Vertebrate Toll-like Receptors Research Articles

Ancient fish lineages illuminate toll-like receptor diversification in early vertebrate evolution.

Since its initial discovery over 50years ago, understanding the evolution of the vertebrate RAG- mediated adaptive immune response has been a major area of research focus for comparative geneticists. However, how the evolutionary novelty of an adaptive immune response impacted the diversity of receptors associated with the innate immune response has received considerably less attention until recently. Here, we investigate the diversification of vertebrate toll-like receptors (TLRs), one of the most ancient and well conserved innate immune receptor families found across the Tree of Life, integrating genomic data that represent all major vertebrate lineages with new transcriptomic data from Polypteriformes, the earliest diverging ray-finned fish lineage. Our analyses reveal TLR sequences that reflect the 6 major TLR subfamilies, TLR1, TLR3, TLR4, TLR5, TLR7, and TLR11, and also currently unnamed, yet phylogenetically distinct TLR clades. We additionally recover evidence for a pulse of gene gain coincident with the rise of the RAG-mediated adaptive immune response in jawed vertebrates, followed by a period of rapid gene loss during the Cretaceous. These gene losses are primarily concentrated in marine teleost fish and synchronous with the mid Cretaceous anoxic event, a period of rapid extinction for marine species. Finally, we reveal a mismatch between phylogenetic placement and gene nomenclature for up to 50% of TLRs found in clades such as ray-finned fishes, cyclostomes, amphibians, and elasmobranchs. Collectively, these results provide an unparalleled perspective of TLR diversity and offer a ready framework for testing gene annotations in non-model species.

Analysis and Prediction of Pathogen Nucleic Acid Specificity for Toll-like Receptors in Vertebrates

Identification of key sequence, expression and function related features of nucleic acid-sensing host proteins is of fundamental importance to understand the dynamics of pathogen-specific host responses. To meet this objective, we considered toll-like receptors (TLRs), a representative class of membrane-bound sensor proteins, from 17 vertebrate species covering mammals, birds, reptiles, amphibians, and fishes in this comparative study. We identified the molecular signatures of host TLRs that are responsible for sensing pathogen nucleic acids or other pathogen-associated molecular patterns (PAMPs), and potentially play important roles in host defence mechanism. Interestingly, our findings reveal that such host-specific features are directly related to the strand (single or double) specificity of nucleic acid from pathogens. However, during host-pathogen interactions, such features were unable to explain the pathogenic PAMP (i.e., DNA, RNA or other) selectivity, suggesting a more complex mechanism. Using these features, we developed a number of machine learning models, of which Random Forest achieved a high performance (94.57% accuracy) to predict strand specificity of TLRs from protein-derived features. We applied the trained model to propose strand specificity of some previously uncharacterized distinct fish-specific novel TLRs (TLR18, TLR23, TLR24, TLR25, TLR27).

Bivalves Present the Largest and Most Diversified Repertoire of Toll-Like Receptors in the Animal Kingdom, Suggesting Broad-Spectrum Pathogen Recognition in Marine Waters.

Toll-like receptors (TLRs) are the most widespread class of membrane-bound innate immune receptors, responsible of specific pathogen recognition and production of immune effectors through the activation of intracellular signaling cascades. The repertoire of TLRs was analyzed in 85 metazoans, enriched on molluscan species, an underrepresented phylum in previous studies. Following an ancient evolutionary origin, suggested by the presence of TLR genes in Anthozoa (Cnidaria), these receptors underwent multiple independent gene family expansions, the most significant of which occurred in bivalve molluscs. Marine mussels (Mytilus spp.) had the largest TLR repertoire in the animal kingdom, with evidence of several lineage-specific expanded TLR subfamilies with different degrees of orthology conservation within bivalves. Phylogenetic analyses revealed that bivalve TLR repertoires were more diversified than their counterparts in deuterostomes or ecdysozoans. The complex evolutionary history of TLRs, characterized by lineage-specific expansions and losses, along with episodic positive selection acting on the extracellular recognition domains, suggests that functional diversification might be a leading evolutionary force. We analyzed a comprehensive transcriptomic data set from Mytilus galloprovincialis and built transcriptomic correlation clusters with the TLRs expressed in gills and in hemocytes. The implication of specific TLRs in different immune pathways was evidenced, as well as their specific modulation in response to different biotic and abiotic stimuli. We propose that, in a similar fashion to the remarkable functional specialization of vertebrate TLRs, the expansion of the TLR gene family in bivalves attends to a functional specification motivated by the biological particularities of these organisms and their living environment.

