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

doi:10.1016/j.fsi.2017.01.020

Fish & Shellfish Immunology

Volume 62, March 2017, Pages 276-290

https://doi.org/10.1016/j.fsi.2017.01.020 Get rights and content

Highlights

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Oftlr5m and Ofmyd88 genes in rock bream display quinquepartite structure.
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While gene structure of tlr5m is evolved with rearrangements, myd88 is preserved.
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Oftlr5m and Ofmyd88 showed similar tissue mRNA profile with highest level in liver.
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Oftlr5m and Ofmyd88 were induced by ultrapure flagellin in gill and head kidney.
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They individually and synergetically activated NF-κB upon flagellin-stimulation.

Abstract

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.

Introduction

Pattern recognition receptors (PRRs) that recognize a group of conserved microbial components, known as pathogen-associated molecular patterns (PAMPs) [1], are part of the ancient innate branch of the immune system. Toll-like receptor (TLR) family is the thoroughly investigated of the PRRs that detect invading pathogens, and are conserved from nematodes to human [2]. These transmembrane proteins sense a diverse range of PAMPs to activate a well-coordinated signaling cascade leading to anti-pathogen responses and subsequently orchestrate the adaptive immunity [3].

TLRs are structured with an ectodomain (ECD) featured by leucine-rich repeat regions (LRRs) that binds PAMPs [4], a transmembrane domain, and an endodomain (END) harboring a Toll/IL-1 receptor (TIR) domain, which transduces the signals to downstream adaptor molecules [5]. To date, at least 21 distinct TLR types have been identified from a wide range of vertebrate species [6]. TLRs are classified based on different perspectives. There are two major subfamilies based on their cellular localization: cell surface subfamily (TLRs: 1, 2, 4, 5, 6 and 10) that recognizes a variety of microbial metabolites including microbial lipids, proteomes and sugars, and the intracellular subfamily (TLRs: 3, 7, 8 and 9) that generally senses the derivatives of microbial nucleic acids [2]. In functional perspective, they are operated through either the Myeloid differentiation primary response protein 88 (MyD88)-dependent or -independent pathways, where the MyD88 is a central adaptor molecule. All the known mammalian TLRs, except TLR3, adopt the MyD88-dependent pathway. In TLR signaling cascade, recognition of PAMPs by TLRs triggers the subsequent recruitment of MyD88, members of interleukin-1 receptor-associated kinases (IRAKs) and TNF receptor-associated factor 6 (TRAF6), which in turn activates the transforming-growth-factor-β-activated kinase (TAK1). This finally activates two distinct pathways, which culminates in the activation of NF-κB and AP1 transcription factors, leading to the production of inflammatory cytokines [2].

TLR5 recognizes both Gram-positive and Gram-negative flagellated bacteria by sensing their flagellin [7]. In mammals, TLR5 is anchored in cellular membrane and is responsible for the flagellin-mediated NF-κB activation through the MyD88-dependent pathway. Unlike in mammals, the flagellin recognition machinery in (some) teleost species was demonstrated to be uniquely equipped with the membrane (TLR5M) and soluble (TLR5S) forms of TLR5 [8], [9], [10], which operate via a positive-loop synergetic mechanism [9]. Whereas, in other fish species, only TLR5M [11], [12], [13] or TLR5S [14], [15] has been isolated and reported.

Rock bream (Oplegnathus fasciatus) is one of the major species engaged in the offshore mariculture industry of South Korea. However, unlike other cultured Korean species, the practical production of rock bream is unsatisfactory, mainly because of the infectious diseases caused by bacterial and viral infections leading to the mass mortalities [16], [17]. To understand the host immune defense and host-pathogen relationships at the molecular level, we engaged a combined transcriptomic- and genomic-approach to characterize the immune genes of rock bream, and a few of our recent findings provided insights into molecules of TLR signaling in teleosts [18], [19], [20], [21]. In this study, we report our investigation on the molecular, genomic and transcriptional characterization of a membrane-anchored TLR5 of O. fasciatus. We also present evidence for the NF-κB activation by TLR5M and its adaptor protein MyD88 [22] upon flagellin treatment using a luciferase reporter assay.

