Characterization of the interleukin 1 receptor-associated kinase 4 (IRAK4)-encoding gene in salmonid fish: The functional copy is rearranged in Oncorhynchus mykiss and that factor can impair TLR signaling in mammalian cells

The interleukin 1 receptor-associated kinase 4 (IRAK4) is an essential factor for TLR-mediated activation of the host's immune functions subsequent to pathogen contact. We have characterized the respective cDNA and gene sequences from three salmonid species, salmon, rainbow trout and maraena whitefish. The gene from salmon is structured into eleven exons, as is the mammalian homologue, while exons have been fused in the genes from the two other salmonid species. Rainbow trout expresses also a pseudogene at low levels. Its basic structure resembles more closely the primordial gene than the functional copy does. The N-terminal death domain and the C-terminal protein kinase domain of the factors are better conserved throughout evolution than the linker domain. The deduced amino acid sequences of the factors from all three species group together in an evolutionary tree of IRAK4 factors. Scrutinizing expression and function of IRAK4 from rainbow trout, we found its highest expression in head kidney and spleen and lowest expression in muscle tissue. Infecting fish with Aeromonas salmonicida did not modulate its expression during 72 h of observation. Expression of a GFP-tagged trout IRAK4 revealed, expectedly, its cytoplasmic localization in human HEK-293 cells. However, this factor significantly quenched in a dose-dependent fashion not only the pathogen-induced stimulation of NF-κB factors in the HEK-293 reconstitution system of TLR2 signaling, but also the basal NF-κB levels in unstimulated control cells. Our data unexpectedly imply that IRAK4 is involved in establishing threshold levels of active NF-κB in resting cells.     

Divergent Toll-like receptors in catfish (Ictalurus punctatus): TLR5S, TLR20, TLR21

Toll-like receptors (TLR) mediate pathogen recognition in vertebrate species through detection of conserved microbial ligands. Families of TLR molecules have been described from the genomes of the teleost fish model species zebrafish and Takifugu, but much research remains to characterize the full length sequences and pathogen specificities of individual TLR members in fish. While the majority of these pathogen receptors are conserved among vertebrate species with clear orthologues present in fish for most mammalian TLRs, several interesting differences are present in the TLR repertoire of teleost fish when compared to that of mammals. A soluble form of TLR5 has been reported from salmonid fish and Takifugu rubripes which is not present in mammals, and a large group of TLRs (arbitrarily numbered 19-23) was identified from teleost genomes with no easily discernible orthologues in mammals. To better understand these teleost adaptations to the TLR family, we have isolated, sequenced, and characterized the full-length cDNA and gene sequences of TLR5S, TLR20, and TLR21 from catfish as well as studied their expression pattern in tissues. We also mapped these genes to bacterial artificial chromosome (BAC) clones for genome analysis. While TLR5S appeared to be common in teleost fish, and TLR21 is common to birds, amphibians and fish, TLR20 has only been identified in zebrafish and catfish. Phylogenetic analysis of catfish TLR20 indicated that it is closely related to murine TLR11 and TLR12, two divergent TLRs about which little is known. All three genes appear to exist in catfish as single copy genes.     

Production of recombinant leptin and its effects on food intake in rainbow trout (Oncorhynchus mykiss)

Leptin is a key factor for the regulation of food intake and energy homeostasis in mammals, but information regarding its role in teleosts is still limited. There are large differences between mammalian and teleost leptin at both gene and protein levels, and in order to characterize the function of leptin in fish, preparation of species-specific leptin is therefore a key step. In this study, full-length cDNA coding for rainbow trout leptin was identified. In spite of low amino acid sequence similarity with other animals, leptin is highly conserved between trout and salmon (98.7%). Based on the cDNA, we produced pure recombinant trout leptin (rt-leptin) in E. coli, with a final yield of 20 mg/L culture medium. We then examined the effects of intraperitoneal (IP) injection of rt-leptin on feeding behavior and gene expression of hypothalamic NPY and POMCs (POMC A1, A2 and B) in a short-term (8 h) experiment. The rt-leptin suppressed food intake and led to transient reduction of NPY mRNA levels, while the expression of POMCs A1 and A2, was elevated compared with vehicle-injected controls. These results for rainbow trout are the first that describe a physiological role of leptin using a species-specific orthologue in teleosts, and they suggest that leptin suppresses food intake mediated by hypothalamic regulation. This anorexic effect is similar to that observed in mammals and frogs and supports that the neuroendocrine pathways that control feeding by leptin are ancient and have been conserved through evolution.     