Evolutionary History of the Toll-Like Receptor Gene Family across Vertebrates.

Adaptation to a wide range of pathogenic environments is a major aspect of the ecological adaptations of vertebrates during evolution. Toll-like receptors (TLRs) are ancient membrane-bound sensors in animals and are best known for their roles in detecting and defense against invading pathogenic microorganisms. To understand the evolutionary history of the vertebrate TLR gene family, we first traced the origin of single-cysteine cluster TLRs that share the same protein architecture with vertebrate TLRs in early-branching animals and then analyzed all members of the TLR family in over 200 species covering all major vertebrate clades. Our results indicate that although the emergence of single-cysteine cluster TLRs predates the separation of bilaterians and cnidarians, most vertebrate TLR members originated shortly after vertebrate emergence. Phylogenetic analyses divided 1,726 vertebrate TLRs into 8 subfamilies, and TLR3 may represent the most ancient subfamily that emerged before the branching of deuterostomes. Our analysis reveals that purifying selection predominated in the evolution of all vertebrate TLRs, with mean dN/dS (ω) values ranging from 0.082 for TLR21 in birds to 0.434 for TLR11 in mammals. However, we did observe patterns of positive selection acting on specific codons (527 of 60,294 codons across all vertebrate TLRs, 8.7‰), which are significantly concentrated in ligand-binding extracellular domains and suggest host–pathogen coevolutionary interactions. Additionally, we found stronger positive selection acting on nonviral compared with viral TLRs, indicating the more essential nonredundant function of viral TLRs in host immunity. Taken together, our findings provide comprehensive insight into the complex evolutionary processes of the vertebrate TLR gene family, involving gene duplication, pseudogenization, purification, and positive selection.

Northeast Chinese lamprey (Lethenteron morii) MyD88: Identification, expression, and functional characterization

Myeloid differentiation factor 88 (MyD88) is a key adaptor of Toll-like receptors (TLR), an important pattern recognition receptor of the innate immune system. To study the origin and evolution of the vertebrate TLR signaling pathway in innate immune systems, we analyzed the biological characteristics and functions of the MyD88 gene in Northeast Chinese lamprey (Lethenteron morii) using PCR amplification, real-time PCR analysis, dual luciferase reporter gene assay, immunofluorescence assay, and other methods. Bioinformatics analysis showed that LmMyD88 has a modular structure consisting of Toll/IL-1R domain (TIR) and death domain (DD), which is typical of the MyD88 family. A phylogenetic tree showed that the homology of LmMyD88 was consistent with the phylogenetic status of lampreys. Tissue expression analysis indicated that the mRNA expression was expressed in some normal tissues of larval and adult L. morii. Real-time PCR analysis showed that the expression of LmMyD88 in tissues, such as gill and kidney, of the adult increased significantly after infection by Pseudomonas aeruginosa. Subcellular localization results showed that LmMyD88 was expressed in the nucleus, cytoplasm, and other parts. A dual luciferase reporter assay indicated that LmMyD88 activated nuclear factor kappa B downstream of the TLR signaling pathway. This study suggested that LmMyD88 might play an important role in the innate immune signal transduction process of L. morii.