Section snippets

Determination of genomic sequences encoding rock bream tlr5m and myd88

Three individual partial cDNA segments of a tlr5-like sequence were identified from a previously constructed multi-tissue cDNA library of rock bream [23] using homology BLAST. In order to obtain the complete coding sequence (CDS) of this gene, and the genomic sequence of the myd88 gene [22], a custom constructed bacterial artificial chromosome (BAC) library was screened using a two-step PCR protocol as described elsewhere [24]. Gene-specific primers designed based on respective cDNA segments

Molecular and genomic characterization of Oftlr5m and Ofmyd88

The complete coding sequence of rock bream membrane-anchored tlr5 counterpart (Oftlr5m) and the genomic sequence of myd88 homolog (Ofmyd88) that we previously reported at cDNA level [22]) were determined using a combination of genomic and transcriptomic approaches. The putative coding sequence of Oftlr5 and exon-intron organizations of both Oftlr5m and Ofmyd88 were inferred by aligning the respective cDNA segments with gDNA sequences obtained from the BAC library.

As illustrated in Fig. 1,

Discussion

TLR5 is believed to be the only TLR that binds to a protein PAMP called bacterial flagellin [7]. In contrast to mammalian flagellin sensing machinery, which possesses a single membrane-anchored TLR5, the teleost flagellin recognition system is furnished with two distinct TLR5 variants localized in membrane and cytosol [9], implying the existence of an efficient recognition mechanism of flagellated bacteria in teleosts. To date, this dual-receptor driven flagellin sensing system has been

Acknowledgments

Authors are grateful to Bong-Soo Lim, Hyung-Bok Jung, W.D.N. Wickramaarachchi and Yucheol Kim, who helped in FLA-ST challenge experiment and to Ilandarage Menu Neelaka Molagoda who assisted in luciferase assay. We thank Dr. Teruyuki Nakanishi (Department of Veterinary Medicine, College of Bioresource Sciences, Nihon University) for scientific comments and Johnna Hayward Muniz for language editing and proofreading. This research was a part of the project titled ‘Fish Vaccine Research Center’,

