Structure and biological activity in vitro of Flagellin and its mutants from Escherichia coli Nissle 1917.

Bacterial flagellin is a potent immunomodulatory agent. Previously, we successfully obtained flagellin from Escherichia coli Nissle 1917 (FliCEcN) and constructed two mutants with varying degrees of deletion in its highly variable regions (HVRs). We found that there was a difference in immune stimulation levels between the two mutants, with the mutant lacking the D2-D3 domain pair of FliCEcN having a better adjuvant effect. Therefore, this study further analyzed the structural characteristics of the aforementioned FliCEcN and its two mutants and measured their levels of Caco-2 cell stimulation to explore the impact of different domains in the HVRs of FliCEcN on its structure and immune efficacy. This study utilized AlphaFold2, SERS (Surface-enhanced Raman spectroscopy), and CD (circular dichroism) techniques to analyze the structural characteristics of FliCEcN and its mutants, FliCΔ174-506 and FliCΔ274-406, and tested their immune effects by stimulating Caco-2 cells in vitro. The results indicate that the D2 and D3 domains of FliCEcN have more complex interactions compared to the D1-D2 domain pair., and these domains also play a role in molecular docking with TLR5 (Toll-like receptor 5). Furthermore, FliCΔ274-406 has more missing side chain and characteristic amino acid peaks than FliCΔ174-506. The FliCEcN group was found to stimulate higher levels of IL-10 (interleukin 10) secretion, while the FliCΔ174-506 and FliCΔ274-406 groups had higher levels of IL-6 (interleukin 6) and TNF-α (tumor necrosis factor-α) secretion. In summary, the deletion of different domains in the HVRs of FliCEcN affects its structural characteristics, its interaction with TLR5, and the secretion of immune factors by Caco-2 cells.

Anesthetics isoflurane and sevoflurane attenuate flagellin-mediated inflammation in the lung.

Isoflurane and sevoflurane are volatile anesthetics (VA) widely used in clinical practice to provide general anesthesia. We and others have previously shown that VAs have immunomodulatory effects and may have a significant impact on the progression of disease states. Flagellin is a component of Gram negative bacteria and plays a significant role in the pathophysiology of bacterial pneumonia through its binding to Toll-like Receptor 5 (TLR5). Our results showed that VAs, not an intravenous anesthetic, significantly attenuated the activation of TLR5 and the release of the neutrophil chemoattractant IL-8 from lung epithelial cells. Furthermore, flagellin-induced lung injury was significantly attenuated by VAs by inhibiting neutrophil migration to the bronchoalveolar space. The lungs of cystic fibrosis (CF) patients are highly colonized by Pseudomonas aeruginosa, which causes inflammation. The retrospective study of oxygenation in patients with CF who had received VA versus intravenous anesthesia suggested that VAs might have the protective effect for gas exchange. To understand the interaction between VAs and TLR5, a docking simulation was performed, which indicated that isoflurane and sevoflurane docked into the binding interphase between TLR5 and flagellin.

Insights into structure and dynamics of extracellular domain of Toll-like receptor 5 in Cirrhinus mrigala (mrigala): A molecular dynamics simulation approach.

The toll-like receptor 5 (TLR5) is the most conserved important pattern recognition receptors (PRRs) often stimulated by bacterial flagellins and plays a major role in the first-line defense against invading pathogenic bacteria and in immune homeostasis. Experimental crystallographic studies have shown that the extracellular domain (ECD) of TLR5 recognizes flagellin of bacteria and functions as a homodimer in model organism zebrafish. However, no structural information is available on TLR5 functionality in the major carp Cirrhinus mrigala (mrigala) and its interaction with bacterial flagellins. Therefore, the present study was undertaken to unravel the structural basis of TLR5-flagellin recognition in mrigala using structural homodimeric TLR5-flagellin complex of zebrafish as reference. Integrative structural modeling and molecular dynamics simulations were employed to explore the structural and mechanistic details of TLR5 recognition. Results from structural snapshots of MD simulation revealed that TLR5 consistently formed close interactions with the three helices of the D1 domain in flagellin on its lateral side mediated by several conserved amino acids. Results from the intermolecular contact analysis perfectly substantiate with the findings of per residue-free energy decomposition analysis. The differential recognition mediated by flagellin to TLR5 in mrigala involves charged residues at the interface of binding as compared to the zebrafish complex. Overall our results shows TLR5 of mrigala involved in innate immunity specifically recognized a conserved site on flagellin which advocates the scientific community to explore host-specific differences in receptor activation.

