The effect of anesthetics on toll like receptor 9.

Toll like receptors (TLRs) are critical receptors to respond to danger signals, and their functions are relevant in the perioperative period. We previously reported that volatile anesthetics directly bound to TLR2 and TLR4 and attenuated their functions. Given that TLR9 can respond to mitochondrial DNA, a danger signal that is released upon tissue injury, we examined the role of anesthetics on TLR9 function. Our reporter assay showed that volatile anesthetics isoflurane and sevoflurane increased the activation of TLR9, while propofol attenuated it. TLR9 activation occurs via its dimerization. The dimerization is facilitated by unmethylated cytosine-phosphate-guanine (CpG) DNA as well as DNA containing cytosine at the second position from 5'-end (5'-xCx DNA). Our structural analysis using photoactivable anesthetics and rigid docking simulation showed that isoflurane and sevoflurane bound to both TLR9 dimer interface and 5'-xCx DNA binding site. Propofol bound to the TLR9 antagonist binding site. This is the first illustration that anesthetics can affect the binding of nucleic acids to their receptor. This study sets the foundation for the effect of anesthetics on TLR9 and will pave the way for future studies to determine the significance of such interactions in the clinical setting.

Structure based design and synthesis of novel Toll-like Receptor 2 (TLR 2) lipid antagonists.

Toll-like receptors (TLRs) play key role in innate immune response to Damage Associated Molecular Patterns (DAMPs) and Pathogen Associated Molecular Patterns (PAMPs). DAMP/PAMP-mediated activation of TLRs triggers NFκB signaling resulting in pro-inflammatory cytokine release. Using TLR2-Pam2CSK4 agonist co-crystal structure information, we designed and synthesized a novel series of Toll-like Receptor 2 (TLR2) lipid antagonists and identified compounds 14, 15 and 17 with sub-micromolar potency. TLR2 antagonists that we identified are stable for > 1.0 h in both gastric juice and PBS buffer and could be used as research tools.

Structural basis of CpG and inhibitory DNA recognition by Toll-like receptor 9.

Innate immunity serves as the first line of defence against invading pathogens such as bacteria and viruses. Toll-like receptors (TLRs) are examples of innate immune receptors, which sense specific molecular patterns from pathogens and activate immune responses. TLR9 recognizes bacterial and viral DNA containing the cytosine-phosphate-guanine (CpG) dideoxynucleotide motif. The molecular basis by which CpG-containing DNA (CpG-DNA) elicits immunostimulatory activity via TLR9 remains to be elucidated. Here we show the crystal structures of three forms of TLR9: unliganded, bound to agonistic CpG-DNA, and bound to inhibitory DNA (iDNA). Agonistic-CpG-DNA-bound TLR9 formed a symmetric TLR9-CpG-DNA complex with 2:2 stoichiometry, whereas iDNA-bound TLR9 was a monomer. CpG-DNA was recognized by both protomers in the dimer, in particular by the amino-terminal fragment (LRRNT-LRR10) from one protomer and the carboxy-terminal fragment (LRR20-LRR22) from the other. The iDNA, which formed a stem-loop structure suitable for binding by intramolecular base pairing, bound to the concave surface from LRR2-LRR10. This structure serves as an important basis for improving our understanding of the functional mechanisms of TLR9.

Equilibrium Binding Model for CpG DNA-Dependent Dimerization of Toll-like Receptor 9 Ectodomain.

Microbial nucleic acids in the extracellular milieu are recognized in vertebrates by Toll-like receptors (TLRs), one of the most important families of innate immune receptors. TLR9 recognizes single-stranded unmethylated CpG DNA in endosomes. DNA binding induces TLR9 dimerization and activation of a potent inflammatory response. To provide insights on how DNA ligands induce TLR9 dimerization, we developed a detailed theoretical framework for equilibrium ligand binding, modeling the binding of the ssDNA at the two main sites on the TLR9 ectodomain. Light scattering and fluorescence anisotropy assays performed with recombinant TLR9 ectodomain and a panel of agonistic and antagonistic DNA ligands provide data that restrain the binding parameters, identify the likely ligand binding intermediates, and suggest cooperative modes of binding. This work brings us one step closer to establishing a rigorous biochemical understanding of how TLRs are activated by their ligands.