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Toll-like receptor 5 (TLR5), serving as a sensor of bacterial flagellin, mediates the innate immune response to actively engage in the host's immune processes against pathogen invasion. However, the mechanism underlying TLR5-mediated immune response in fish remains unclear. Despite the presumed cell surface expression of TLR5 member form (TLR5M), its trafficking dynamics remain elusive. Here, we have identified Epinephelus coioides TLR5M as a crucial mediator of Vibrio flagellin-induced cytokine expression in grouper cells. EcTLR5M facilitated the activation of NF-κB signaling pathway in response to flagellin stimulation and exerted a modest influence on the mitogen-activated protein kinase (MAPK)-extracellular regulated kinase (ERK) signaling. The trafficking chaperone Unc-93 homolog B1 (EcUNC93B1) participated in EcTLR5M-mediated NF-κB signaling activation and downstream cytokine expression. In addition, EcUNC93B1 combined with EcTLR5M to mediate its exit from the endoplasmic reticulum, and also affected its post-translational maturation. Collectively, these findings first discovered that EcTLR5M mediated the flagellin-induced cytokine expression primarily by regulating the NF-κB signaling pathway, and EcUNC93B1 mediated EcTLR5M function through regulating its trafficking and post-translational maturation. This research expanded the understanding of fish innate immunity and provided a novel concept for the advancement of anti-vibrio immunity technology.
Identification and characterization of toll-like receptor genes in silver pomfret (Pampus argenteus) and their involvement in the host immune response to Photobacterium damselae subsp. Damselae and Nocardia seriolae infection
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Toll-like receptors (TLRs) are vital pattern recognition receptors that play a critical role in the innate immune response against pathogenic attack. Among the bacteria commonly found in the culture process of silver pomfret, Photobacterium damselae subsp. Damselae (PDD, gram-negative) and Nocardia seriolae (NS, gram-positive), can cause large-scale mortality in this fish species. However, there is currently no research on the role of TLRs in mediating the immune response of silver pomfret to these two bacterial infections. Therefore, in this study, we identified nine PaTLRs family members, including several fish-specific TLRs (TLR14 and TLR21). Phylogenetic analysis revealed that these PaTLRs genes could be classified into five subfamilies, namely TLR1, TLR3, TLR5, TLR7, and TLR11, indicating their evolutionary conservation. To further explore the interactions of TLR genes with immune-related mediators, protein and protein interaction network (PPI) results were generated to explain the association of TLR genes with TNF receptor-associated factor 6 (TRAF6) and other relevant genes in the MyD88-dependent pathway and NF-κb signaling pathway. Subsequently, RT-qPCR was conducted to verify the expression patterns of the nine TLR genes in the gills, skin, kidney, liver, and spleen of healthy fish, with most of the TLRs showing high expression levels in the spleen. Following infection with PDD and NS, these PaTLRs exhibited different expression patterns in the spleen, with PaTLR2, PaTLR3, PaTLR5, PaTLR7, PaTLR9, and PaTLR14 being significantly up-regulated. Furthermore, when spleen cells were treated with bacterial compositions, the majority of PaTLRs expression was up-regulated in response to Lipopolysaccharide (LPS) and lipophosphorylcholic acid (LTA) treatment, except for PaTLR21. Finally, changes in the expression levels of TLR-interacting genes were also observed under the stimulation of bacteria and bacterial compositions. The results of this study provide a preliminary reference for further understanding the mechanism of the innate immune response of the TLR gene family in silver pomfret and offer theoretical support for addressing the disease problems encountered during large-scale fish breeding.
TLR5M cooperates with TLR5S to activate NF-κB in Nile tilapia (Oreochromis niloticus)
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Moreover, their simultaneous presence could further amplify the signal of NF-κB. Similar data have been found in rock bream, which show that transfection with OfTLR5M exhibited a higher NF-κB activity than the control vector, and the luciferase activity was intensified when the cells were stimulated with flagellin (Umasuthan et al., 2017). In miiuy croaker, the ability of mmiTLR5S to activate NF-κB was increased in a concentration-dependent manner (Huo et al., 2018).