The Vertebrate TLR Supergene Family Evolved Dynamically by Gene Gain/Loss and Positive Selection Revealing a Host–Pathogen Arms Race in Birds

The vertebrate toll-like receptor (TLRs) supergene family is a first-line immune defense against viral and non-viral pathogens. Here, comparative evolutionary-genomics of 79 vertebrate species (8 mammals, 48 birds, 11 reptiles, 1 amphibian, and 11 fishes) revealed differential gain/loss of 26 TLRs, including 6 (TLR3, TLR7, TLR8, TLR14, TLR21, and TLR22) that originated early in vertebrate evolution before the diversification of Agnatha and Gnathostomata. Subsequent dynamic gene gain/loss led to lineage-specific diversification with TLR repertoires ranging from 8 subfamilies in birds to 20 in fishes. Lineage-specific loss of TLR8-9 and TLR13 in birds and gains of TLR6 and TLR10-12 in mammals and TLR19-20 and TLR23-27 in fishes. Among avian species, 5–10% of the sites were under positive selection (PS) (omega 1.5–2.5) with radical amino-acid changes likely affecting TLR structure/functionality. In non-viral TLR4 the 20 PS sites (posterior probability PP > 0.99) likely increased ability to cope with diversified ligands (e.g., lipopolysaccharide and lipoteichoic). For viral TLR7, 23 PS sites (PP > 0.99) possibly improved recognition of highly variable viral ssRNAs. Rapid evolution of the TLR supergene family reflects the host–pathogen arms race and the coevolution of ligands/receptors, which follows the premise that birds have been important vectors of zoonotic pathogens and reservoirs for viruses.

Ectodomain Architecture Affects Sequence and Functional Evolution of Vertebrate Toll-like Receptors.

Toll-like receptors (TLRs) are crucial components of innate immunity that specifically recognize diverse pathogen-associated molecular patterns from pathogens. The continuous hydrogen-bond network (asparagine ladder) formed among the asparagine residues on the concave surfaces of neighboring leucine-rich repeat modules assists in stabilizing the overall shape of TLR ectodomains responsible for ligand recognition. Analysis of 28 types of vertebrate TLRs showed that their ectodomains possessed three types of architectures: a single-domain architecture with an intact asparagine ladder, a three-domain architecture with the ladder interrupted in the middle, and a trans-three-domain architecture with the ladder broken in both termini. Based on a phylogenetic analysis, the three vertebrate TLR architectures arose during early evolution. The 1428 vertebrate TLRs can be divided into eight families based on sequence and structural differences. TLRs ligand specificities are affected by their ectodomain architectures. Three-domain TLRs bind hydrophobic ligands, whereas single-domain and trans-three-domain TLRs mainly recognize hydrophilic ligands. Analysis of 39 vertebrate genomes suggested that the number of single-domain TLR genes in terrestrial vertebrate genomes decreased by half compared to aquatic vertebrate genomes. Single-domain TLR genes underwent stronger purifying selective pressures than three-domain TLR genes in mammals. Overall, ectodomain architecture influences the sequence and functional evolution of vertebrate TLRs.

Evolutionary redesign of the Atlantic cod (Gadus morhua L.) Toll-like receptor repertoire by gene losses and expansions.

Genome sequencing of the teleost Atlantic cod demonstrated loss of the Major Histocompatibility Complex (MHC) class II, an extreme gene expansion of MHC class I and gene expansions and losses in the innate pattern recognition receptor (PRR) family of Toll-like receptors (TLR). In a comparative genomic setting, using an improved version of the genome, we characterize PRRs in Atlantic cod with emphasis on TLRs demonstrating the loss of TLR1/6, TLR2 and TLR5 and expansion of TLR7, TLR8, TLR9, TLR22 and TLR25. We find that Atlantic cod TLR expansions are strongly influenced by diversifying selection likely to increase the detectable ligand repertoire through neo- and subfunctionalization. Using RNAseq we find that Atlantic cod TLRs display likely tissue or developmental stage-specific expression patterns. In a broader perspective, a comprehensive vertebrate TLR phylogeny reveals that the Atlantic cod TLR repertoire is extreme with regards to losses and expansions compared to other teleosts. In addition we identify a substantial shift in TLR repertoires following the evolutionary transition from an aquatic vertebrate (fish) to a terrestrial (tetrapod) life style. Collectively, our findings provide new insight into the function and evolution of TLRs in Atlantic cod as well as the evolutionary history of vertebrate innate immunity.

