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Conservation and divergence of gene families encoding components of innate immune response systems in zebrafish - PubMed

Conservation and divergence of gene families encoding components of innate immune response systems in zebrafish

Cornelia Stein et al. Genome Biol. 2007.

Abstract

Background: The zebrafish has become a widely used model to study disease resistance and immunity. Although the genes encoding many components of immune signaling pathways have been found in teleost fish, it is not clear whether all components are present or whether the complexity of the signaling mechanisms employed by mammals is similar in fish.

Results: We searched the genomes of the zebrafish Danio rerio and two pufferfish for genes encoding components of the Toll-like receptor and interferon signaling pathways, the NLR (NACHT-domain and leucine rich repeat containing) protein family, and related proteins. We find that most of the components known in mammals are also present in fish, with clearly recognizable orthologous relationships. The class II cytokines and their receptors have diverged extensively, obscuring orthologies, but the number of receptors is similar in all species analyzed. In the family of the NLR proteins, the canonical members are conserved. We also found a conserved NACHT-domain protein with WD40 repeats that had previously not been described in mammals. Additionally, we have identified in each of the three fish a large species-specific subgroup of NLR proteins that contain a novel amino-terminal domain that is not found in mammalian genomes.

Conclusion: The main innate immune signaling pathways are conserved in mammals and teleost fish. Whereas the components that act downstream of the receptors are highly conserved, with orthologous sets of genes in mammals and teleosts, components that are known or assumed to interact with pathogens are more divergent and have undergone lineage-specific expansions.

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Figures

Figure 1
Figure 1

Components of the TLR and IFN signaling pathways and intracellular pattern recognition receptors. The molecules analyzed in this study are shown in color. For simplicity, not all members of each protein family are shown. IFN, interferon; TLR, Toll-like receptor.

Figure 2
Figure 2

Phylogenetic trees of the innate immune signaling adaptors and diagrams of their protein structures. The fish protein names are highlighted in blue (Dr [Danio rerio]) or green (Fr [Takifugu rubripes] and Tn [Tetraodon nigroviridis]). The numbers in the tree indicate the bootstrap values. Scale: interval of 0.1 amino acid substitutions. Hs, Homo sapiens, Mm, Mus musculus. Protein domains are shown as boxes based on identification by Pfam [55] or Smart [56]. Some domains were not recognized by these programs, although manual inspection indicated clear conservation of the domain within the protein family. These domains are also shown as boxes in the diagrams. The identities of the domains are listed at the bottom. Scale bar = 100 amino acids. The Tetraodon version of the Ikap (IKK [Inhibitor of nuclear factor-κB kinase] complex associated protein) gene contains two full repeats of the IKI3 domain. It is not clear whether this prediction is due to an error in the genome assembly or whether the gene does indeed contain an internal duplication covering the whole length of the gene found in other species. The two halves of the predicted gene were treated as separate peptides in the phylogenetic tree and the diagram.

Figure 3
Figure 3

Phylogenetic tree of the kinases. Details of the tree are as in Figure 2.

Figure 4
Figure 4

Phylogenetic tree of the interferon response factors. Details are as in Figure 2. The chicken (Gg [Gallus gallus]) IRF10 was included to show its relationship to fish IRF10, because no ortholog for this gene is found in mammals. IRF, interferon response factor.

Figure 5
Figure 5

Phylogenetic tree of the STAT proteins. Details are as in Figure 2. STAT, signal transducer and activator of transcription.

Figure 6
Figure 6

Phylogenetic tree of the TRAFs and diagrams of their protein domain structure. Details are as in Figure 2, except that the scale shows 0.2 amino acid substitutions. TRAF, tumor necrosis factor receptor-associated factor.

Figure 7
Figure 7

Synteny between regions containing STAT4 and STAT1 genes on human chromosome 2 and Danio rerio chromosomes 22 and 9. Genes transcribed on the top or bottom strands are shown above and below the lines representing the chromosomes. Homologous regions are shown by colored arrows. A further duplication of the region containing HIBCH and GDF8 is found on Danio rerio chromosome 11. Numbers represent nucleotide positions in the genome in megabases based on the Zv6 assembly. Gene names are Swissprot, Zebrafish Information Network, or Ensembl identifiers.

Figure 8
Figure 8

Syntenic organization of classII cytokine receptor genes. (a) Diagram of the structures of the mammalian receptor chains, with the blue and green rectangles representing the S100A and S100B domains, the red rectangle the intracellular domain of the ligand binding chains, and the gray rectangle the intracellular domain of the non-ligand-binding chains and TF (after Renauld [57]). (b) Synteny between regions containing class II cytokine receptor genes in mammals and fish. Fat horizontal lines indicate chromosomes in the four species. The brackets above the human genes show evolutionary relationships between the paralogs. Vertical broken lines indicate suggested evolutionary relationships between the genes in the different species, based on the tree in Figure 9. Color coding of names: red = long intracellular domain; black = short intracellular domain; blue = no intracellular domain; and pink = intermediate length intracellular domain. Circled names indicate ligand binding chains. Round brackets denote groups of genes in cases where there are no clear orthologous relationships of individual members with genes in the other species.

Figure 9
Figure 9

Phylogenetic tree of the class II cytokine receptors. Details are as in figure 2.

Figure 10
Figure 10

Phylogenetic tree for the classII cytokines. Details are as in Figure 2. See text for gene names.