Toll-Like Receptor 5 of Golden Pompano Trachinotus ovatus (Linnaeus 1758): Characterization, Promoter Activity and Functional Analysis.

Toll-like receptors (TLRs), as important pattern recognition receptors, represent a significant component of fish immune systems and play an important role in resisting the invasion of pathogenic microorganisms. The TLR5 subfamily contains two types of TLR5, the membrane form of TLR5 (TLR5M) and the soluble form of TLR5 (TLR5S), whose detailed functions have not been completely elucidated. In the present study, we first identified two genes, TLR5M (ToTLR5M) and TLR5S (ToTLR5S), from golden pompano (Trachinotus ovatus). The full-length ToTLR5M and ToTLR5S cDNA are 3644 bp and 2329 bp, respectively, comprising an open reading frame (ORF) of 2673 bp, encoding 890 amino acids, and an ORF of 1935 bp, encoding 644 amino acids. Both the ToTLR5s possess representative TLR domains; however, only ToTLR5M has transmembrane and intracellular TIR domains. Moreover, the transcription of two ToTLR5s was significantly upregulated after stimulation by polyinosinic:polycytidylic acid (poly (I:C)), lipopolysaccharide (LPS), and flagellin in both immune-related tissues (liver, intestine, blood, kidney, and skin) and nonimmune-related tissue (muscle). Furthermore, the results of bioinformatic and promoter analysis show that the transcription factors GATA-1 (GATA Binding Protein 1), C/EBPalpha (CCAAT Enhancer Binding Protein Alpha), and ICSBP (Interferon (IFN) consensus sequence binding protein) may play a positive role in moderating the expression of two ToTLR5s. Overexpression of ToTLR5M and ToTLR5S notably increases NF-κB (nuclear factor kappa-B) activity. Additionally, the binding assay revealed that two rToTLR5s can bind specifically to bacteria and pathogen-associated molecular patterns (PAMPs) containing Vibrio harveyi, Vibrio anguillarum, Vibrio vulnificus, Escherichia coli, Photobacterium damselae, Staphylococcus aureus, Aeromonas hydrophila, LPS, poly(I:C), flagellin, and peptidoglycan (PGN). In conclusion, the present study may help to elucidate the function of ToTLR5M/S and clarify their possible roles in the fish immune response to bacterial infection.

Development of epitope-based peptide vaccine against novel coronavirus 2019 (SARS-COV-2): Immunoinformatics approach.

Recently, a novel coronavirus (SARS-COV-2) emerged which is responsible for the recent outbreak in Wuhan, China. Genetically, it is closely related to SARS-CoV and MERS-CoV. The situation is getting worse and worse, therefore, there is an urgent need for designing a suitable peptide vaccine component against the SARS-COV-2. Here, we characterized spike glycoprotein to obtain immunogenic epitopes. Next, we chose 13 Major Histocompatibility Complex-(MHC) I and 3 MHC-II epitopes, having antigenic properties. These epitopes are usually linked to specific linkers to build vaccine components and molecularly dock on toll-like receptor-5 to get binding affinity. Therefore, to provide a fast immunogenic profile of these epitopes, we performed immunoinformatics analysis so that the rapid development of the vaccine might bring this disastrous situation to the end earlier.