Identification and functional characterization of nonmammalian Toll-like receptor 20

Like other vertebrate Toll-like receptors (TLRs), the TLRs of teleost fish can be subdivided into six major families, each of which recognize a general class of molecular patterns. However, there also are a number of Tlrs with unknown function, the presence of which seems unique to the bony fish, among which is Tlr20. We identified full-length complementary DNA (cDNA) sequences for tlr20 of zebrafish and common carp, two closely related fish species. Zebrafish have six copies of tlr20, whereas carp express only a single copy. Both zebrafish Tlr20 (at least Tlr20a-d) and carp Tlr20 have 26 leucine-rich repeats (LRRs). Three-dimensional modeling indicates a best fit to the crystal structure of TLR8. Phylogenetic analyses place Tlr20 in the TLR11 family closest to Tlr11 and Tlr12, which sense ligands from protozoan parasites in the mouse. Conservation of genes on zebrafish chromosome 9, which carries tlr20, with genes on mouse chromosome 14, which carries tlr11, indicates Tlr11 could be a possible ortholog of Tlr20. Confocal microscopy suggests a subcellular localization of Tlr20 at the endoplasmatic reticulum. Although in vitro reporter assays could not identify a ligand unique to Tlr20, in vivo infection experiments indicate a role for Tlr20 in the immune response of carp to protozoan parasites (Trypanoplasma borreli). Carp tlr20 is mainly expressed in peripheral blood leukocytes (PBL) with B lymphocytes, in particular, expressing relatively high levels of Tlr20. In vitro stimulation of PBL with T. borreli induces an upregulation of tlr20, supportive of a role for Tlr20 in the immune response to protozoan parasites.

Central role of liver in anticancer and radioprotective activities of Toll-like receptor 5 agonist.

Vertebrate Toll-like receptor 5 (TLR5) recognizes bacterial flagellin proteins and activates innate immune responses to motile bacteria. In addition, activation of TLR5 signaling can inhibit growth of TLR5-expressing tumors and protect normal tissues from radiation and ischemia-reperfusion injuries. To understand the mechanisms behind these phenomena at the organismal level, we assessed nuclear factor kappa B (NF-κB) activation (indicative of TLR5 signaling) in tissues and cells of mice treated with CBLB502, a pharmacologically optimized flagellin derivative. This identified the liver and gastrointestinal tract as primary CBLB502 target organs. In particular, liver hepatocytes were the main cell type directly and specifically responding to systemic administration of CBLB502 but not to that of the TLR4 agonist LPS. To assess CBLB502 impact on other pathways, we created multireporter mice with hepatocytes transduced in vivo with reporters for 46 inducible transcription factor families and found that along with NF-κB, CBLB502 strongly activated STAT3-, phenobarbital-responsive enhancer module (PREM), and activator protein 1 (AP-1-) -driven pathways. Livers of CBLB502-treated mice displayed induction of numerous immunomodulatory factors and massive recruitment of various types of immune cells. This led to inhibition of growth of liver metastases of multiple tumors regardless of their TLR5 status. The changed liver microenvironment was not, however, hepatotoxic, because CBLB502 induced resistance to Fas-mediated apoptosis in normal liver cells. Temporary occlusion of liver blood circulation prevented CBLB502 from protecting hematopoietic progenitors in lethally irradiated mice, indicating involvement of a factor secreted by responding liver cells. These results define the liver as the key mediator of TLR5-dependent effects in vivo and suggest clinical applications for TLR5 agonists as hepatoprotective and antimetastatic agents.

Sequence specific detection of bacterial 23S ribosomal RNA by TLR13

Toll-like receptors (TLRs) detect microbial infections and trigger innate immune responses. Among vertebrate TLRs, the role of TLR13 and its ligand are unknown. Here we show that TLR13 detects the 23S ribosomal RNA of both gram-positive and gram-negative bacteria. A sequence containing 13 nucleotides near the active site of 23S rRNA ribozyme, which catalyzes peptide bond synthesis, was both necessary and sufficient to trigger TLR13-dependent interleukin-1β production. Single point mutations within this sequence destroyed the ability of the 23S rRNA to stimulate the TLR13 pathway. Knockout of TLR13 in mice abolished the induction of interleukin-1β and other cytokines by the 23S rRNA sequence. Thus, TLR13 detects bacterial RNA with exquisite sequence specificity.DOI:http://dx.doi.org/10.7554/eLife.00102.001.