Transmembrane mutations in Toll-like receptor 9 bypass the requirement for ectodomain proteolysis and induce fatal inflammation.

Recognition of nucleic acids as a signature of infection by Toll-like receptors (TLRs) 7 and 9 exposes the host to potential self-recognition and autoimmunity. It has been proposed that intracellular compartmentalization is largely responsible for reliable self versus nonself discrimination by these receptors. We have previously shown that TLR9 and TLR7 require processing prior to activation, which may further reinforce receptor compartmentalization and tolerance to self, yet this possibility remains untested. Here we report that residues within the TLR9 transmembrane (TM) region conferred the requirement for ectodomain proteolysis. TLR9 TM mutants responded to extracellular DNA, and mice expressing such receptors died from systemic inflammation and anemia. This inflammatory disease did not require lymphocytes and appeared to require recognition of self-DNA by dendritic cells. To our knowledge, these results provide the first demonstration that TLR-intrinsic mutations can lead to a break in tolerance.

Bacterial amyloid curli acts as a carrier for DNA to elicit an autoimmune response via TLR2 and TLR9.

Bacterial biofilms are associated with numerous human infections. The predominant protein expressed in enteric biofilms is the amyloid curli, which forms highly immunogenic complexes with DNA. Infection with curli-expressing bacteria or systemic exposure to purified curli-DNA complexes triggers autoimmunity via the generation of type I interferons (IFNs) and anti-double-stranded DNA antibodies. Here, we show that DNA complexed with amyloid curli powerfully stimulates Toll-like receptor 9 (TLR9) through a two-step mechanism. First, the cross beta-sheet structure of curli is bound by cell-surface Toll-like receptor 2 (TLR2), enabling internalization of the complex into endosomes. After internalization, the curli-DNA immune complex binds strongly to endosomal TLR9, inducing production of type I IFNs. Analysis of wild-type and TLR2-deficient macrophages showed that TLR2 is the major receptor that drives the internalization of curli-DNA complexes. Suppression of TLR2 internalization via endocytosis inhibitors led to a significant decrease in Ifnß expression. Confocal microscopy analysis confirmed that the TLR2-bound curli was required for shuttling of DNA to endosomal TLR9. Structural analysis using small-angle X-ray scattering revealed that incorporation of DNA into curli fibrils resulted in the formation of ordered curli-DNA immune complexes. Curli organizes parallel, double-stranded DNA rods at an inter-DNA spacing that matches up well with the steric size of TLR9. We also found that production of anti-double-stranded DNA autoantibodies in response to curli-DNA was attenuated in TLR2- and TLR9-deficient mice and in mice deficient in both TLR2 and TLR9 compared to wild-type mice, suggesting that both innate immune receptors are critical for shaping the autoimmune adaptive immune response. We also detected significantly lower levels of interferon-stimulated gene expression in response to purified curli-DNA in TLR2 and TLR9 deficient mice compared to wild-type mice, confirming that TLR2 and TLR9 are required for the induction of type I IFNs. Finally, we showed that curli-DNA complexes, but not cellulose, were responsible elicitation of the immune responses to bacterial biofilms. This study defines the series of events that lead to the severe pro-autoimmune effects of amyloid-expressing bacteria and suggest a mechanism by which amyloid curli acts as a carrier to break immune tolerance to DNA, leading to the activation of TLR9, production of type I IFNs, and subsequent production of autoantibodies.

Diurnal rhythm and pathogens induced expression of toll-like receptor 9 (TLR9) in Pelteobagrus vachellii.

