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Identification and characterization of three TLR1 subfamily members from the orange-spotted grouper, Epinephelus coioides
Developmental and Comparative Immunology 61 (2016) 180e189
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Short communication
Identification and characterization of three TLR1 subfamily members from the orange-spotted grouper, Epinephelus coioides Yan-Wei Li a, b, Dong-Dong Xu c, Xia Li b, Ze-Quan Mo a, Xiao-Chun Luo c, An-Xing Li b, *, Xue-Ming Dan a, ** a b c
College of Animal Science, South China Agricultural University, Guangzhou 510642, Guangdong Province, PR China State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, Guangdong Province, PR China School of Bioscience and Biotechnology, South China University of Technology, Guangzhou 510006, Guangdong Province, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 March 2016 Received in revised form 27 March 2016 Accepted 27 March 2016 Available online 30 March 2016
Toll-like receptors (TLRs), which play important roles in host defense against pathogen infection, are the most intensively studied pattern recognition receptors (PRRs). In this study, we identified three novel TLR1 subfamily members, including TLR1 (EcTLR1b), TLR2 (EcTLR2b) and TLR14 (EcTLR14), from the orange-spotted grouper (Epinephelus coioides). EcTLR1b and EcTLR2b displayed low sequence identity with the previously reported grouper TLR1 (EcTLR1a) and TLR2 (EcTLR2a), respectively. The open reading frames (ORFs) of EcTLR1b, EcTLR2b and EcTLR14 contain 2484 bp, 2394 bp and 2640 bp, which encode the corresponding 827 amino acids (aa), 797 aa and 879 aa, respectively. All three TLRs have leucine-rich repeat (LRR) domains (including an LRR-NT (except for EcTLR1b), several LRR motifs and an LRR-CT), a trans-membrane region and a Toll/interleukin-1 receptor (TIR) domain. The TIR domains of the three TLRs exhibited conserved boxes, namely box1, box2 and box3, and their 3D models were similar to those of human TLR1 or TLR2. Sequence alignment demonstrated that the TIR domains of the three TLRs shared higher sequence identity with those of other species than the full-length receptors. Phylogenetic analysis indicated that EcTLR1s and EcTLR2s are characterized by their differing evolutionary status, whereas EcTLR14 was found to be in the same group as other piscine TLR14/18s. The three TLRs were ubiquitously expressed in seven tested tissues of healthy groupers, although their expression profiles were different. Post Cryptocaryon irritans infection, TLR1s expression was up-regulated in the gills. The expression of TLR2b was mainly increased in the spleen, but decreased in the gills, which was similar to the expression pattern of TLR2a post C. irritans infection. Unlike EcTLR1b and EcTLR2b, however, the grouper TLR14 transcript was substantially induced in both tissues post challenge. These findings may be helpful in understanding the innate immune mechanism of host anti-parasite infection. Crown Copyright © 2016 Published by Elsevier Ltd. All rights reserved.
Keywords: Epinephelus coioides Toll-like receptor Innate immune Cryptocaryon irritans
1. Introduction Toll-like receptor (TLR) is a crucial pattern recognition receptor (PRR) that recognizes specific molecules that are conserved among microbes, called pathogen associated molecular patterns (PAMPs), to trigger the host innate immune response (Kawai and Akira,
* Corresponding author. State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, 135 Xingang West Street, Haizhu District, Guangzhou 510275, Guangdong Province, PR China. ** Corresponding author. College of Animal Science, South China Agricultural University, 483 Wushan Street, Tianhe District, Guangzhou 510642, PR China. E-mail addresses: [email protected] (A.-X. Li), [email protected] (X.-M. Dan). http://dx.doi.org/10.1016/j.dci.2016.03.028 0145-305X/Crown Copyright © 2016 Published by Elsevier Ltd. All rights reserved.
