Identification, characterization and genetic mapping of TLR7, TLR8a1 and TLR8a2 genes in rainbow trout (Oncorhynchus mykiss)

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Abstract

Induction of the innate immune pathways is critical for early anti-viral defense but there is limited understanding of how teleost fish recognize viral molecules and activate these pathways. In mammals, Toll-like receptors (TLR) 7 and 8 bind single-stranded RNA of viral origin and are activated by synthetic anti-viral imidazoquinoline compounds. Herein, we identify and describe the rainbow trout (Oncorhynchus mykiss) TLR7 and TLR8 gene orthologs and their mRNA expression. Two TLR7/8 loci were identified from a rainbow trout bacterial artificial chromosome (BAC) library using DNA fingerprinting and genetic linkage analyses. Direct sequencing of two representative BACs revealed intact omTLR7 and omTLR8a1 open reading frames (ORFs) located on chromosome 3 and a second locus on chromosome 22 that contains an omTLR8a2 ORF and a putative TLR7 pseudogene. We used the omTLR8a1/2 nomenclature for the two trout TLR8 genes as phylogenetic analysis revealed that they and all the other teleost TLR8 genes sequenced to date are similar to the zebrafish TLR8a, but are distinct from the zebrafish TLR8b. The duplicated trout loci exhibit conserved synteny with other fish genomes extending beyond the tandem of TLR7/8 genes. The trout TLR7 and 8a1/2 genes are composed of a single large exon similar to all other described TLR7/8 genes. The omTLR7 ORF is predicted to encode a 1049 amino acid (aa) protein with 84% similarity to the Fugu TLR7 and a conserved pattern of predicted leucine-rich repeats (LRR). The omTLR8a1 and omTLR8a2 are predicted to encode 1035- and 1034-aa proteins, respectively, and have 86% similarity to each other. omTLR8a1 is likely the ortholog of the only Atlantic salmon TLR8 gene described to date as they have 95% aa sequence similarity. The tissue expression profiles of omTLR7, omTLR8a1 and omTLR8a2 in healthy trout were highest in spleen tissue followed by anterior and then posterior kidney tissues. Rainbow trout anterior kidney leukocytes produced elevated levels of pro-inflammatory and type I interferon cytokines mRNA in response to stimulation with the human TLR7/8 agonist R848 or the TLR3 agonist poly I:C. Only poly I:C-induced IFN2 transcription was significantly suppressed in the presence of chloroquine, a compound known to block endosomal acidification and inhibit endosomal maturation. The effect of chloroquine on R848-induced cytokine expression was equivocal and so it remains questionable whether rainbow trout recognition of R848 requires endosomal maturation. TLR7 and TLR8a1 expression levels in rainbow trout anterior kidney leukocytes were not affected by poly I:C or R848 treatments, but surprisingly, TLR8a2 expression was moderately down-regulated by R848. The down-regulation of omTLR8a2 may imply that this gene has evolved to a new or altered function in rainbow trout, as often occurs when the two duplicated genes remain active.

Introduction

Receptors that recognize conserved pathogen molecules are part of the ancient innate arm of the immune system and are conserved in both invertebrate and vertebrate lineages. Toll-like receptors (TLRs) are a family of transmembrane proteins that recognize conserved pathogen structures to induce immune effector molecules. In vertebrates, TLRs can distinguish among classes of pathogens and serve an important role in orchestrating the appropriate adaptive immune responses [1]. TLRs are type I membrane proteins that contain an extracellular N-terminus with leucine-rich repeat region (LRR) and an intracellular C-terminus with a Toll/IL-1 receptor domain (TIR). The cytoplasmatic TIR domain harbors conserved amino acids that have been shown to be involved in the signaling as well as in the localization of the TLR [2], [3], while the LRR region is involved in pathogen recognition [4]. Approximately 10 TLRs have been described in several species, and in mammals most of the TLRs have been shown to identify distinct pathogen associated components [5], [6], [7], [8].