Toll-like receptor 9 (TLR9) is activated by bacterial DNA and induces the production of inflammatory cytokines. In this study, the darkbarbel catfish Pelteobagrus vachellii TLR9 cDNA was cloned and sequenced. The daily expression pattern of TLR9 mRNA was investigated in various tissues. Furthermore, its expression was analyzed following exposure to the pathogen Aeromonas hydrophila. The 4249 bp cDNA includes a 3201 bp open reading frame (ORF) encoding 1067 amino acids. The predicted amino acid sequence comprises a leucine-rich domain (LRD), a toll/interleukin-1 receptor (TIR), and a transmembrane domain. P. vachellii TLR9 showed 42-87% amino acid sequence identity with TLR9 sequences of Ictalurus punctatus, Rhincodon typus, and Miichthys miiuy. The P. vachellii TLR9 mRNA was highly expressed in intestines, head kidney, and spleen in an apparently healthy fish. Following pathogen challenge, TLR9 expression increased significantly (P < 0.05) and peaked at 48 h post-exposure in the liver, at 24 in the head kidney, and at 12 h in the spleen. In addition, the pattern of TLR9 expression over a 24-h period showed a circadian rhythm in the head kidney, spleen, and intestine, with the acrophase at 20:34, 18:45, and 3:50, respectively. This result provided the basis for further study of the rhythm of innate immunity against bacteria in catfish.

Class B CpG-ODN2006 is highly associated with IgM and antimicrobial peptide gene expression through TLR9 pathway in yellowtail Seriola lalandi.

The purpose of this study was to characterize the TLR9 gene from yellowtail (Seriola lalandi) and evaluate its functional activity using the class B Cytosine-phosphate-guanine-oligodeoxynucleotide2006 (CpG-ODN2006) in an in vivo experiment after one-week immunostimulation. The gene expressions of TLR9, Immunoglobulin M (IgM), antimicrobial peptides and cytokines were evaluated by real time PCR, and humoral immune parameters were analyzed in serum. The TLR9 nucleotide sequence from yellowtail was obtained using the whole-genome shotgun sequencing method and bioinformatics tools. The yellowtail full-length cDNA sequence of SlTLR9 was 3789 bp in length, including a 66-bp 5'-untranslated region (UTR), a 3'-UTR of 528 bp, and an open reading frame (ORF) of 3192 bp translatable to 1064 amino acid showing a high degree of similarity with the counterparts of other fish species and sharing common structural architecture of the TLR family, including LRR domains, one C-terminal LRR region, and a TIR domain. Gene expression studies revealed the constitutive expression of TLR9 mRNA in all analyzed tissues; the highest levels were observed in intestine, liver and spleen where they play an important role in the fish immune system. The expression levels of TLR9 after B class CpG-ODN2006 (the main TLR9-agonist) was significantly up-regulated in all analyzed tissues, with the high expression observed in spleen followed by intestine and skin. The CpG-B has been shown as a potent B cell mitogen, and interestingly, IgM mRNA transcript was up-regulated in spleen and intestine, which was highly correlated with TLR9 after CpG-ODN2006 stimulation. The antimicrobial peptides, piscidin and NK-lysine, were up-regulated in spleen and gill after CpG-ODN2006 injection with a high correlation (r ≥ 0.82) with TLR9 gene expression. Cytokine genes were up-regulated in spleen, intestine and skin after CpG-ODN was compared with the control group. No significant correlation was observed between TLR9 and IL-1ß, TNF-α and Mx gene expressions. The results showed that CpG-ODN2006 intraperitoneal injection enhanced lysozyme, peroxidase and superoxide dismutase activities in serum and demonstrated that CpG-ODN2006 can induce a specific immune response via TLR9 in which IgM and antimicrobial peptides must have an important role in the defense mechanisms against infections in yellowtail.

Rapid Complexation of Aptamers by Their Specific Antidotes.