Analysis of substitution rates showed that TLR5 is evolving at different rates among mammalian groups.

BACKGROUND: Toll-like receptors (TLRs) are the most widely studied innate immunity receptors responsible for recognition of invading pathogens. Among the TLR family, TLR5 is the only that senses and recognizes flagellin, the major protein of bacterial flagella. TLR5 has been reported to be under overall purifying selection in mammals, with a small proportion of codons under positive selection. However, the variation of substitution rates among major mammalian groups has been neglected. Here, we studied the evolution of TLR5 in mammals, comparing the substitution rates among groups. RESULTS: In this study we analysed the TLR5 substitution rates in Euungulata, Carnivora, Chiroptera, Primata, Rodentia and Lagomorpha, groups. For that, Tajima's relative rate test, Bayesian inference of evolutionary rates and genetic distances were estimated with CODEML's branch model and RELAX. The combined results showed that in the Lagomorpha, Rodentia, Carnivora and Chiroptera lineages TLR5 is evolving at a higher substitution rate. The RELAX analysis further suggested a significant relaxation of selective pressures for the Lagomorpha (K = 0.22, p < 0.01), Rodentia (K = 0.58, p < 0.01) and Chiroptera (K = 0.65, p < 0.01) lineages and for the Carnivora ancestral branches (K = 0.13, p < 0.01). CONCLUSIONS: Our results show that the TLR5 substitution rate is not uniform among mammals. In fact, among the different mammal groups studied, the Lagomorpha, Rodentia, Carnivora and Chiroptera are evolving faster. This evolutionary pattern could be explained by 1) the acquisition of new functions of TLR5 in the groups with higher substitution rate, i.e. TLR5 neofunctionalization, 2) by the beginning of a TLR5 pseudogenization in these groups due to some redundancy between the TLRs genes, or 3) an arms race between TLR5 and species-specific parasites.

Nonconservation of TLR5 activation site in Edwardsiella tarda flagellin decreases expression of interleukin-1ß and NF-κB genes in Japanese flounder, Paralichthys olivaceus.

Flagellin is the subunit protein that composes bacterial flagella and is recognized by toll-like receptor 5 (TLR5) as a ligand. Flagellin protein (e.g., FliC and FlaA) contains the D1, D2, and D3 domains; the D1 domain is important for recognition by TLR5 for activation of the innate immune system. In teleosts, there are two types of TLR5, the membrane form (TLR5M) and soluble form (TLR5S), the latter of which is not present in mammals. In this study, the potential of flagellin from Edwardsiella tarda (EtFliC) to induce inflammation-related genes interleukin (IL)-1ß and NF-κB-p65 through TLR5S in Japanese flounder (Paralichthys olivaceus) was elucidated. A transient overexpression system was developed in flounder natural embryonic (HINAE) cells using constructs encoding two flagellin genes derived from E. tarda (pEtFliC) and Escherichia coli (pEcoFliC) and the flounder TLR5S gene (pPoTLR5S). Expression of inflammation-related genes in EtFliC- and PoTLR5S-overexpressing HINAE cells was significantly lower than in EcoFliC- and PoTLR5S-overexpressing cells. To clarify the difference between EtFliC and EcoFliC potency, the amino acid sequence of EtFliC was compared with that of other bacterial flagellin. The 91st arginine residue, known as the mammalian TLR5 activation site, was conserved in the flagellin of E. coli and other bacteria but not in EtFliC. To reveal the importance of the 91st arginine residue in FliC, a pEtFliC construct in which the 91st asparagine was mutated to arginine (pEtFliC_N91R) was generated. Expression of the IL-1ß and NF-κB-p65 genes in the HINAE cells co-transfected with pEtFliC_N91R and pPoTLR5S was significantly higher than that in cells co-transfected with pEtFliC and pPoTLR5S. The results suggested that the 91st arginine residue of bacterial flagellin is involved in inflammatory response through TLR5S in teleosts. Thus, EtFliC improved by site-directed mutagenesis could be an effective adjuvant against E. tarda infection in Japanese flounder.