Identification,characterization and genetic mapping of TLR1 loci in rainbow trout (Oncorhynchus mykiss)

Induction of innate immune pathways is critical for early anti-microbial defense but there is limited understanding of how teleosts recognize microbial molecules and activate these pathways. In mammals, Toll-like receptors (TLR) 1 and 2 form a heterodimer involved in recognizing peptidoglycans and lipoproteins of microbial origin. Herein, we identify and describe the rainbow trout (Oncorhynchus mykiss) TLR1 gene ortholog and its mRNA expression. Two TLR1 loci were identified from a rainbow trout bacterial artificial chromosome (BAC) library using DNA sequencing and genetic linkage analyses. Full length cDNA clone and direct sequencing of four BACs revealed an intact omTLR1 open reading frame (ORF) located on chromosome 14 and a second locus on chromosome 25 that contains a TLR1 pseudogene. The duplicated trout loci exhibit conserved synteny with other fish genomes that extends beyond the TLR1 gene sequences. The omTLR1 gene includes a single large coding exon similar to all other described TLR1 genes, but unlike other teleosts it also has a 5′ UTR exon and intron preceding the large coding exon. The omTLR1 ORF is predicted to encode an 808 amino-acid protein with 69% similarity to the Fugu TLR1 and a conserved pattern of predicted leucine-rich repeats (LRR). Phylogenetic analysis grouped omTLR1 with other fish TLR1 genes on a separate branch from the avian TLR1 and mammalian TLR1, 6 and 10. omTLR1 expression levels in rainbow trout anterior kidney leukocytes were not affected by the human TLR2/6 and TLR2/1 agonists diacylated lipoprotein (Pam₂CSK₄) and triacylated lipoprotein (Pam₃CSK₄). However, due to the lack of TLR6 and 10 genes in teleost genomes and up-regulation of TLR1 mRNA in response to LPS and bacterial infection in other fish species we hypothesize an important role for omTLR1 in anti-microbial immunity. Therefore, the identification of a TLR2 ortholog in rainbow trout and the development of assays to measure ligand binding and downstream signaling are critical for future elucidation of omTLR1 functions.     

Fish gonadotropin-releasing hormone gene and molecular approaches for control of sexual maturation: development of a transgenic fish model.

The prepro-GnRH gene and mRNA primary structure were fully established from Atlantic salmon (Salmo salar) and partially from rainbow trout (Oncorhynchus mykiss). Results show that the GnRH coding region of 30 base pairs is well conserved during evolution. In contrast, the GnRH-associated peptide (GAP) sequence shows very limited homology when the GnRH genes from mammalian and teleost species are compared. A simple method for selecting transgenic fish after transfer of the firefly luciferase gene was developed. The method involves bioluminescent measurement of live animals in a scintillation counter.     

Human intestinal epithelial cells are broadly unresponsive to Toll-like receptor 2-dependent bacterial ligands: implications for host-microbial interactions in the gut