Nucleic acid ligands, aptamers, harbor the unique characteristics of small molecules and antibodies. The specificity and high affinity of aptamers enable their binding to different targets, such as small molecules, proteins, or cells. Chemical modifications of aptamers allow increased bioavailability. A further great benefit of aptamers is the antidote (AD)-mediated controllability of their effect. In this study, the AD-mediated complexation and neutralization of the thrombin binding aptamer NU172 and Toll-like receptor 9 (TLR9) binding R10-60 aptamer were determined. Thereby, the required time for the generation of aptamer/AD-complexes was analyzed at 37 °C in human serum using gel electrophoresis. Afterwards, the blocking of aptamers' effects was analyzed by determining the activated clotting time (ACT) in the case of the NU172 aptamer, or the expression of immune activation related genes IFN-1ß, IL-6, CXCL-10, and IL-1ß in the case of the R10-60 aptamer. Gel electrophoresis analyses demonstrated the rapid complexation of the NU172 and R10-60 aptamers by complementary AD binding after just 2 min of incubation in human serum. A rapid neutralization of anticoagulant activity of NU172 was also demonstrated in fresh human whole blood 5 min after addition of AD. Furthermore, the TLR9-mediated activation of PMDC05 cells was interrupted after the addition of the R10-60 AD. Using these two different aptamers, the rapid antagonizability of the aptamers was demonstrated in different environments; whole blood containing numerous proteins, cells, and different small molecules, serum, or cell culture media. Thus, nucleic acid ADs are promising molecules, which offer several possibilities for different in vivo applications, such as antagonizing aptamer-based drugs, immobilization, or delivery of oligonucleotides to defined locations.

Identification and expression analysis of toll-like receptor genes (TLR8 and TLR9) in mucosal tissues of turbot (Scophthalmus maximus L.) following bacterial challenge.

Mucosal immune system is one of the most important components in the innate immunity and constitutes the front line of host defense against infection, especially for teleost, which are living in the pathogen-rich aquatic environment. The pathogen recognition receptors (PRRs), which can recognize the conserved pathogen-associated molecular patterns (PAMPs) of bacteria, are considered as one of the most important component for pathogen recognition and immune signaling pathways activation in mucosal immunity. In this regard, we sought to identify TLR8 and TLR9 in turbot (Scophthalmus maximus), as well as their mucosal expression patterns following different bacterial infection in mucosal tissues for the first time. The full-length TLR8 transcript consists of an open reading frame (ORF) of 3108 bp encoding the putative peptide of 1035 amino acids. While the TLR9 was 6730 bp long, containing a 3168 bp ORF that encodes 1055 amino acids. The phylogenetic analysis revealed both TLR8 and TLR9 showed the closest relationship to large yellow croaker. Moreover, both TLR8 and TLR9 could be detected in all examined healthy turbot tissues, with the lowest expression level in liver and a relatively moderate expression pattern in healthy mucosal tissues. Distinct expression patterns of TLR8 and TLR9 were comparatively observed in the mucosal tissues (intestine, gill and skin) following Vibrio anguillarum and Streptococcus iniae infection, suggesting their different roles for mucosal immunity. Further functional studies are needed to better characterize TLR8 and TLR9 and their family members, to better understand the ligand specificity and to identify their roles in different mucosal tissues in protecting fish from the pathogenically hostile environment.

Endosomal localization of TLR8 confers distinctive proteolytic processing on human myeloid cells.

Nucleic acid-sensing TLRs are involved in both antimicrobial immune responses and autoimmune inflammation. TLR8 is phylogenetically and structurally related to TLR7 and TLR9, which undergo proteolytic processing in the endolysosomes to generate functional receptors. Recent structural analyses of human TLR8 ectodomain and its liganded form demonstrated that TLR8 is also cleaved, and both the N- and C-terminal halves contribute to ligand binding. However, the structures and ssRNA recognition mode of endogenous TLR8 in human primary cells are largely unknown. In this study, we show that proteolytic processing of TLR8 occurs in human monocytes and macrophages in a different manner compared with TLR7/9 cleavage. The insertion loop between leucine-rich repeats 14 and 15 in TLR8 is indispensable for the cleavage and stepwise processing that occurs in the N-terminal fragment. Both furin-like proprotein convertase and cathepsins contribute to TLR8 cleavage in the early/late endosomes. TLR8 recognizes viral ssRNA and endogenous RNA, such as microRNAs, resulting in the production of proinflammatory cytokines. Hence, localization sites of the receptors are crucial for the nucleic acid-sensing mode and downstream signaling.

Cross talk between Leishmania donovani CpG DNA and Toll-like receptor 9: an immunoinformatics approach.