Extension and refinement of the recognition motif for Toll-like receptor 5 activation by flagellin.

TLRs sense conserved and essential molecular components of microbes that invade multicellular organisms. The wide range of TLR agonists, differing in size and shape, is recognized either through a single or a pair of binding sites on the ectodomains of TLRs. TLR5 recognizes bacterial flagellin through two distinct binding sites on the ectodomain, the first facilitating primary binding of flagellin and the second guiding receptor dimerization necessary for signaling. The regions of flagellin recognized by TLR5 encompass key functional regions within the D1 domain of flagellin, which is also required for the assembly of functional flagella. In addition to previously identified binding sites at the N-terminal and central segment of the TLR5 ectodomain, we extended the TLR5'-D1 interaction interface on TLR5 and showed a species-specific recognition relevance of this extended region. In addition, we showed that the loop and following ß-hairpin region of flagellin, previously proposed to participate in the TLR5-flagellin dimerization interface, is not accountable for these species-specific differences. We further identified residues that contribute to the interaction between two TLR5 ectodomains in an active signaling complex. Our work demonstrates that flagellin is recognized by TLR5 through a more extensive interaction surface than previously characterized.

Duplicated TLR5 of zebrafish functions as a heterodimeric receptor.

Toll-like receptor 5 (TLR5) of mammals, birds, and reptiles detects bacterial flagellin and signals as a homodimeric complex. Structural studies using truncated TLR5b of zebrafish confirm the homodimeric TLR5-flagellin interaction. Here we provide evidence that zebrafish (Danio rerio) TLR5 unexpectedly signals as a heterodimer composed of the duplicated gene products drTLR5b and drTLR5a. Flagellin-induced signaling by the zebrafish TLR5 heterodimer increased in the presence of the TLR trafficking chaperone UNC93B1. Targeted exchange of drTLR5b and drTLR5a regions revealed that TLR5 activation needs a heterodimeric configuration of the receptor ectodomain and cytoplasmic domain, consistent with ligand-induced changes in receptor conformation. Structure-guided substitution of the presumed principal flagellin-binding site in human TLR5 with corresponding zebrafish TLR5 residues abrogated human TLR5 activation, indicating a species-specific TLR5-flagellin interaction. Our findings indicate that the duplicated TLR5 of zebrafish underwent subfunctionalization through concerted coevolution to form a unique heterodimeric flagellin receptor that operates fundamentally differently from TLR5 of other species.

VLR Recognition of TLR5 Expands the Molecular Characterization of Protein Antigen Binding by Non-Ig-based Antibodies.

Variable lymphocyte receptors (VLRs) are unconventional adaptive immune receptors relatively recently discovered in the phylogenetically ancient jawless vertebrates, lamprey and hagfish. VLRs bind antigens using a leucine-rich repeat fold and are the only known adaptive immune receptors that do not utilize an immunoglobulin fold for antigen recognition. While immunoglobulin antibodies have been studied extensively, there are comparatively few studies on antigen recognition by VLRs, particularly for protein antigens. Here we report isolation, functional and structural characterization of three VLRs that bind the protein toll-like receptor 5 (TLR5) from zebrafish. Two of the VLRs block binding of TLR5 to its cognate ligand flagellin in functional assays using reporter cells. Co-crystal structures revealed that these VLRs bind to two different epitopes on TLR5, both of which include regions involved in flagellin binding. Our work here demonstrates that the lamprey adaptive immune system can be used to generate high-affinity VLR clones that recognize different epitopes and differentially impact natural ligand binding to a protein antigen.

Molecular characterization and expression of toll-like receptor 5 genes from Pelteobagrus vachellii.

