Abstract Toll‐like receptors (TLRs) are an important family of pattern‐recognition receptors that recognize a series of pathogen‐associated molecular patterns to trigger the host immune response. In ...the present study, we identified three TLR13 genes (i.e., LcLTR13a, LcTLR13b, and LcTLR13c) in American bullfrogs, Lithobates catesbeianus , and explored their constitutive and pathogen‐induced expression profile in different tissues. The open reading frames of LcLTR13a, LcTLR13b and LcTLR13c are 2811, 2844, and 2814 bp long, encoding 955, 947, and 933 amino acids, respectively. Phylogenetic analysis showed that three LcTLR13 genes grouping in different subclades indicate the gene duplication of bullfrog TLR13 as in teleost. The three LcTLR13 genes were expressed in all the tissues examined, with the liver and spleen expressing the higher levels. Moreover, the expression levels of LcLTR13a, LcTLR13b, and LcTLR13c were significantly induced in the liver and spleen after bullfrogs were challenged with bacterial (i.e., Citrobacter freundii , Aeromonas hydrophila , and Edwardsiella tarda ) pathogens. These results indicate that LcTLR13 genes play an essential role in the bullfrog antibacterial innate immune response, providing new insights into immune mechanisms of bullfrogs.
Congenital immunity mediated by Toll-like receptor (TLR) family is the first line of defense for disease-resistant immunity of fish and plays a vital role as a bridge between innate immunity and ...acquired immunity. As a less known member of the TLR family TLR13 can participate in the immune and inflammatory reactions of the body for recognizing the conserved sequence of 23S rRNA in bacteria and induce immune response. In this study, the full-length cDNA of TLR13 from Nibea albiflora (named as NaTLR13) was cloned and was functionally characterized. It was 4210bp (GenBank accession no. MT701899) including an open reading frame (ORF) of 2886bp to encode 962 amino acids with molecular weight of 110.37 kDa and the theoretical isoelectric point of 9.08. There were several conservative structures in NaTLR13 such as 15 leucine-rich repeat sequences (LRRs), a Toll-IL-1 receptor domain (TIR), an LRR-CT terminal domain, two LRR-TYP structures and two transmembrane domains. The multiple sequence alignment and phylogenetic analysis manifested that NaTLR13 had high similarity with Larimichthys crocea and Collichthys lucidus (88.79% and 87.02%, respectively) and they fell into the same branch. The Real-time PCR showed that NaTLR13 was expressed in all selected tissues, with the highest in the spleen, followed by the liver, kidney, gill, heart and muscle. After being challenged by Vibrio alginolyticus, Vibrio parahaemolyticus or Poly (I:C), the expression of NaTLR13 increased firstly, then decreased and finally stabilized with time for its immune defense function. Subcellular localization analysis revealed that NaTLR13 was unevenly distributed in the cytoplasm with green fluorescence and MyD88 was evenly spread in the cytoplasm with red signals. When NaTLR13 and MyD88 were co-transfected, they obviously overlapped and displayed orange-yellow color, which showed that the homologous TLR13 might interact with MyD88 for NFκB signaling pathway transmission. The functional domains of NaTLR13 (named NaTLR13-TIR and NaTLR13-LRR) were expressed in E.coli BL21 (DE3) and purified by Ni-NAT Superflow Resin conforming to the expected molecular weights, and the recombinant proteins could bind to three Vibrios (V.alginolyticus, V.parahaemolyticus and Vibrio harveyi), indicating that NaTLR13 could be bounden to bacteria through its functional domain. These results suggested that NaTLR13 might play an important role in the defense of N.albiflora against bacteria or viral infection and the data would provide some information for further understanding the regulatory mechanism of the innate immune system in fish.
•A TLR13 homologue was identified from Nibea albiflora (NaTLR13).•NaTLR13 had highly conserved functional domains and located in cytoplasm.•There might be the interaction between NaTLR13 and MyD88 for Toll/NF-κB pathway.•NaTLR13 could be significantly induced in response to bacteria and viruses.•NaTLR13-TIR and NaTLR13-LRR could couple to the pathogenic bacteria.
Toll‐like receptor 13 (TLR13) recognizes a conserved 10‐nucleotide sequence from bacterial 23S ribosomal RNA, and binding of TLR13 to the target rRNA molecule triggers immune responses. Recently, the ...crystal structure of the TLR13 ectodomain bound by a 13‐nucleotide single‐stranded RNA (ssRNA13) was determined by using the initial phases provided by a cryo‐electron microscopy map of TLR13 in complex with a 25‐nucleotide ssRNA (ssRNA25). This structural snapshot describes a unique method for solving the crystal structure of the TLR13–ssRNA13 complex based on medium‐resolution reconstruction of TLR13–ssRNA25 cryo‐electron microscopy images.