The precise and potential contribution of Toll-like receptors (TLRs) signaling pathways in fighting parasitic infections of Leishmania spp., an intracellular protozoan parasite, has gained significant attention during the last decades. Although it is well established that TLR9 recognizes CpG motifs in microbial genomes, the specificity of the CpG DNA pattern of Leishmania parasite interacting with endosomal TLR9 is still unknown. Hence in our study to identify the CpG DNA pattern of Leishmania donovani acting as ligand for TLR9, consecutive homology searches were performed using known CpG ODN 2216 as initial template until a consistent CpG pattern in L. donovani was found. A reliable model of TLR9 ectodomains (ECDs) as well as CpG DNA patterns was predicted to develop the 3D structural complexes of TLR9 ECD-CpG DNA utilizing molecular modeling and docking approaches. The results revealed the preferential specificity of L. donovani CpG DNA to TLR9 compared to control ODN and other CpG patterns. The interface between TLR9 and L. donovani CpG DNA was also found to be geometrically complementary with the LRR11 region of TLR9, acting as the critical region for ligand recognition. The L. donovani CpG pattern identified can be employed to derive a platform for development of an innate immunomodulatory agent for deadly disease.

Acidic amino acid residues in the juxtamembrane region of the nucleotide-sensing TLRs are important for UNC93B1 binding and signaling.

TLRs are divided into two groups based on their subcellular localization patterns. TLR1, 2, 4, 5, and 6 are expressed on the cell surface, whereas the nucleotide-sensing TLRs, such as TLR3, 7, 8, and 9 stay mainly inside cells. The polytopic membrane protein UNC93B1 physically interacts with the nucleotide-sensing TLRs and delivers them from the endoplasmic reticulum to endolysosomes, where the TLRs recognize their ligands and initiate signaling. In cells with nonfunctional UNC93B1, the nucleic acid-sensing TLRs fail to exit the endoplasmic reticulum and consequently do not signal. However, the detailed molecular mechanisms that underlie the UNC93B1-mediated TLR trafficking remain to be clarified. All nucleotide-sensing TLRs contain acidic amino acid residues in the juxtamembrane region between the leucine-rich repeat domain and the transmembrane segment. We show that the D812 and E813 residues of TLR9 and the D699 and E704 residues of TLR3 help to determine the interaction of these TLRs with UNC93B1. Mutation of the acidic residues in TLR3 and TLR9 prevents UNC93B1 binding, as well as impairs TLR trafficking and renders the mutant receptors incapable of transmitting signals. Therefore, the acidic residues in the juxtamembrane region of the nucleotide-sensing TLRs have important functional roles.

Crystal structure of the C-terminal domain of mouse TLR9.

Toll-like receptors (TLRs) are important pattern recognition receptors that function in innate immunity. Elucidating the structure and signaling mechanisms of TLR9, a sensor of foreign and endogenous DNA, is essential for understanding its key role in immunity against microbial pathogens as well as in autoimmunity. Abundant evidence suggests that the TLR9-CTD (C-terminal domain) by itself is capable of DNA binding and signaling. The crystal structure of unliganded mouse TLR9-CTD is presented. TLR9-CTD exhibits one unique feature, a cluster of stacked aromatic and arginine side chains on its concave face. Overall, its structure is most related to the TLR8-CTD, suggesting a similar mode of ligand binding and signaling.

Identification and expression analysis of cobia (Rachycentron canadum) Toll-like receptor 9 gene.

Cobia culture is hindered by bacterial infection (Photobacterium damselae subsp. piscicida) and in order to study the effect of P. damselae subsp. piscicida challenge and CpG ODN stimulation on cobia Toll like receptor 9 (RCTLR9), we used PCR to clone RCTLR9 gene and qRT-PCR to quantify gene expression. The results indicated that RCTLR9 cDNA contains 3141 bp. It encodes 1047 amino acids containing 16 typical structures of leucine-rich repeats (LRRs) including an LRRTYP, LRRCT and a motif involved in PAMP binding was identified at position 240-253 amino acid. Broad expression of RCTLR9 was found in larval, juvenile and adult stages irrespective of the tissues. In larval stage, RCTLR9 mRNA expression decreased at 5 d and then increased at 10 dph. At juvenile stage cobia, the expression was significantly high (p < 0.05) in spleen and intestine compared to gill, kidney, liver and skin. However, at adult stage, the significant high expression was found in gill and intestine. Cobia challenged with P. damselae subsp. piscicida showed significant increase in RCTLR9 expression at 24 h post challenge in intestine, spleen and liver, while in kidney the expression was peak at 12 h and later it decreased at 24 h. The highest expression was 40 fold increase in spleen and the lowest expression was ∼3.6 fold increase in liver. Cobia stimulated with CpG oligonucleotides showed that the induction of these genes was CpG ODN type and time dependent. In spleen and liver, CpG ODNs 1668 and 2006 injected group showed high expression of RCTLR9, IL-1ß, chemokine CC compared to other groups. Meanwhile, CpG ODN 2006 has induced high expression of IgM. The CpG ODNs 2395 have induced significant high expression of Mx in spleen and liver. These results demonstrates the potential of using CpG ODN to enhance cobia resistance to P. damselae subsp. piscicida infection and use as an adjuvant in vaccine development.