Toll-like receptor 5 (TLR5) is an important pathogen recognition receptor (PRR) that recognizes the flagellin protein of pathogenic bacteria and plays a fundamental role in activating the innate immune response. In this study, full-length pvTLR5m (membrane) and pvTLR5s (soluble) genes were cloned from darkbarbel catfish Pelteobagrus vachellii, and their expression and that of downstream genes were analyzed following exposure to the Aeromonas hydrophila pathogen. The 3009 bp pvTLR5m cDNA includes a 2652 bp open reading frame (ORF) encoding 884 amino acids. The 2422 bp pvTLR5s cDNA includes a 1944 bp ORF encoding a predicted protein of 648 amino acids. The genes are most closely related to TLR5m (75%) and TLR5s (69%) from Ictalurus punctatus, respectively, and both have a typical TLR structure. Both genes were constitutively expressed in all examined tissues, and most abundantly in the head kidney and spleen. Following pathogen challenge, pvTLR5m and pvTLR5s expression was increased significantly (P <0.05) and peaked at 24 and 12 h post-exposure in the liver, 24 and 12 h in the head kidney, and 48 and 24 h in the spleen, respectively. The downstream genes interleukin-1ß (IL-1ß), IL-12 and tumor necrosis factor-alpha (TNF-α) were significantly up-regulated following pathogen exposure in spleen, and the NF-kB inhibitor (IκB) was down-regulated. These findings indicated that pvTLR5 may play an important role in the immune responses to A. hydrophila. These results provide new insight to elucidate the immune signalling pathways of fish TLR.

Identification of Individual Bacterial Cells through the Intermolecular Interactions with Peptide-Functionalized Solid-State Pores.

Bioinspired pore sensing for selective detection of flagellated bacteria was investigated. The Au micropore wall surface was modified with a synthetic peptide designed from toll-like receptor 5 (TLR5) to mimic the pathogen-recognition capability. We found that intermolecular interactions between the TLR5-derived recognition peptides and flagella induce ligand-specific perturbations in the translocation dynamics of Escherichia coli, which facilitated the discrimination between the wild-type and flagellin-deletion mutant (ΔfliC) by the resistive pulse patterns thereby demonstrating the sensing of bacteria at a single-cell level. These results provide a novel concept of utilizing weak intermolecular interactions as a recognition probes for single-cell microbial identification.

Molecular characterization and expression analyses of toll like receptor-5 induced by Vibrio parahaemolyticus antigens in Pacific red snapper.

Toll-like receptor 5 (TLR5) is a member of TLRs family responsible for the bacterial flagellin recognition in vertebrates. Herein, the TLR5M gene structure of Pacific red snapper (Lutjanus peru) was characterized. The full-length cDNA of LpTLR5M comprises an open reading frame (ORF) of 2715 bp, encoding a polypeptide of 904 amino acids including 9 LRRs (residues 119-562) and one LRR-CT domain (residues 593-646) at the extracellular region, and a TIR domain (residues 710-904) in the cytoplasmic region. The amino acid sequence in L. peru TLR5 showed high identity (66-69%) with TLR5 from Paralichthys olivaceus and Scophthalmus maximus. Quantitative real-time PCR (qPCR) analysis demonstrated the constitutive expression of LpTLR5M mRNA in all the examined tissues, with higher levels in intestine, liver, and head-kidney. Furthermore, expression of LpTLR5M and five cytokine genes was also investigated 24 h and one week post-stimulation in fish intraperitoneally injected with ToxA, live V. parahaemolyticus (Vp) or V. parahaemolyticus Lysate antigens. TLR5M was significantly induced in fish infected with Vp. The pro-inflammatory cytokines IL-6, IL8 and IL-12 were significantly up-regulated in head-kidney in fish stimulated with Vp, while in intestine upregulation was observed following ToxA or Lysate injection. In contrast, IL-17 mRNA was significantly up-regulated in the intestine from fish infected with live Vp at 24 h post-injection. The results indicate that Lysate and Vp antigens can induce an immune response via TLR5M and that cytokines have an important role in the defense mechanisms against V. parahaemolyticus.