The binding of flagellin to receptor TLR5 (Toll-like receptor 5) on the cell surface is a prerequisite for the pathogen infection to the body, which has important implication for disease prevention. Unlike mammals, teleost TLR5 exists in two different forms: membrane-bound TLR5M (membrane TLR5, TLR5M) and secreted TLR5S (soluble TLR5, TLR5S). In this study, TLR5M and TLR5S from Nile tilapia (Oreochromis niloticus), named OnTLR5M and OnTLR5S, were cloned and identified. The open reading frame (ORF) of OnTLR5M was 2658 base pair (bp), encoding 885 amino acids (aa), while the ORF of OnTLR5S was 1926 bp, encoding 641 aa. Under normal physiological conditions, both OnTLR5M and OnTLR5S are expressed in the tested tissues, among which OnTLR5M has the highest expression in the liver and the lowest expression in the brain, while OnTLR5S has the highest expression in peripheral blood and the lowest expression in thymus. After Aeromonas hydrophila stimulation in vivo, OnTLR5M and OnTLR5S were significantly up-regulated in peripheral blood and head kidney, and relatively insignificant in spleen. After A. hydrophila stimulation in monocytes/macrophages, OnTLR5M and OnTLR5S were also significantly up-regulated. The results of subcellular localization analysis showed that OnTLR5M was distributed on the cell membrane, while OnTLR5S was distributed in the whole cell. Both OnTLR5M and OnTLR5S can significantly activate NF-κB signal, and the activation degree is further increased after the synergy of A. hydrophila or flagellin. In addition, co-transfection of OnTLR5M and OnTLR5S showed a stronger ability to activate NF-κB than expression of OnTLR5M alone. Further, the response to NF-κB after transferring the hotspot site of zebrafish TLR5 into tilapia TLR5M and TLR5S was not significantly different from that of wild type, suggesting that the hotspot site may be conserved in teleost fish. Moreover, OnTLR5M interacts with OnTLR5S and OnMyD88, respectively. It is speculated that OnTLR5S cooperates with OnTLR5M to detect pathogens, and then OnTLR5M recruits OnMyD88, which activates NF-κB through a signaling cascade, prompting cells to express cytokines, thereby resisting pathogens. This study may provide a theoretical support for the understanding of the flagellin recognition system and TLR5-mediated signaling pathway in teleost fish.
Identification and functional characteristics of two TLR5 subtypes in S. grahami
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Flagellin, a structural protein which forms the filaments of bacterial flagella, is the specific ligand of TLR5 in mammals [8]. In this study, sgTLR5a and sgTLR5b strongly responded to the in vivo and in vitro stimulation of flagellin and A. hydrophila (Figs. 5 and 6), which is consistent with results in Pelteobagrus vachelli [25], Oplegnathus fasciatus [12] and Oncorhynchus mykiss [29], implying that fish TLR5 conservatively recognizes flagellin. Remarkably, although there is no evidence that LPS is the ligand of TLR5, our results showed that sgTLR5b responded to the in vivo stimulation of LPS (Figs. 5 and 6), providing the possibility for sgTLR5b to sense LPS.
TLR5, as a member of Toll-like receptors (TLRs) family in mammals, is responsible for recognizing bacterial flagellin and initiating innate immunity, but its function is still unclear in fish species. In this study, two family members of TLR5 were cloned and identified from Sinocyclocheilus grahami (S. grahami), named sgTLR5a and sgTLR5b. The length of coding sequence of sgTLR5a and sgTLR5b is 2,622 bp and 2,658 bp, encoding 873 and 885 amino acids, respectively. Molecular phylogenetic analysis indicates that sgTLR5a and sgTLR5b have the closest genetic relationship with TLR5M (membrane-type) of Cyprinus carpio and Schizothorax prenanti, respectively. sgTLR5a and sgTLR5b were widely expressed in various tested tissues, of which the expression levels were the highest in skin tissue. After stimulations of Aeromonas hydrophila (A. hydrophila) and flagellin, the expression levels of sgTLR5a and sgTLR5b in liver, spleen and head kidney tissues were strongly up-regulated, but LPS stimulation only increased the expression of sgTLR5b in these tissues. The luciferase reporter assay displayed that sgTLR5a and sgTLR5b could specifically recognize bacterial flagellin and A. hydrophila and activate the downstream NF-κB signaling pathway in HEK293T cells. Moreover, the overexpression of sgTLR5a and sgTLR5b in EPC cells up-regulated the expression levels of IL-8 and TNF. sgTLR5a and sgTLR5b were observed to locate in the intracellular region by confocal microscope. Interestingly, we found that the NF-κB signaling pathway was positively regulated by co-transfecting sgTLR5a or sgTLR5b with TLR trafficking chaperone sgUNC93B1. In conclusion, our results reveal sgTLR5a and sgTLR5b may play an important role in antibacterial response by activating the NF-κB signaling pathway.