The use of low‐to‐medium‐resolution three‐dimensional reconstructions as molecular replacement models to phase X‐ray data is becoming a more feasible tool due to advances in cryo‐electron microscopic single‐particle analysis methodology. The whole solution procedure is briefly divided into two consecutive steps: molecular replacement and phase extension. The crystal structure of the Toll‐like receptor 13 ectodomain was recently determined using this method.
•TLR13a and TLR13b genes were identified from tilapia (Oreochromis niloticus).•Expressions of TLR13a and TLR13b mRNAs were altered after Streptococcus agalactiae challenge.•The TLR13a and TLR13b ...mRNAs were detected in various embryonic developmental stages.•Both OnTLR13a and OnTLR13b could interact with OnMyd88.•Both OnTLR13a and TLR13b can combine with S. agalactiae 23S rRNA.
Toll-like receptors (TLRs) play a crucial role in the innate immune system, which is the first line of defence against pathogens and pathogenic products in fish. In the present study, we cloned the full-length cDNA and genome sequences of two TLR13 s (OnTLR13a, OnTLR13b) from Nile tilapia (Oreochromis niloticus). TLR family motifs, i.e., the leucine-rich repeat (LRR) domains and Toll/interleukin (IL)-1 receptor (TIR) domains, were conserved in the putative proteins OnTLR13a and OnTLR13b, with fifteen LRR domains and one TIR domain. Four exons and three introns were identified in the OnTLR13a genome sequence, and three exons and two introns were identified in the OnTLR13b genome sequence. In healthy Nile tilapia tissues, OnTLR13a and OnTLR13b were ubiquitously expressed in all 11 tested tissues/organs. The highest expression levels were observed in the spleen (OnTLR13a) and blood (OnTLR13b), and the lowest expression levels were observed in the liver (OnTLR13a) and stomach (OnTLR13b). The expression level of OnTLR13b at 5.5 days postfertilization (dpf) was significantly higher than that at the other 8 time points (2.5, 3.5, 4.5, 5, 6, 6.5, 7.5 and 8.5 dpf). Upon stimulation with an intraperitoneal injection of 200 μL (107 CFU/mL) Streptococcus agalactiae, the expression levels of OnTLR13a and OnTLR13b were significantly upregulated in the intestine and gill. After cotransfection with MyD88, OnTLR13a significantly increased MyD88-dependent NF-κB activation in 293 T cells. However, OnTLR13b significantly impaired MyD88-dependent NF-κB activation. In addition, TLR13a slightly increased MyD88-dependent AP-1 activation, and TLR13b significantly increased MyD88-dependent AP-1 activation. TLR13a significantly increased MyD88-dependent interferon-β (IFN-β) activation, and TLR13b had no effect on MyD88-dependent IFN-β activation. These findings suggest that although the deduced protein structure of OnTLR13 is evolutionarily conserved between OnTLR13 and other TLR members, its signal transduction function is markedly different. Co-immunoprecipitation (Co-IP) assays showed that both OnTLR13a and OnTLR13b could interact with OnMyD88. RNA pulldown assays showed that TLR13a and TLR13b could combine with the 23S rRNA of S. agalactiae. These results indicate that TLR13a and TLR13b play important roles in the innate immune response against bacterial infection in Nile tilapia.
Background Bacterial cowshed isolates are allergy protective in mice; however, the underlying mechanisms are largely unknown. We examined the ability of Lactococcus lactis G121 to prevent allergic ...inflammatory reactions. Objective We sought to identify the ligands and pattern recognition receptors through which L lactis G121 confers allergy protection. Methods L lactis G121–induced cytokine release and surface expression of costimulatory molecules by untreated or inhibitor-treated (bafilomycin and cytochalasin D) human monocyte-derived dendritic cells (moDCs), bone marrow–derived mouse dendritic cells (BMDCs), and moDC/naive CD4+ T-cell cocultures were analyzed by using ELISA and flow cytometry. The pathology of ovalbumin-induced acute allergic airway inflammation after adoptive transfer of BMDCs was examined by means of microscopy. Results L lactis G121–treated murine BMDCs and human moDCs released TH 1-polarizing cytokines and induced TH 1 T cells. Inhibiting phagocytosis and endosomal acidification in BMDCs or moDCs impaired the release of TH 1-polarizing cytokines, costimulatory molecule expression, and T-cell activation on L lactis G121 challenge. In vivo allergy protection mediated by L lactis G121 was dependent on endosomal acidification in dendritic cells (DCs). Toll-like receptor ( Tlr ) 13 −/− BMDCs showed a weak response to L lactis G121 and were unresponsive to its RNA. The TH 1-polarizing activity of L lactis G121–treated human DCs was blocked by TLR8-specific inhibitors, mediated by L lactis G121 RNA, and synergistically enhanced by activation of nucleotide-binding oligomerization domain-containing protein (NOD) 2. Conclusion Bacterial RNA is the main driver of L lactis G121–mediated protection against experimentally induced allergy and requires both bacterial uptake by DCs and endosomal acidification. In mice L lactis G121 RNA signals through TLR13; however, the most likely intracellular receptor in human subjects is TLR8.
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