Molecular characterization and expression analysis of toll-like receptors 5 and 22 from natural triploid Carassius auratus.

Innate immunity, as the most primitive and universal host defense in fish, constitutes an efficient first line of defense to combat invading microbes. Toll-like receptors (TLRs) play essential roles in the innate immunity, and TLR5 and TLR22 are two important TLRs that can recognize flagellin and double stranded RNA (dsRNA), respectively. In this study, we identified and characterized two TLRs genes of Qihe crucian carp (Carassius auratus) (designated as CaTLR5 and CaTLR22). The full-length cDNA sequence of CaTLR5 was cloned with 2972 bp including a 140 bp 5'-terminal untranslated region (UTR), a 183 bp 3'-UTR, and a 2649 bp open reading frame (ORF) encoding a deduced protein with 882 amino acids. The full-length cDNA of CaTLR22 was identified to be 3613 bp, consisting of a 228 bp 5'-UTR, a 547 bp 3'-UTR, and a 2838 bp ORF encoding a predicted protein of 945 amino acids. A typical TLR structure (an extracellular leucine-rich repeat domain, a transmembrane domain, and an intracellular Toll/IL-1 receptor domain) was found in CaTLR5 and CaTLR22. For either CaTLR5 or CaTLR22 gene, the mRNA expression levels varied in the different periods during the early stages of development. It was suggested that expression changes of gene CaTLR5 and CaTLR22 at mRNA levels were involved in developmental regulation in the early stages, and it was postulated that CaTLR5 and CaTLR22 play the important roles in immune defense in the early development stages of fish. Quantitative Real-Time PCR (qRT-PCR) revealed that CaTLR5 and CaTLR22 were constitutively expressed in all eleven tissues examined, although the mRNA expression level varied considerably among the different tissues. Following exposure to polyI:C, flagellin, and Aeromonas hydrophila, CaTLR5 and CaTLR22 were up-regulated in different tissues, and it was suggested that CaTLR5 and CaTLR22 were involved in the immune response of Qihe crucian carp against pathogenic invasions. The present findings will provide the valuable information for understanding the structure, function, expression, and the immune defense process of CaTLR5 and CaTLR22 in Qihe crucian carp, and provide new insights for developing the new strategies of disease control to protect fish against pathogens infection.

Studies on expression pattern of toll-like receptor 5 (TLR5) in Edwardsiella tarda infected Pangasianodon hypophthalmus.

TLR5 is one of the important PRR (pathogen recognition receptors) and plays a fundamental role in pathogen recognition and activation of innate immune responses. It recognizes bacterial flagellin and stimulates the production of proinflammatory cytokines, through signalling via the adaptor protein MyD88. In this study, we characterized partial TLR5 (soluble form) gene from Pangasianodon hypophthalmus and analysed its expression profile upon challenge by Edwardsiella tarda. Bioinformatic analysis of gene sequence revealed a putative protein of 266 amino acids with four Leucine rich repeats. Quantitative expression analysis of TLR 5S showed its wide distribution in various organs and tissues. However, significant expression of TLR5S was observed in liver and spleen at 12 h (∼207.8 fold, p < 0.05). Significant upregulation was observed in kidney at 72 h.p.i. (50 folds, p < 0.05) indicating that the kidney provides longer protection almost till the activation of the adaptive immune system. This study enriches the knowledge of TLR5S in boosting the innate immunity against bacterial invasion in fish.

Identification of a gene encoding a membrane-anchored toll-like receptor 5 (TLR5M) in Oplegnathus fasciatus that responds to flagellin challenge and activates NF-κB.