Intestinal epithelial cells (IEC) interact with a high density of Gram-positive bacteria and are active participants in mucosal immune responses. Recognition of Gram-positive organisms by Toll-like receptor (TLR)2 induces proinflammatory gene expression by diverse cells. We hypothesized that IEC are unresponsive to Gram-positive pathogen-associated molecular patterns and sought to characterize the functional responses of IEC to TLR2-specific ligands. Human colonic epithelial cells isolated by laser capture microscopy and IEC lines (Caco-2, T84, HT-29) were analyzed for expression of TLR2, TLR6, TLR1, and Toll inhibitory protein (Tollip) mRNA by RT-PCR and quantitative real-time PCR. Response to Gram-positive bacterial ligands was measured by NF-kappa B reporter gene activation and IL-8 secretion. TLR2 protein expression was analyzed by immunofluorescence and flow cytometry. Colonic epithelial cells and lamina propria cells from both uninflamed and inflamed tissue demonstrate low expression of TLR2 mRNA compared with THP-1 monocytes. IECs were unresponsive to TLR2 ligands including the staphylococcal-derived Ags phenol soluble modulin, peptidoglycan, and lipotechoic acid and the mycobacterial-derived Ag soluble tuberculosis factor. Transgenic expression of TLR2 and TLR6 restored responsiveness to phenol soluble modulin and peptidoglycan in IEC. In addition to low levels of TLR2 protein expression, IEC also express high levels of the inhibitory molecule Tollip. We conclude that IEC are broadly unresponsive to TLR2 ligands secondary to deficient expression of TLR2 and TLR6. The relative absence of TLR2 protein expression by IEC and high level of Tollip expression may be important in preventing chronic proinflammatory cytokine secretion in response to commensal Gram-positive bacteria in the gut.     

Prediction of the prototype of the human Toll-like receptor gene family from the pufferfish,<Emphasis Type="Italic"> Fugu rubripes</Emphasis>, genome

The insect Toll family of proteins and their mammalian counterparts seemingly shared one common ancestor and evolved independently. Here we demonstrated that the prototype of the mammalian-type (M-type) Toll family is shared by the fish and humans. According to the draft of the pufferfish Fugu genome project, the signature Toll-IL-1 receptor homology domain (TIR domain) has been conserved during evolution. FuguTLR2, 3, 5, 7, 8 and 9 members correspond structurally to respective mammalian TLRs. One Fugu TLR showed equally high amino acid identity to human TLR1, 6 and 10, and we named it FuguTLR1. Fugu rubripes has genes for TLR21 and 22, which are unique to fish. One possible interpretation of these findings is that TLR1, 2, 3, 4, 5, 7, 8, 9, 21 and 22 existed in the ancestral genome common to fish and mammals, and that TLR4 was lost in the fish lineage, while TLR21 and 22 were lost in the mammalian lineage. Strikingly, a solitary ascidian, Halocynthia roretzi, has only a few Toll-like proteins, which, like Caenorhabditis elegans Toll, represent primitive ones before the expansion of the Toll family. Therefore, the expansion of TLR genes should have occurred earlier than fish, but not C. intestinalis, separated evolutionarily from mammals. These results infer that the appearance of the M-type innate system was completed before or concomitant with the appearance of acquired immunity. We interpret the present data to mean that the differences of TLRs identified in this study between fishes and humans may be rather peripheral, partially due to selection pressure exerted by pathogens in distinct environments.     

Characterization and comprehensive analysis of the miiuy croaker TLR2 reveals a direct evidence for intron insert and loss

Toll-like receptor 2 (TLR2) is a member of an ancient pattern recognition receptor family, conserved from insects to mammals and it is best known as a receptor for recognizing conserved components of Gram-positive bacteria. In present study, the genomic structure of TLR2 gene from miiuy croaker was identified and characterized. It comprises twelve exons and eleven introns. The lengths of exons 3 to 10 of miiuy croaker TLR2 and exons 2 to 9 of fugu and pufferfish TLR2 are exactly the same, but most importantly, both of fugu and pufferfish have only eleven exons and ten introns. An intron insert event probably happened on exon 1 of miiuy croaker TLR2 after its divergence from ancestor of zebrafish, and an intron loss event probably happened on those of Tetraodontiformes TLR2 after the divergence with ancestor of miiuy croaker. Our study showed the direct evidence and strongly supported the intron insert and loss on fish TLR2. The pathogen injection experiments indicated that TLR2 might not be an important responder to Gram-negative bacteria in miiuy croaker. Molecular evolutionary analyses indicated TLR2 genes were under strong purifying selection pressure, showing a quite strong functional constraint in both of fish and mammals, despite of their distinct living environment conditions.     