MyD88-deficient Hydra reveal an ancient function of TLR signaling in sensing bacterial colonizers

Toll-like receptor (TLR) signaling is one of the most important signaling cascades of the innate immune system of vertebrates. Studies in invertebrates have focused on the fruit fly Drosophila melanogaster and the nematode Caenorhabditis elegans, and there is little information regarding the evolutionary origin and ancestral function of TLR signaling. In Drosophila, members of the Toll-like receptor family are involved in both embryonic development and innate immunity. In C. elegans, a clear immune function of the TLR homolog TOL-1 is controversial and central components of vertebrate TLR signaling including the key adapter protein myeloid differentiation primary response gene 88 (MyD88) and the transcription factor NF-κB are not present. In basal metazoans such as the cnidarians Hydra magnipapillata and Nematostella vectensis, all components of the vertebrate TLR signaling cascade are present, but their role in immunity is unknown. Here, we use a MyD88 loss-of-function approach in Hydra to demonstrate that recognition of bacteria is an ancestral function of TLR signaling and that this process contributes to both host-mediated recolonization by commensal bacteria as well as to defense against bacterial pathogens.

Molecular evolution of vertebrate Toll-like receptors: Evolutionary rate difference between their leucine-rich repeats and their TIR domains

Toll-like receptors (TLRs) that initiate an innate immune response contain an extracellular leucine rich repeat (LRR) domain and an intracellular Toll IL-receptor (TIR) domain. There are fifteen different TLRs in vertebrates. The LRR domains, which adopt a solenoid structure, usually have higher rates of evolution than do the TIR globular domains. It is important to understand the molecular evolution and functional roles of TLRs from this standpoint. Both pairwise genetic distances and Ka/Ks's (the ratios between non synonymous and synonymous substitution rates) were compared between the LRR domain and the TIR domain of 366 vertebrate TLRs from 96 species (from fish to primates). In fourteen members (TLRs 1, 2, 3, 4, 5, 6, 7, 8, 9, 11/12, 13, 14, 21, and 22/23) the LRR domains evolved significantly more rapidly than did the corresponding TIR domains. The evolutionary rates of the LRR domains are significantly different among these members; LRR domains from TLR3 and TLR7 from primates to fishes have the lowest rate of evolution. In contrast, the fifteenth member, TLR10, shows no significant differences; its TIR domain is not highly conserved. The present results suggest that TLR10 may have a different function in signaling from those other members and that a higher conservation of TLR3 and TLR7 may reflect a more ancient mechanism and/or structure in the innate immune response system. Gene conversions are suggested to have occurred in platypus TLR6 and TLR10. This study provides new insight about structural and functional diversification of vertebrate TLRs.

Toll-Like Receptors of Deuterostome Invertebrates

Defensive systems against pathogens are responsible not only for survival or lifetime of an individual but also for the evolution of a species. Innate immunity is expected to be more important for invertebrates than mammals, given that adaptive immunity has not been acquired in the former. Toll-like receptors (TLRs) have been shown to play a crucial role in host defense of pathogenic microbes in innate immunity of mammals. Recent genome-wide analyses have suggested that TLR or their related genes are conserved in invertebrates. In particular, numerous TLR-related gene candidates were detected in deuterostome invertebrates, including a sea urchin (222 TLR-related gene candidates) and amphioxus (72 TLR-related gene candidates). Molecular phylogenetic analysis verified that most of sea urchin or amphioxus TLR candidates are paralogous, suggesting that these organisms expanded TLR-related genes in a species-specific manner. In contrast, another deuterostome invertebrate, the ascidian Ciona intestinalis, was found to possess only two TLR genes. Moreover, Ciona TLRs, Ci-TLR1 and Ci-TLR2, were shown to possess “hybrid” functionality of mammalian TLRs. Such functionality of Ci-TLRs could not be predicted by sequence comparison with vertebrate TLRs, indicating confounding evolutionary lineages of deuterostome invertebrate TLRs or their candidates. In this review article, we present recent advances in studies of TLRs or their candidates among deuterostome invertebrates, and provide insight into an evolutionary process of TLRs.

