Toll-Like Receptor Evolution: Does Temperature Matter?

Abstract

Toll-like receptors (TLRs) recognize conserved pathogen-associated molecular patterns (PAMPs) and are an ancient and well-conserved group of pattern recognition receptors (PRRs). The isolation of the Antarctic continent and its unique teleost fish and microbiota prompted the present investigation into Tlr evolution. Gene homologues of tlr members in teleosts from temperate regions were present in the genome of Antarctic Nototheniidae and the non-Antarctic sister lineage Bovichtidae. Overall, in Nototheniidae apart from D. mawsoni, no major tlr gene family expansion or contraction occurred. Instead, lineage and species-specific changes in the ectodomain and LRR of Tlrs occurred, particularly in the Tlr11 superfamily that is well represented in fish. Positive selective pressure and associated sequence modifications in the TLR ectodomain and within the leucine-rich repeats (LRR), important for pathogen recognition, occurred in Tlr5, Tlr8, Tlr13, Tlr21, Tlr22, and Tlr23 presumably associated with the unique Antarctic microbiota. Exposure to lipopolysaccharide (Escherichia coli O111:B4) Gram negative bacteria did not modify tlr gene expression in N. rossii head-kidney or anterior intestine, although increased water temperature (+4°C) had a significant effect.

Keywords: Antarctic fish; TLR; cold temperature; evolution; immune challenge; innate immunity.

Figure 1

Detailed dendrogram of the tlr genes/transcripts in Nototheniidae. Genes/transcripts identified in Nototheniidae were distributed within five vertebrate TLR superfamilies and are indicated by the colored circles. When multiple genes were identified for a given tlr isoform, gene number is indicated inside the colored circles. Genes that were not identified are indicated with a white circle. C. gobio was used as a representative of the Nototheniidae sister lineage and G. aculeatus was used as a representative of the other Perciform species outside the Notothenioidei sub-order. Tlr gene family members from all species analyzed were obtained by searching their genome assemblies. The exception was N. rossii where an “in house” de novo multi-tissue transcriptome assembly was used. The figure was drawn considering the relative evolutionary relationship between the six Antarctic fish species and the sub-Antarctic fish (35, 64). Gene/transcript accession numbers are available in Supplementary Table 1 .

Figure 2

Simplified phylogenetic tree of the Tlrs from Nototheniidae and other vertebrates. The phylogenetic tree was constructed using the BI method and the full tree is available as Supplementary Figure 3 . Branches corresponding to the six vertebrate TLR superfamilies are identified: S1 indicates the TLR1 superfamily (orange); S3 identifies the TLR3 superfamily (red); S4 identifies the TLR4 superfamily (pink); S5 identifies the TLR5 superfamily; S7 identifies the TLR7 superfamily (blue); and S11 identifies the TLR11 superfamily (green). Some branches of the phylogenetic tree are collapsed to facilitate visualization and three subsections of the same phylogenetic tree are shown: (A) the TLR1 superfamily, (B) the TLR11 and TLR3 superfamilies, and (C) the TLR5, TLR7, and TLR4 superfamilies. The tetrapod branches were also collapsed. The accession numbers of the sequences used to construct the phylogenetic tree are available in Supplementary Table 1 . The teleost tlr2 duplicates were named, tlr2a and tlr2b, and TLR15 was not included in the phylogenetic tree since it was only found in chicken and lizard. The phylogenetic tree was rooted with the Cnidarian Tlr clade (31). The sea lamprey tlrs within the vertebrate TLR1 and TLR7 superfamilies are named Pma_X and Pma_Y, respectively, since their assignment to the Tlr subfamilies was ambiguous. Only the posterior probability values for the main branches are indicated. A phylogenetic tree generated by the ML method is available as Supplementary Figure 2 . Members from Antarctic species are indicated with squares and with a Notothenia cartoon to facilitate identification. The other member of the Perciform order, G. aculeatus, is indicated by a black dot and the Pleuronectiformes is indicated by a black star.

Figure 3

Gene synteny analysis of tlr1, tlr2, tlr5, tlr5S, tlr8, and tlr23 in Antarctic Nototheniidae and other vertebrates. Presented are the Antarctic Nototheniidae P. georgianus, T. bernacchii, and D. mawsoni. Other vertebrates include C. gobio as the representative of the sister lineage, G. aculeatus as a non-Antarctic representative of the Perciform order, one representative of the Pleuronectiformes order, C. semilaevis, and H. sapiens as the tetrapod. Genome regions analyzed are indicated by a line and predicted genes are represented by arrows and the arrowhead indicates gene orientation and the gene symbol is given. The gene positions in the genome assemblies analyzed (Mega base pairs, Mbp) are indicated below each synteny map. Tlr genes are represented by full colored arrows. Extended analysis including more species are available as Supplementary Figures 6–8 . The red cross indicates gene absence.

Figure 4

Evidence of positive selection in the LRR ectodomains of Nototheniidae Tlrs. Receptors represented are Tlr8 and Tlr21 and Tlr22. (A) Positive selection identified with the branch-site model is represented by colored shapes on the ancestral Nototheniidae branch and on the Tlr21 duplicate in G. acuticeps (Tlr21_2) and PSS were only found for Tlr21_2 and are boxed. (B) PSS identified with the branch-site and site models for Nototheniidae sequences. PSS were colored according to their physicochemical properties: acidic in red, basic in blue, neutral in purple, polar in green, and hydrophobic in black using Weblogo vs3 annotation. The amino acid positions given refer to the edited multiple sequence alignment used for the selective pressure analysis. The PSS represented were mapped in the sequences and are marked in red ( Supplementary Figures 4–8 ).

Figure 5

Explaining TLR evolution in Nototheniidae. The Antarctic fish speciation and associated unique adaptations to extreme cold occurred 10–20 million years ago (Mya) but did not result in profound modifications of the TLR gene complement. Representatives of the five vertebrate TLR superfamilies exist in Nototheniids, and gene number and evolutionary origin were common with those in other teleosts and vertebrates. Notable changes in TLRs (Tlr5, Tlr8, Tlr13, Tlr21, Tlr22, and Tlr23) arose from positive evolutionary pressure acting on the LRR motifs that determine receptor function and pattern recognition due presumably to the unique Antarctic microbiome. Tlr expression was temperature sensitive and indicates that pathogen detection may be modified under future climate change scenarios. The five TLR superfamilies (S) are represented by numbers and with colored circles. The positive selective pressure sites are highlighted on the receptor conceptual structure with red lines and red circles. LRR: leucine-rich repeat, TM: transmembrane, and TIR: Toll-interleukin 1 receptor. Speciation times for fish lineages were obtained from (35). The scheme is not drawn to scale.