Conservation of Toll-Like Receptor Signaling Pathways in Teleost Fish

In mammals, toll-like receptors (TLR) recognize ligands, including pathogen-associated molecular patterns (PAMPs), and respond with ligand-specific induction of genes. In this study, we establish evolutionary conservation in teleost fish of key components of the TLR-signaling pathway that act as switches for differential gene induction, including MYD88, TIRAP, TRIF, TRAF6, IRF3, and IRF7. We further explore this conservation with a molecular phylogenetic analysis of MYD88. To the extent that current genomic analysis can establish, each vertebrate has one ortholog to each of these genes. For molecular tree construction and phylogeny inference, we demonstrate a methodology for including genes with only partial primary sequences without disrupting the topology provided by the high-confidence full-length sequences. Conservation of the TLR-signaling molecules suggests that the basic program of gene regulation by the TLR-signaling pathway is conserved across vertebrates. To test this hypothesis, leukocytes from a model fish, rainbow trout (Oncorhynchus mykiss), were stimulated with known mammalian TLR agonists including: diacylated and triacylated forms of lipoprotein, flagellin, two forms of LPS, synthetic double-stranded RNA, and two imidazoquinoline compounds (loxoribine and R848). Trout leukocytes responded in vitro to a number of these agonists with distinct patterns of cytokine expression that correspond to mammalian responses. Our results support the key prediction from our phylogenetic analyses that strong selective pressure of pathogenic microbes has preserved both TLR recognition and signaling functions during vertebrate evolution.     

Genomic organization of the IGF1, IGF2, MYF5, MYF6 and GRF/PACAP genes across Salmoninae genera

Whole-genome duplication in the ancient ray-finned fish and subsequent tetraploidization in the ancestor to the salmonids have complicated genomic and candidate gene studies in these organisms as many genes with multiple copies are present throughout their genomes. In an attempt to identify genes with a potential influence on growth and development, we investigated the genomic positions of insulin-like growth factors 1 and 2 (IGF1, IGF2), myogenic factors 5 and 6 (MYF5, MYF6) and growth hormone-releasing factor/pituitary adenylate cyclase-activating polypeptide (GRF/PACAP) in three salmonid species: rainbow trout (Oncorhynchus mykiss), Atlantic salmon (Salmo salar) and Arctic charr (Salvelinus alpinus). Our results suggest a tight association between the IGF1, MYF5 and MYF6 genes in all three species. We further localized the duplicated copies of IGF1 to the homeologous linkage groups RT-7/15 in rainbow trout and AC-3/24 in Arctic charr, and the two copies of MYF6 to homeologous linkage groups AS-22/24 in Atlantic salmon. Localization of GRF/PACAP to RT-7, AS-31 and AC-27 and IGF2 to RT-27, AS-2 and AC-4 in rainbow trout, Atlantic salmon and Arctic charr respectively is consistent with previously reported homologies among these chromosomal segments identified using other genetic markers. However, localization of the second copy of GRF/PACAP to RT-19 and AC-14 and the duplicated copy of IGF2 to AC-19 suggest a possible new homology/homeology between these chromosomes. These results might also be an indication of a more ancient polyploidization event that occurred deep in the ray-finned fish lineage.     

Two arylalkylamine N-acetyltransferase genes mediate melatonin synthesis in fish

Serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AANAT, EC 2.3.1.87) is the first enzyme in the conversion of serotonin to melatonin. Large changes in AANAT activity play an important role in the daily rhythms in melatonin production. Although a single AANAT gene has been found in mammals and the chicken, we have now identified two AANAT genes in fish. These genes are designated AANAT-1 and AANAT-2; all known AANATs belong to the AANAT-1 subfamily. Pike AANAT-1 is nearly exclusively expressed in the retina and AANAT-2 in the pineal gland. The abundance of each mRNA changes on a circadian basis, with retinal AANAT-1 mRNA peaking in late afternoon and pineal AANAT-2 mRNA peaking 6 h later. The pike AANAT-1 and AANAT-2 enzymes (66% identical amino acids) exhibit marked differences in their affinity for serotonin, relative affinity for indoleethylamines versus phenylethylamines and temperature-activity relationships. Two AANAT genes also exist in another fish, the trout. The evolution of two AANATs may represent a strategy to optimally meet tissue-related requirements for synthesis of melatonin: pineal melatonin serves an endocrine role and retinal melatonin plays a paracrine role.