Domain combination of the vertebrate-like TLR gene family: implications for their origin and evolution

Domain shuffling, which is an important mechanism in the evolution of multi-domain proteins, has shaped the evolutionary development of the immune system in animals. Toll and Toll-like receptors (TLRs) are a class of proteins that play a key role in the innate and adaptive immune systems. Draft genome sequences provide the opportunity to compare the Toll/TLR gene repertoire among representative metazoans. In this study, we investigated the combination of Toll/interleukin-1 receptor (TIR) and leucine-rich repeat (LRR) domains of metazoan Toll/TLRs. Before Toll with both domains occurred in Cnidaria (sea anemone, Nematostella vectensis), through domain combinations, TIR-only and LRR-only proteins had already appeared in sponges (Amphimedon queenslandica). Although vertebrate-like TIR (V-TIR) domain already appeared in Cnidaria, the vertebrate-like TLR (V-TLR) with both domains appeared much later. The first combination between V-TIR domain and vertebrate-like LRR (V-LRR) domain for V-TLR may have occurred after the divergence of Cnidaria and bilateria. Then, another combination for V-TLR, a recombination of both domains, possibly occurred before or during the evolution of primitive vertebrates. Taken together, two rounds of domain combinations may thus have co-shaped the vertebrate TLRs.

Comparative Overview of Toll-Like Receptors in Lower Animals

Toll-like receptors (TLRs) have been shown to play a crucial role in host defense against pathogenic microbes in innate immunity in mammals. Recent genome-wide analyses have suggested that TLRs or related genes are conserved in the genome of non-mammalian organisms such as fishes, cyclostomes, ascidians, cephalochordates, sea urchins, and hydras. However, neither active forms nor functions of authentic invertebrate TLRs had been elucidated. Quite recently, we verified the structures, localization, ligand recognition, activities, and inflammatory cytokine production of two TLRs in the ascidian Ciona intestinalis, designated Ci-TLR1 and Ci-TLR2. Both Ci-TLRs possess a unique structural organization, with moderate sequence similarity to functionally characterized vertebrate TLRs, and are expressed predominantly in the stomach and intestine as well as in hemocytes. Unlike vertebrate TLRs, Ci-TLR1 and Ci-TLR2 are present in both the plasma membrane and endosomes. Furthermore, both Ci-TLR1 and Ci-TLR2 stimulate NF-kappaB induction in response to multiple pathogenic ligands that are differentially recognized by respective vertebrate TLRs. Pathogenic ligands that stimulate the Ci-TLRs also induce the expression of Ci-TNFalpha in the intestine and stomach, where the Ci-TLRs are abundantly expressed. These data reveal the conservation of the TLR-triggered innate immune system in C. intestinalis, and both common and unique biological and immunological functions of the Ci-TLRs. Based on the latest findings, we review recent advances in studies of TLRs or related receptors in fish, cyclostomes, deuterostome invertebrates, and hydra, and also the significance of studies of lower organism TLRs.

Toll-like Receptors of the Ascidian Ciona intestinalis: PROTOTYPES WITH HYBRID FUNCTIONALITIES OF VERTEBRATE TOLL-LIKE RECEPTORS

Key transmembrane proteins in the innate immune system, Toll-like receptors (TLRs), have been suggested to occur in the genome of non-mammalian organisms including invertebrates. However, authentic invertebrate TLRs have been neither structurally nor functionally investigated. In this paper, we originally present the structures, localization, ligand recognition, activities, and inflammatory cytokine production of all TLRs of the ascidian Ciona intestinalis, designated as Ci-TLR1 and Ci-TLR2. The amino acid sequence of Ci-TLR1 and Ci-TLR2 were found to possess unique structural organization with moderate sequence similarity to functionally characterized vertebrate TLRs. ci-tlr1 and ci-tlr2 genes were expressed predominantly in the stomach and intestine as well as in hemocytes. Ci-TLR1 and Ci-TLR2 expressed in HEK293 cells, unlike vertebrate TLRs, were localized to both the plasma membrane and endosomes. Intriguingly, both Ci-TLR1 and Ci-TLR2 stimulate NF-kappaB induction in response to multiple pathogenic ligands such as double-stranded RNA, and bacterial cell wall components that are differentially recognized by respective vertebrate TLRs, revealing that Ci-TLRs recognize broader pathogen-associated molecular patterns than vertebrate TLRs. The Ci-TLR-stimulating pathogenic ligands also induced the expression of Ci-TNFalpha in the intestine and stomach where Ci-TLRs are expressed. These results provide evidence that the TLR-triggered innate immune systems are essentially conserved in ascidians, and that Ci-TLRs possess "hybrid" biological and immunological functions, compared with vertebrate TLRs. Moreover, it is presumed that chordate TLR ancestors also acquired the Ci-TLR-like multiple cellular localization and pathogen-associated molecular pattern recognition.

