll-like receptor signaling in bony fish. Vet Immunol Immunopathol 134: 139-150.

29. Elfeil WK (2012): Newcastle-Avian flu recombinant vaccine in embryonated eggs and chicks. LAP LAMBERT Academic Publishing, Germany.

30. Matsumoto M, Funami K, Oshiumi H, et al. (2004): Toll-like receptor 3: A link between Toll-like receptor, interferon and viruses. Microbiol Immunol 48: 147-154.

31. Rajendran KV, Zhang J, Liu S, et al. (2012): Pathogen recognition receptors in channel catfish: I. Identification, phylogeny and expression of NOD-like receptors. Dev Comp Immunol 37: 77-86.

32. Acevedo-Whitehouse K, Cunningham AA (2006): Is MHC enough for understanding wildlife immunogenetics? Trends Ecol Evol 21: 433-438.

33. Zhang J, Liu S, Rajendran KV, et al. (2013): Pathogen recognition receptors in channel catfish: capital SHA, Cyrillic. Phylogeny and expression analysis of Toll-like receptors. Dev Comp Immunol 40: 185-194.

34. Palti Y (2011): Toll-like receptors in bony fish: from genomics to function. Dev Comp Immunol 35: 1263-1272.

35. Huang R, Dong F, Jang S, et al. (2012): Isolation and analysis of a novel grass carp Toll-like receptor 4 (TLR4) gene cluster involved in the response to grass carp reovirus. Dev Comp Immunol 38: 383-388.

36. Lv J, Huang R, Li H, et al. (2012): Cloning and characterization of the grass carp (Ctenopharyngodon idella) Toll-like receptor 22 gene, a fish-specific gene. Fish Shellfish Immunol 32: 1022-1031.

37. Tamura K, Peterson D, Peterson N, et al. (2011): MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28: 2731-2739.