The ectodomain of Toll-like receptor 9 is cleaved to generate a functional receptor.

Mammalian Toll-like receptors (TLRs) 3, 7, 8 and 9 initiate immune responses to infection by recognizing microbial nucleic acids; however, these responses come at the cost of potential autoimmunity owing to inappropriate recognition of self nucleic acids. The localization of TLR9 and TLR7 to intracellular compartments seems to have a role in facilitating responses to viral nucleic acids while maintaining tolerance to self nucleic acids, yet the cell biology regulating the transport and localization of these receptors remains poorly understood. Here we define the route by which TLR9 and TLR7 exit the endoplasmic reticulum and travel to endolysosomes in mouse macrophages and dendritic cells. The ectodomains of TLR9 and TLR7 are cleaved in the endolysosome, such that no full-length protein is detectable in the compartment where ligand is recognized. Notably, although both the full-length and cleaved forms of TLR9 are capable of binding ligand, only the processed form recruits MyD88 on activation, indicating that this truncated receptor, rather than the full-length form, is functional. Furthermore, conditions that prevent receptor proteolysis, including forced TLR9 surface localization, render the receptor non-functional. We propose that ectodomain cleavage represents a strategy to restrict receptor activation to endolysosomal compartments and prevent TLRs from responding to self nucleic acids.

Heterozygous carriage of a dysfunctional Toll-like receptor 9 allele affects CpG oligonucleotide responses in B cells.

Toll-like receptors (TLR) are employed by the innate immune system to detect microbial pathogens based on conserved microbial pathogen molecules. For example, TLR9 is a receptor for CpG-containing microbial DNA, and its activation results in the production of cytokines and type I interferons from human B cells and plasmacytoid dendritic cells, respectively. Both are required for mounting an efficient antibacterial or antiviral immune response. These effects are mimicked by synthetic CpG oligodeoxynucleotides (ODN). Although several hyporesponsive TLR9 variants have been reported, their functional relevance in human primary cells has not been addressed. Here we report a novel TLR9 allele, R892W, which is hyporesponsive to CpG ODN and acts as a dominant-negative in a cellular model system. The R892W variant is characterized by increased MyD88 binding and defective co-localization with CpG ODN. Whereas primary plasmacytoid dendritic cells isolated from a heterozygous R892W carrier responded normally to CpG by interferon-α production, carrier B cells showed impaired IL-6 and IL-10 production. This suggests that heterozygous carriage of a hyporesponsive TLR9 allele is not associated with complete loss of TLR9 function but that TLR9 signals elicited in different cell types are regulated differently in human primary cells.

Maintenance of colonic homeostasis by distinctive apical TLR9 signalling in intestinal epithelial cells.

The mechanisms by which commensal bacteria suppress inflammatory signalling in the gut are still unclear. Here, we present a cellular mechanism whereby the polarity of intestinal epithelial cells (IECs) has a major role in colonic homeostasis. TLR9 activation through apical and basolateral surface domains have distinct transcriptional responses, evident by NF-kappaB activation and cDNA microarray analysis. Whereas basolateral TLR9 signals IkappaBalpha degradation and activation of the NF-kappaB pathway, apical TLR9 stimulation invokes a unique response in which ubiquitinated IkappaB accumulates in the cytoplasm preventing NF-kappaB activation. Furthermore, apical TLR9 stimulation confers intracellular tolerance to subsequent TLR challenges. IECs in TLR9-deficient mice, when compared with wild-type and TLR2-deficient mice, display a lower NF-kappaB activation threshold and these mice are highly susceptible to experimental colitis. Our data provide a case for organ-specific innate immunity in which TLR expression in polarized IECs has uniquely evolved to maintain colonic homeostasis and regulate tolerance and inflammation.