Figure 11
Figure 11

Genomic organization of two class II cytokine gene clusters. Chromosomes are shown as lines with the positions of the region marked in megabase pairs underneath. Genes transcribed on the top strand are shown above the line, and those transcribed in the opposite direction are shown below. Class II cytokine encoding genes are shaded in gray. In the left diagram the syntenic regions and duplications, and inversions surrounding the IL-10 locus are shaded in red and blue. The human IL-10 gene is located on chromosome 1 and the region shows the same arrangement as in the mouse. The current zebrafish genome assembly Zv7 does not yet contain the recently sequenced clone CU459075, which places IL-34 into the interval between IL-10 and prolargin (IL-34 is included in Zv7 on the unplaced contig Zv7_NA1656). There are therefore no coordinates for the right end of the interval. The two pufferfish show the same arrangement both for the region around IL-10 and for the MDM1/cytokine/IFN-γ region. The names for the fish genes are explained in the text. IFN, interferon; IL, interleukin.

Figure 13
Figure 13

Phylogenetic tree of NACHT proteins shared by mammals and fish and diagram of their protein structures. In addition to the known proteins Nod1, Nod2, Nod3, Nod9, CIITA, and Apaf, this tree shows that a new protein is shared by all five species, which we have named NACHT-P1. The protein domain structure diagram shown next to NACHT-P3 is representative of the majority of the novel fish proteins. Apaf, apoptotic protease activating factor; CIITA, major histocompatibility complex class II, transactivator; Nod, nucleotide oligomerization domain containing protein.

Figure 14
Figure 14

The fish-specific domain upstream of the NACHT domain. (a) Alignment of a representative subset of the Fisna domain (the region upstream of the NACHT domain that is shared by all of the novel fish NLR proteins. The group names on the right refer to the subdivision of the Danio rerio groups according to similarities in the NACHT-domain and the Fisna domain (also see Figure 15) or indicate which species form the group. Peptide motifs with similarity to Nod2 and Nod3 are underlined. (b) Hidden Markov model (HMM) logo representing the consensus sequence of the Fisna domain in all three fish species. The logo has been generated using the software HMMER [58,59] and visualized using the HMM-Logo web server [60,61]. Peptide sequences from human Nod2 and Nod3 with similarity to short stretches of the Fisna consensus, color coded to highlight conserved residues, are listed underneath, as are stretches from the regions upstream of the NACHT domain present in 140 sea urchin NLR proteins. NLR, nucleotide-binding domain/NACHT domain and leucine rich repeat containing family; Nod, nucleotide oligomerization domain containing protein.

Figure 12
Figure 12

Overview of a phylogenetic tree of 277 NLR proteins. Each sequence is assigned a background color to illustrate species relationships: pink = human, yellow = mouse, blue = zebrafish, green = Takifugu, and turquoise = Tetraodon. The 'canonical' proteins Nod1, Nod2, Nod3, Nod9, CIITA, and Apaf, which show clear homologous relationships between the five species, cluster at the top (rainbow colors). The mammalian Nalp proteins cluster together (pink/yellow region). Each fish has a large group of species-specific proteins (blue, green, and turquoise regions). In addition, Takifugu and Tetraodon share several apparently orthologous gene pairs (green and turquoise region). Apaf, apoptotic protease activating factor; CIITA, major histocompatibility complex class II, transactivator; Nalp, NACHT, leucine rich repeat and PYD containing protein; NLR, nucleotide-binding domain/NACHT domain and leucine rich repeat containing family; Nod, nucleotide oligomerization domain containing protein.

Figure 15
Figure 15

Structure of the amino-termini of the new zebrafish NLR proteins. ClustalW alignment of the set of 70 predicted NLR proteins was truncated four amino acids downstream of the start of the Fisna domain, and the alignment of the remaining amino-terminal sequences was edited manually using Jalview [62]. Sequences that did not extend significantly beyond the Fisna domain were deleted, as were some sequences in groups with many similar or identical sequences. The remaining sequences represent a set of characteristic compositions of motifs found in the amino-terminal part of this family of proteins. (a) Overview of the alignment with characteristic sequence motifs shaded in color: green = pyrin-like domain in groups 1 and 2; blue = repeated motif (different shades of blue mark different versions of the repeat); yellow/orange tones = conserved amino-terminal amino acids; and pink = specific amino-terminal peptide of 14 amino acids. (b) Details of the alignment in panel a in which amino acid similarities and identities are highlighted in ClustalW colors. A set of mammalian PYD domains are aligned above the zebrafish group 1 and group 2 pyrin-like domains to illustrate the similarity. NLR, nucleotide-binding domain/NACHT domain and leucine rich repeat containing family.

Figure 16
Figure 16

Chromosomal locations of zebrafish NLR proteins. The 11 chromosomes containing the main clusters of NLR genes are shown. The number of NLR genes on each chromosome is listed below the chromosome number. A further 42 genes) are distributed on 11 other chromosomes, and 20 genes are on as yet unplaced contigs. This list includes a compilation of all predictions (automated as well as manually annotated) and locations of hits from a TBLASTN search for NACHT domains. Future improvements of the genome assembly and further manual annotations will most likely result in minor changes of this map. Genes are denoted by lines on the right of the chromosome irrespective of orientation. NLR, nucleotide-binding domain/NACHT domain and leucine rich repeat containing family.

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