2011). TLRs are type I trans-membrane glycoproteins that contain two typical domains, namely an N-terminal leucine-rich repeat (LRR) domain and a C-terminal Toll/IL-1R homology (TIR) domain, and the two domains are linked by a single trans-membrane helix (Sasai and Yamamoto, 2013; Takeuchi and Akira, 2010). The LRR domain binds specifically the conserved ligand(s) and the TIR domain is responsible for signal transduction. To date, 10 and 12 TLRs have been found in humans and mice, respectively, and these TLRs are categorized into six subfamilies according to their sequence identities and function (Roach et al., 2005). The TLR1 subfamily, including TLR1, TLR2, TLR6 and TLR10, localizes on the cell surface, and senses pathogen associated molecular patterns (PAMPs) mainly on the surfaces or membranes of microbes (Akira et al., 2006). In mammals, TLR2 recognizes a broad range of
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PAMPs, including lipoteichoic acid (LTA), peptidoglycans (PGN), lipoarabinomannan, zymosan, glycosylphosphatidylinositols (GPI) and glycoproteins from bacteria, yeasts, parasites or viruses (Aravalli et al., 2005; Krishnegowda et al., 2005; Sato et al., 2003; Schwandner et al., 1999; Underhill et al., 1999). In addition, TLR2 can specifically recognize triacylated or diacylated lipopeptides via heterodimerization with TLR1 or TLR6, and this heterodimerization is indispensible in expanding the ligand spectrum of TLR2 but not for differential signaling (Farhat et al., 2008). To date, however, the ligand responsible for the activation of TLR10 has not been absolutely identified (Govindaraj et al., 2010). Upon interaction with ligands, TLR1 subfamily members directly recruit adaptor protein MyD88 via their TIR domain to trigger the MyD88-dependent signaling pathway and subsequently regulate the expression of pro-inflammatory cytokines (Takeuchi and Akira, 2010). In fish, more than 20 TLRs have been discovered, and several of these TLRs are common to both mammals and fish. However, the TLR repertoire in fish is more diverse than that in mammals (Rebl et al., 2010; Takano et al., 2010). For the TLR1 subfamily, counterparts of mammalian TLR1 and TLR2 have been found in fish, and piscine TLR1 displays equally high sequence identity to mammalian TLR1, TLR6 and TLR10. However, thus far, no TLR6 or TLR10 sequence has been identified in any fish species. Ribeiro et al. (2010) have reported that, using p38 MAPK phosphorylation as the output, common carp TLR2 can be activated by LTA and PGN from Staphylococcus aureus, as well as synthetic triacylated lipopeptide (Pam3CSK4), but found no response to the diacylated lipopeptide (MALP-2). This may be due to the lack of TLR6 in fish, which prevents the heterodimerization of TLR2 with TLR6 that is required to sense MALP-2 (Pietretti and Wiegertjes, 2014). However, Brietzke et al. (2016) recently demonstrated that classical ligands of mammalian TLR2 cannot activate rainbow trout TLR2 to induce the activation of NF-kB. In addition to TLR1 and TLR2, a nonmammalian receptor TLR14 (sometimes called TLR18) has been discovered that is a new TLR1 subfamily member unique to fish. Huang et al. (2015) found that overexpression of grass carp TLR18 increased NF-kB activation, induced the expression of IL-8, IFN-1 and TNF-a and, more importantly, reduced the Aeromonas hydrophila infection in CIK cells. However, the ligand specifically recognized by TLR18 is so far unclear. In recent years, TLRs have been reported in many teleost fish (Palti, 2011). However, to date, only six TLRs, including TLR1, TLR2, TLR3, TLR9, TLR21 and TLR22 (Ding et al., 2012; Li et al., 2012; Qiao et al., 2012; Wei et al., 2011), have been identified and characterized from the orange-spotted grouper (Epinephelus coioides), which is one of the more important mari-cultured species reared in South China. In the present study, three TLR1 subfamily members, including a novel TLR1 and TLR2 and a non-mammalian TLR14, were identified. The structure conservation and phylogenetic status of these receptors were analyzed, and subsequently the expressions of these TLRs in healthy groupers and Cryptocaryon irritans-infected fish were determined. C. irritans is a protozoan ectoparasite, which can infect most farmed marine fish and causes heavy economic losses in South China. The results obtained here contribute to an understanding of the innate immune mechanism of host defense against infection by this parasite. 2. Materials and methods 2.1. Screening of cDNA and ORF amplification Five EST sequences were identified from a transcriptome database of the grouper, and they each shared high sequence identity with one of the piscine TLR1 subfamily members (unpublished data). Among them, two ESTs shared 100% sequence identity with
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grouper TLR1 and TLR2 reported by Wei et al. (2011), respectively, and are here referred to as EcTLR1a and EcTLR2a. However, two other ESTs displayed sequence identity with other piscine TLR1s or TLR2s, and we refer to them as EcTLR1b and EcTLR2b. A 408 bp EcTLR2b has been amplified in our lab, and the expression level of this receptor in C. irritans-infected grouper tissues has been determined (Li et al., 2011). The last EST sequence with high sequence identity with other piscine TLR14/18 is referred to as EcTLR14. All three novel TLRs contain complete open reading frames (ORFs). Subsequently, primers of TLR1bFLF/R, TLR2bFLF/R and TLR14FLF/R (STable 1) were designed to amplify the ORFs of EcTLR1b, EcTLR2b and EcTLR14, respectively, with PrimeSTAR® max DNA polymerase (Takara, Japan). After insertion into the pEASYblunt simple cloning vector (TransGen Biotech, China) and transformation into Escherichia coli Trans1-T1 phage resistant chemically competent cells (TransGen Biotech), positive clones were identified using colony PCR and sequenced by the Life Technologies Corporation (Guangdong, China). 