Toll-like receptor 9 interaction with CpG ODN--an in silico analysis approach.

BACKGROUND: Toll-like receptor 9 (TLR9) recognises unmethylated CpG DNA and activates a signalling cascade, leading to the production of inflammatory cytokines such as TNF-α, IL-1, IL-6 and IL-12 via the adaptor protein MyD88. However, the specific sequence and structural requirements of the CpG DNA for the recognition of and binding to TLR9 are unknown. Moreover, the 3D structures of TLR9 and the TLR9-ODN complex have not been determined. In this study, we propose a reliable model of the interaction of the TLR9 ECD with CpG ODN using bioinformatics tools. RESULTS: The three-dimensional structures of two TLR9 ECD-CpG ODN complexes were constructed using a homology modelling and docking strategy. Based on the models of these complexes, the TLR9 ECD-CpG ODN interaction patterns were calculated. The results showed that the interface between the human TLR9 and the CpG ODN molecule is geometrically complementary. The computed molecular interactions indicated that LRR11 is the main region of TLR9 that binds to CpG ODN and that five positively charged residues within LRR11 are involved in the binding of the TLR9 ECD to the CpG ODN. Observations in the close-up view of these interactions indicated that these five positively charged residues contribute differently to the binding region within the TLR9 ECD-CpG ODN complex. 337Arg and 338Lys reside in the binding sites of ODN, forming hydrogen bonds and direct contacts with the CpG ODN, whereas 347Lys, 348Arg, and 353His do not directly contact the CpG ODN. These results are in agreement with previously reported experimental data. CONCLUSION: In this study, we present two structural models for the human and mouse TLR9 ECD in a complex with CpG ODN. Some features predicted by this model are consistent with previously reported experimental data. This complex model may lead to a better understanding of the function of TLR9 and its interaction with CpG ODN and will improve our understanding of TLR9-ligand interaction in general.

Evolutionary analysis of TLR9 genes reveals the positive selection of extant teleosts in Perciformes.

The innate immune system can recognize non-self through pattern recognition receptors. Toll-like receptors were the best-known members of these receptors, and they could sense, recognize, and bind pathogen-associated molecular patterns. TLRs played an important role in innate immune system and were conserved in both invertebrate and vertebrate lineages. Thereinto, TLR9 could detect unmethylated CpG motifs in dsDNA and was expected to undergo coevolution with its microbial ligands. It was known that aquatic and terrestrial organisms dwelled in different environments which contained different pathogens, and they had to adapt to their local environmental conditions. Therefore, we collected TLR9 genes from invertebrate to vertebrate to further explore whether the huge differences between aquatic and terrestrial environments affected the TLR9s evolution between aquatic and terrestrial organisms. Molecular evolution analysis detected positively selected sites in the ancestral lineages of vertebrates, teleosts, and Perciformes but not in the ancestral lineage of mammals. In PAML, site model revealed that extant mammalian TLR9 genes underwent positive selection. However, the positive selection of extant teleosts appeared primarily in Perciformes in which there were 14 positively selected sites. Among these sites, two of them were located on the amino acid insertions of the leucine-rich repeats which could create DNA binding sites, three were found on the convex surface which might possibly affect the flexibility of the TLR solenoids, and six were located on the ß-face of concave surface which contained the ligand-binding sites of the TLR solenoids. In other ML methods, we also found three sites under selection that coincided with the codons identified by M8 and these sites were all located in LRRs. The diverse aquatic and terrestrial environments might possess different pathogens to make the living organisms adapt to their local environmental conditions. The positive selection on LRRs in TLR9s of Perciformes might be associated with the adaptation to the rapidly evolving pathogens in the water.