TLR3, 7, 8 and 9 are the members of the nucleic acid subgroup of the TLR gene family. In mammals, TLR3 has been shown to respond to double-stranded RNA (dsRNA), TLR9 to unmethylated CpG DNA and TLR7 and TLR8 were shown to be activated by synthetic anti-viral imidazoquinoline compounds and were implicated in recognizing single-stranded RNA [9], [10], [11], [12], [13]. These TLRs are primarily located in the endoplasmic reticulum and in lysosomal-like vesicles and are thought to have an important role in anti-viral immunity [14]. The TLR7, 8 and 9 genes form a phylogenetically related cluster based on sequence similarities and genomic structures [7], [8]. Upon activation they recruit myeloid differentiation primary response protein 88 (MyD88) that through several effector molecules initiates the activation of two major signaling pathways resulting in the production of pro-inflammatory cytokines and/or type I interferons [1], [15]. The teleost specific TLR22 was recently shown to recognize long-sized dsRNA, but unlike the other members of this TLR gene family subgroup it is primarily located on the cell surface [16].

TLR orthologs have been described in several fish species. A complete repertoire of 10 or more TLRs was identified in the Fugu and the zebrafish genomes [17], [18], [19]. A number of TLR genes were identified, characterized and mapped in rainbow trout including TLR3, TLR5, TLR5S, TLR9, TLR20 and TLR22 [20], [21], [22], [23], [24], [25]. Although found in all the teleost genomes sequenced to date, rainbow trout TLR7 and TLR8 orthologs have not been reported. Due to their important role in anti-viral immunity we hypothesized that the rainbow trout genome contains TLR7 and TLR8 orthologs.

In this paper, we report the TLR7 and TLR8 orthologs in rainbow trout (Oncorhynchus mykiss) and describe their genomic location, gene organization, expression pattern, and regulation. Rainbow trout are widely used in basic research [26] and they are economically important for aquaculture and sport fishing. A better understanding of anti-viral immunity is necessary to reduce viral disease loss in aquaculture and for comparative study of immune system evolution in teleost fish.

Section snippets

Genes identification and sequencing

The rainbow trout gene index (http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/gimain.pl?gudb=r_trout) [27] and Atlantic salmon gene index (http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/gimain.pl?gudb=salmon) [28] were screened by BLAST for ESTs with high homology to Fugu TLR8 (Accession AC156438) [7]. Two Atlantic salmon ESTs were identified (Accessions DY707798 and DW540423). PCR primers were designed from conserved gene segments (Supplement Table 1) for screening of the NCCCWA Swanson 10×

The duplicated rainbow trout TLR7/TLR8 genomic region displays conserved synteny with other vertebrates

Screening of the rainbow trout BAC library identified a total of seven positive BACs that formed two contigs (five and two BACs) by DNA fingerprint analysis. DNA sequencing and genetic linkage mapping were conducted on BAC clones 141K11 and 318O13 which represented the five and two BACs contigs, respectively. A 5106 bp TLR8-like fragment was sequenced by primer walking from BAC 141K11, revealing a single-exon putative ORF of 1034 amino acid (Genbank Accession no. GQ422120), and a 3658 bp fragment

Discussion

In mammals, TLR7 and TLR8 are activated by synthetic anti-viral imidazoquinoline compounds and recognize single-stranded RNA (ssRNA) of viral origin, resulting in the induction of signaling pathways that activate production of various inflammatory cytokines [9], [54], [55], [59]. In fish, TLR7 and TLR8 orthologs were identified in the zebrafish and Fugu genomes [7], [17], [18], [19] and recently a TLR8 ortholog was identified in Atlantic salmon [50]. In this study we identified two putative

Acknowledgements

The authors thank Dr. Roger Vallejo for his contribution in developing the genetic linkage analysis pipeline and Mrs. M. Renee Fincham for her technical assistance. The use of trade, firm, or corporation names in this publication is for the information and convenience of the reader. Such use does not constitute an official endorsement or approval by the USDA, Agricultural Research Service, U.S. Department of Interior or the U.S. Geological Survey of any product or service to the exclusion of

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    1

    Current address: Biology Department, St. Vincent College, 300 Fraser Purchase Rd., Latrobe, PA 15650, USA.

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    Current address: The Biomedical Research Center, 2222 Health Sciences Mall, Vancouver, BC V6T 1Z3, Canada.

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