TLR5 recognizes Aeromonas hydrophila flagellin and interacts with MyD88 in Nile tilapia
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Cotransfection of OnMyd88 and OnTLR5 significantly upregulated Myd88-mediated NF-κB activation (Fig. 1A). This result is consistent with previous research results showing that TLR5M and MyD88 are responsible for NF-κB activation in Oplegnathus fasciatus (Umasuthan et al., 2017). Cotransfection of Schizothorax prenanti TLR5-1 or TLR5-2 into HEK293T cells induced significantly increased NF-kB luciferase activity in response to flagellin stimulation (Liu et al., 2018).
Toll-like receptor 5 (TLR5) is responsible for bacterial flagellin recognition in vertebrates. In the present study, TLR5M was identified in the Nile tilapia Oreochromis niloticus (OnTLR5), containing a conserved LRR domain, a transmembrane region and a C-terminal TIR domain, similar to that of other fishes and mammals. OnTLR5 was broadly expressed in all the tissues examined, presenting the highest expression levels in the blood and the lowest in the kidney. OnTLR5 was detected from 2 d postfertilization (dpf) to 8 dpf during embryonic development. Moreover, expression levels of OnTLR5 were clearly altered in all five tissues examined in response to Streptococcus agalactiae infection in vivo. Overexpression of OnTLR5 in HEK293T cells revealed that OnTLR5 was distributed in the cytoplasm and significantly increased NF-κB activation. In response to cotransfection with OnMyd88, OnTLR5 significantly upregulated OnMyd88-induced NF-κB activation. Pulldown assays showed that OnTLR5 interacts with OnMyd88 and revealed an interaction between TLR5 and Aeromonas hydrophila flagellin. Taken together, these findings suggest that OnTLR5 plays important roles in TLR/IL-1R signalling pathways and the immune response to pathogen invasion.
Membrane orthologs of TLR5 of tongue sole Cynoglossus semilaevis: Expression patterns, signaling pathway and antibacterial property
2022, Fish and Shellfish Immunology
Mammalian toll-like receptor 5 (TLR5) is crucial for recognizing bacterial flagellin and initiating the inflammatory signaling cascades via myeloid differentiation factor 88 (MyD88) signaling pathway, which plays vital roles in innate immune against pathogenic bacteria. Herein, we reported the signaling pathway and antibacterial property of tongue sole (Cynoglossus semilaevis) membrane forms of TLR5 (i.e. CsTLR5M1and CsTLR5M2). CsTLR5M1/M2 contain 936 and 885 amino acid residues respectively. CsTLR5M1 shares 86.7% overall sequence identities with CsTLR5M2. CsTLR5M1/M2 possess the same extracellular domain (ECD) and transmembrane domain (TMD), but the different toll-interleukin-1 receptor (TIR) domain. CsTLR5M1/M2 expression occurred constitutively in multiple tissues and regulated by bacterial stimulation. Recombinant CsTLR5M1/M2 (rCsTLR5M) could bind to flagellin and Gram-negative/positive bacteria, which could suppress bacterial growth. Stimulation of the CsTLR5M pathway by flagellin resulted in increased expression of MyD88-dependent signaling molecules and inflammatory cytokines. Blocking rCsTLR5M by antibody markedly reduced the phagocytosis and ROS production of peripheral blood leukocytes (PBLs), which in turn in vivo promoted the dissemination of bacteria. Overall, these observations add new insights into the signaling pathway and immune function of teleost TLR5M.

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

Highlights

Abstract

Introduction

Section snippets

Determination of genomic sequences encoding rock bream tlr5m and myd88

Molecular and genomic characterization of Oftlr5m and Ofmyd88

Discussion

Acknowledgments

Cell

Trends Immunol.

J. Biol. Chem.

J. Biol. Chem.

Fish Shellfish Immunol.

Fish Shellfish Immunol.

Mol. Immunol.

Fish Shellfish Immunol.

Veterinary Immunol. Immunopathol.

Fish Shellfish Immunol.

Fish. Shellfish Immunol.

Fish. Shellfish Immunol.

Fish Shellfish Immunol.

Fish Shellfish Immunol.

Dev. Comp. Immunol.

Dev. Comp. Immunol.

Methods

Fish Shellfish Immunol.

Dev. Comp. Immunol.

Immunol. Lett.

J. Biol. Chem.

Fish Shellfish Immunol.

J. Biol. Chem.

Mol. Immunol.

Dev. Comp. Immunol.

Mol. Immunol.

Vaccine

Dev. Comp. Immunol.

Fish. Shellfish Immunol.

Full length article
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