Toll-like receptor 5 (TLR5) recognizes bacterial flagellin and induces the downstream signaling through the myeloid differentiation primary response gene 88 (MyD88) protein to produce proinflammatory cytokines. In this study, we describe a TLR5 membrane form (OfTLR5M) and its adaptor protein MyD88 (OfMyD88) in rock bream, Oplegnathus fasciatus. Both Oftlr5m (6.7 kb) and Ofmyd88 (3.7 kb) genes displayed a quinquepartite structure with five exons and four introns. Protein structure of OfTLR5M revealed the conventional architecture of TLRs featured by an extracellular domain with 22 leucine rich repeats (LRR), a transmembrane domain and an endodomain with TIR motif. Primary OfTLR5M sequence shared a higher homology with teleost TLR5M. The evolutional analysis confirmed that TLR5 identified in the current study is a membrane receptor and the data further suggested the co-evolution of the membrane-anchored and soluble forms of TLR5 in teleosts. Inter-lineage comparison of gene structures in vertebrates indicated that the tlr5m gene has evolved with extensive rearrangement; whereas, the myd88 gene has maintained a stable structure throughout the evolution. Inspection of 5' flanking region of these genes disclosed the presence of several transcription factor binding sites including NF-κB. Quantitative real-time PCR (qPCR) detected Oftlr5m mRNA in eleven tissues with the highest abundance in liver. In vivo flagellin administration strongly induced the transcripts of both Oftlr5m and Ofmyd88 in gills and head kidney tissues suggesting their ligand-mediated upregulation. In a luciferase assay, HEK293T cells transiently transfected with Oftlr5m and Ofmyd88 demonstrated a higher NF-κB activity than the mock control, and the luciferase activity was intensified when cells were stimulated with flagellin. Collectively, our study represents the genomic, evolutional, expressional and functional insights into a receptor and adaptor molecules of teleost origin that are involved in flagellin sensing.

A conserved TLR5 binding and activation hot spot on flagellin.

Flagellin is a bacterial protein that polymerizes into the flagellar filament and is essential for bacterial motility. When flagellated bacteria invade the host, flagellin is recognized by Toll-like receptor 5 (TLR5) as a pathogen invasion signal and eventually evokes the innate immune response. Here, we provide a conserved structural mechanism by which flagellins from Gram-negative γ-proteobacteria and Gram-positive Firmicutes bacteria bind and activate TLR5. The comparative structural analysis using our crystal structure of a complex between Bacillus subtilis flagellin (bsflagellin) and TLR5 at 2.1 Šresolution, combined with the alanine scanning analysis of the binding interface, reveals a common hot spot in flagellin for TLR5 activation. An arginine residue (bsflagellin R89) of the flagellin D1 domain and its adjacent residues (bsflagellin E114 and L93) constitute a hot spot that provides shape and chemical complementarity to a cavity generated by the loop of leucine-rich repeat 9 in TLR5. In addition to the flagellin D1 domain, the D0 domain also contributes to TLR5 activity through structurally dispersed regions, but not a single focal area. These results establish the groundwork for the future design of flagellin-based therapeutics.

Molecular cloning, characterization and expression analysis of Toll-like receptor 5M gene in Japanese sea perch (Lateolabrax japonicas) after bacterial infection.

Toll-like receptor 5M belongs to Toll-like receptors (TLRs) family, which plays a crucial role in innate immunity due to its important role in the recognition of bacteria invasion and in the activation of immune related pathways downstream. In the present study, we firstly cloned the full-length cDNAs of TLR 5M (LjTLR 5M) from Japanese sea perch (Lateolabrax japonicas). The full-length cDNAs of LjTLR 5M include an open reading frame (ORF) of 2676 bp encoding a polypeptide of 891 amino acid residues. The deduced amino acid sequence analysis showed that LiTLR 5M contains LRRs (extracellular leucine rich repeats), transmembrane and TIR (Toll/interleukin-1 receptor) domain. Transcriptional expression analysis indicated that LiTLR 5M mRNAs were ubiquitously expressed in wide array of tissues and the peak level was observed in the head-kidney. The expression patterns of LjTLR 5M after Vibro harveyi and Streptococus agalactiae infection were detected by qRT-PCR, and the results showed that LjTLR 5M was significant up-regulated in spleen, liver and head-kidney. Additionally, the expression patterns of LjTLR 5M in infected spleen and head-kidney were further validated by in situ hybridization (ISH). In summary, these findings indicate that LjTLR 5M is significant induced after different bacterial infection and is involved in immune response. Furthermore, this study will provide foundational information for other TLRs research of L. japonicas against different bacterial pathogens invasion.