Pan-Vertebrate Toll-Like Receptors During Evolution

Human toll-like receptors (TLRs) recognize pathogen-associated molecular patterns (PAMPs) to raise innate immune responses. The human TLR family was discovered because of its sequence similarity to fruit fly (Drosophila) Toll, which is involved in an anti-fungal response. In this review, we focus on the origin of the vertebrate TLR family highlighted through functional and phylogenetic analyses of TLRs in non-mammalian vertebrates. Recent extensive genome projects revealed that teleosts contain almost all subsets of TLRs that correspond to human TLRs (TLR1, 2, 3, 4, 5, 7, 8, and 9), whereas the urochordate Ciona intestinalis contains only a few TLR genes. Therefore, mammals likely obtained almost all TLR family members at the beginning of vertebrate evolution. This premise is further supported by several functional analyses of non-mammalian TLRs. We have summarized several teleost TLRs with unique properties distinct from mammalian TLRs to outline their specific roles. According to Takifugu rubripes genome project, the puffer fish possesses fish-specific TLR21 and 22. Surprisingly, phylogenetic analyses indicate that TLR21 and 22 emerged during an early period of vertebrate evolution in parallel with other TLRs and that the mammalian ancestor lost TLR21 and 22 during evolution. Our laboratory recently revealed that TLR22 recognizes double-strand RNA and induces interferon production through the TICAM-1 adaptor, as in TLR3, but unlike TLR3, TLR22 localizes to the cell surface. Therefore, differential expression of TLR3 and TLR22, rather than simple redundancy of RNA sensors, may explain the effective protection of fish from RNA virus infection in the water. In this review, we summarize the similarities and differences of the TLR family in various vertebrates and introduce these unique TLRs for a possible application to the field of clinical practices for cancer or virus infection.

Little Change, Large Consequence

Occasionally, small changes in sequence change the overall architecture of large protein assemblies. One system for understanding protein assembly is the bacterial flagellar filament, the prototype of which comes from Salmonella and contains 11 protofilaments. Galkin et al. now show that the homologous flagellar filament of Campylobacter contains only 7 protofilaments. The difference may be related to sequence divergence in a region of flagellin that in Salmonella is involved in coiled-coil formation and is recognized by the vertebrate Toll-like receptor 5 (TLR5). Campylobacter is not recognized by TLR5, and its evasion may have driven the change in quaternary structure. V. E. Galkin, X. Yu, J. Bielnicki, J. Heuser, C. P. Ewing, P. Guerry, E. H. Egelman, Divergence of quaternary structures among bacterial flagellar filaments. Science 320 , 382-385 (2008). [Abstract] [Full Text]

Divergence of Quaternary Structures Among Bacterial Flagellar Filaments

It has been widely assumed that the atomic structure of the flagellar filament from Salmonella typhimurium serves as a model for all bacterial flagellar filaments given the sequence conservation in the coiled-coil regions responsible for polymerization. On the basis of electron microscopic images, we show that the flagellar filaments from Campylobacter jejuni have seven protofilaments rather than the 11 in S. typhimurium. The vertebrate Toll-like receptor 5 (TLR5) recognizes a region of bacterial flagellin that is involved in subunit-subunit assembly in Salmonella and many other pathogenic bacteria, and this short region has diverged in Campylobacter and related bacteria, such as Helicobacter pylori, which are not recognized by TLR5. The driving force in the change of quaternary structure between Salmonella and Campylobacter may have been the evasion of TLR5.