Toll-Like Receptor Evolution in Birds: Gene Duplication, Pseudogenization, and Diversifying Selection

Abstract

Toll-like receptors (TLRs) are key sensor molecules in vertebrates triggering initial phases of immune responses to pathogens. The avian TLR family typically consists of ten receptors, each adapted to distinct ligands. To understand the complex evolutionary history of each avian TLR, we analyzed all members of the TLR family in the whole genome assemblies and target sequence data of 63 bird species covering all major avian clades. Our results indicate that gene duplication events most probably occurred in TLR1 before synapsids diversified from sauropsids. Unlike mammals, ssRNA-recognizing TLR7 has duplicated independently in several avian taxa, while flagellin-sensing TLR5 has pseudogenized multiple times in bird phylogeny. Our analysis revealed stronger positive, diversifying selection acting in TLR5 and the three-domain TLRs (TLR10 [TLR1A], TLR1 [TLR1B], TLR2A, TLR2B, TLR4) that face the extracellular space and bind complex ligands than in single-domain TLR15 and endosomal TLRs (TLR3, TLR7, TLR21). In total, 84 out of 306 positively selected sites were predicted to harbor substitutions dramatically changing the amino acid physicochemical properties. Furthermore, 105 positively selected sites were located in the known functionally relevant TLR regions. We found evidence for convergent evolution acting between birds and mammals at 54 of these sites. Our comparative study provides a comprehensive insight into the evolution of avian TLR genetic variability. Besides describing the history of avian TLR gene gain and gene loss, we also identified candidate positions in the receptors that have been likely shaped by direct molecular host-pathogen coevolutionary interactions and most probably play key functional roles in birds.

Keywords: adaptive evolution; amino acid physicochemical properties; convergence; pattern recognition receptors; positive selection; pseudogene.

Fig. 1.

Phylogenetic tree based on nonconverted regions of TLR1 subfamily members. The bootstrap values of maximum likelihood analysis obtained using PhyML and the posterior probability of Bayesian analysis obtained using MrBayes (in percentage per each node) are provided. Birds are represented by zebra finch (TaGu, Taeniopygia guttata) and chicken (GaGa, Gallus gallus), crocodiles by alligator (AlMi, Alligator mississippiensis), turtles by painted turtle (ChPi, Chrysemys picta), mammals by human (HoSa, Homo sapiens) and horse (EqCa, Equus caballus), amphibians by clawed frog (XeTr, Xenopus tropicalis), bony fish by zebrafish (DaRe, Danio rerio) and cartilaginous fish by shark (ChGr, Chiloscyllium griseum). The analysis was performed using a single amino acid sequence per TLR and species. Based on the results, we suggest renaming TLR1A to TLR10 and TLR1B to TLR1 in birds.

Fig. 2.

Avian TLR7 duplication. In the schematic avian phylogenetic tree the species with duplicated TLR7 are highlighted in teal rectangles. The numbers of amino acid substitutions distinguishing the two copies of the duplicated TLR7 are shown behind the species name. The analysis was performed using a single sequence per gene and species.

Fig. 3.

TLR5 pseudogenization in birds. Species possessing only TLR5 pseudogene are highlighted in red rectangles within the schematic representation of avian phylogenetic tree. The position of the first stop codon is indicated by the number provided behind the species name (position numbering according to the chicken reference). The analysis was performed using a single sequence per gene and species.

Fig. 4.

Physicochemical properties of the positively selected sites (PSS). All PSS are shown in all avian TLRs—amino acid substitutions are colored according to their physicochemical properties: acidic in red, basic in blue, neutral in purple, polar in green, and hydrophobic in black. The size of an letter corresponds to the procentual proportion of that particular amino acid within the sequence alignment. The numbering is adopted from reference chicken TLRs (for NCBI IDs see supplementary material S1: table S14, Supplementary Material online). PSS which correspond to functionaly important residues (black dot—ligand binding; gray dot—dimerization) are highlighted in bold and orange (identical site) or yellow (topological proximity closer than 5 Å from a functionaly important residue). Ectodomain (ECD), intracellular domain (ICD), and transmembrane (TM) region are visualized; nonconverted region in TLR1/2 is highlighted in pink; cleavage site in TLR3, TLR7, and TLR15 is indicated by a black line tipped with arrows (supplementary material S1: table S11 and text S1, Supplementary Material online). Nonconservative PSS are marked by stars (supplementary material S1: table S12, Supplementary Material online).

Fig. 5.

Positively selected sites and functionaly important sites visualized on 3D extracellular domain structures of avian TLRs. PSS detected in this study are shown in blue. By orange coloration are highlighted the PSS identified at sites with previously described function. Other previously reported functionaly important residues are highlighted in black (ligand binding residues) or in gray (dimerization residues). The total numbers of PSS for each TLR are shown in blue rectangles, where in parentheses are numbers of PSS detected also in other avian/mammalian/both studies (for references see supplementary material S6: table S27, Supplementary Material online). The numbers of PSS with previously described function are provided in orange rectangles, where the number of PSS in topological proximity <5 Å is shown in parentheses. For more detailed information (including ICD and TM), see supplementary material S9, Supplementary Material online.