Flagellin-induced NADPH oxidase 4 activation is involved in atherosclerosis.

It is widely accepted that bacterial infection-mediated inflammation facilitates development of atherosclerosis by activating toll-like receptor (TLR) signaling system. We reasoned that NADPH oxidases (Nox), required for TLR-mediated inflammatory response, are involved in atherogenesis. Here, we show that the activation of Nox4 through TLR5 regulates the inflammation of the endothelium and in atherogenesis. Flagellin-induced interaction between the COOH region of Nox4 and the TIR domain of TLR5 led to H2O2 generation, which in turn promoted the secretion of pro-inflammatory cytokines including IL-8, as well as the expression of ICAM-1 in human aortic endothelial cells (HAECs). Knockdown of the Nox4 in HAECs resulted in attenuated expressions of IL-8 and ICAM-1 leading to a reduction in the adhesion and trans-endothelial migration of monocytes. Challenge of recombinant FliC (rFliC) to the ApoE KO mice with high-fat diet (HFD) resulted in significantly increased atherosclerotic plaque sizes compared to the saline-injected mice. However, an injection of rFliC into the Nox4ApoE DKO mice with HFDs failed to generate atherosclerotic plaque, suggesting that Nox4 deficiency resulted in significant protections against rFliC-mediated atherogenesis. We conclude that TLR5-dependent Nox4 activation and subsequent H2O2 generation play critical roles for the development of atherosclerosis.

Utilizing a TLR5-Adjuvanted Cytomegalovirus as a Lentiviral Vaccine in the Nonhuman Primate Model for AIDS.

Despite tremendous progress in our understanding of human immunodeficiency virus (HIV) natural history and advances in HIV treatment, there is neither an approved vaccine nor a cure for infection. Here, we describe the development and characterization of a novel replicating vaccine vector utilizing Cytomegalovirus (CMV) and a TLR5 adjuvant. After partial truncation of the central, immunodominant hypervariable domain, flagellin (fliC) from Salmonella was cloned downstream of a codon optimized gag gene from simian immunodeficiency virus (SIV) and transiently expressed in telomerized rhesus fibroblast (TeloRF) cells in culture. Lysates generated from these transfected cells induced the tumor necrosis factor alpha (TNF-α), in a mouse macrophage cell line, in a TLR5-dependent manner. The Gag/FliC expression construct was cloned into a bacterial artificial chromosome encoding the rhesus CMV (RhCMV) genome, and infectious RhCMV was generated following transfection of TeloRF cells. This virus stably expressed an SIV Gag/FliC fusion protein through four serial passages. Lysates generated from infected cells induced TNF-α in a TLR5-dependent manner. Western blot analysis of infected cell lysates verified expression of a Gag/FliC fusion protein using a SIV p27 capsid monoclonal antibody. Lastly, rhesus macaques inoculated with this novel RhCMV virus demonstrated increased inflammatory responses at the site of inoculation seven days post-infection when compared to the parental RhCMV. These results demonstrate that an artificially constructed replicating RhCMV expressing an SIV Gag/FliC fusion protein is capable of activating TLR5 in a macrophage cell line in vitro and induction of an altered inflammatory response in vivo. Ongoing animals studies are aimed at determining vaccine efficacy, including subsequent challenge with pathogenic SIV.