2.2. Bioinformatic analysis Sequence alignment was constructed using NCBI BLAST (http:// blast.ncbi.nlm.nih.gov/Blast.cgi) or Clustal Omega software (http:// www.ebi.ac.uk/Tools/msa/clustalo/). ORFs were identified using the NCBI ORF Finder tool (http://www.ncbi.nlm.nih.gov/gorf/gorf. html). N-glycosylation sites were predicted using NetNGlyc 1.0 Server (http://www.cbs.dtu.dk/services/NetNGlyc/). The theoretical Mw (molecular weight) was computed with the Compute pI/Mw tool (http://web.expasy.org/compute_pi/). Protein structure analysis was performed using SMART software (http://smart.emblheidelberg.de/), and the numbers of LRRs were manually identified through the method described by Matsushima et al. (2007). A three-dimensional (3D) model of the TIR domain was constructed using SWISS-MODLE software (http://swissmodel.expasy.org/ interactive). The phylogenetic tree was constructed using MEGA 5.04 software. 2.3. Fish and sampling Orange-spotted groupers, weighing 12.3 ± 3.3 g, were purchased from the Marine Fisheries Development Center of Guangdong Province, China, where no disease outbreak had occurred during the course of breeding. Fish were then acclimated for 2 weeks at 25 C in a flow-through water system and fed daily with a commercial grouper feed. C. irritans used in the present study was propagated using pompano Trachinotus ovatus as the host following the methods described by Dan et al. (2006). For the challenge, 80 groupers were randomly divided into two groups, a parasiteinfected group and an untreated control group. The former was exposed to C. irritans at a dose of 4800 theronts per fish as previously described, and the latter was treated the same as the infection group with the exception of parasite infection (Li et al., 2014). Samples of gill and spleen tissue were collected from both groups at 6 h, 12 h, 1 d, 2 d and 3 d post challenge, and were used as the template to analyze the expression profiles of the target genes after parasite infection. In addition, samples of skin, gill, thymus, brain, spleen, head kidney and trunk kidney were isolated from healthy groupers to detect the tissue distribution of the target genes. All samples were immediately frozen in liquid nitrogen after collection and then stored at 80 C until RNA isolation. 2.4. RNA isolation and cDNA synthesis Total RNA was isolated from the tissues sampled in Section 2.3 using Trizol Reagent (Invitrogen, USA) following the manual's
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protocols and then dissolved in 20 mL RNase-free water. The quality of the extracted RNA was determined by agarose gel electrophoresis and using an OD260/280 test. After treatment with RNase-free DNase I (ThermoFisher Scientific, USA) at 37 C for 30 min, 1 mg total RNA was used as the template to synthesize the first-strand cDNA by treating with ReverTra Ace-a reverse transcriptase (Toyobo, Japan) at 42 C for 60 min. The cDNA was then stored at 20 C until use. 2.5. Expression analysis Real-time PCR was used to determine the tissue expression profiles of EcTLR1b, EcTLR2b and EcTLR14 in healthy fish, and the temporal expressions of EcTLR1a, EcTLR1b, EcTLR2a and EcTLR14 post C. irritans infection. Primers are listed in STable 1 and b-actin was used as the internal control. Real-time PCR was performed with Maxima SYBR Green qPCR Master Mix (2) on a Roche LightCycler 480 Realtime PCR Detection System (Roche). The PCR cycling conditions were as follows: one cycle of 95 C for 10 min, followed by 40 cycles of 95 C for 15 s, 57 C for 30 s and 72 C for 30 s. The specificities of the PCR products were confirmed by melting curve analysis and sequencing. Each sample was amplified in triplicate. The mRNA expression of each target gene relative to the reference gene was calculated using the 2△△Ct method (Livak and Schmittgen, 2001). Data were analyzed using SPSS software and results are expressed as the mean ± standard error (SE). The significance of differences was determined by Duncan's test, and the level of statistically significant difference was set at P < 0.05. 3. Results and discussion 3.1. Sequence and domain analysis The ORFs of EcTLR1b, EcTLR2b and EcTLR14 were submitted to the NCBI GenBank under accession nos. KM282519, KM282521 and KM282520, respectively. The ORFs of EcTLR1b, EcTLR2b and EcTLR14 contained 2484 bp, 2394 bp and 2640 bp, which encode the corresponding 827 amino acids (aa), 797 aa and 879 aa, respectively (Table 1 and SFig. 1). The calculated Mws were about 90 kDa for TLR1s and TLR2s and 100 kDa for EcTLR14 (Table 1) (Wei et al., 2011). Weber et al. (2004) indicated that N-glycosylation is required for TLR function in humans, and each TLR has its unique conserved N-glycosylation sites. In this study, several putative Nglycosylation sites were identified from the LRR domains of EcTLR1a (at positions 73, 105, 122, 152, 376 and 429), EcTLR1b (at positions 62, 116, 233, 268, 300, 349 and 567), EcTLR2a (at positions 189, 218, 246, 294 and 400), EcTLR2b (at positions 39, 225 and 439) and EcTLR14 (at positions 96 and 576), and the amounts and positions of N-glycosylation sites between TLR1s or TLR2s were different (SFig. 1). However, these N-glycosylation sites are predicted by software, and whether they really contain N-linked oligosaccharides at these sites is unclear. TLR has a characteristic structure with an LRR domain, a short trans-membrane region and a TIR domain, which is normally located on the cell surface or in intracellular compartments such as
endosomes, using its trans-membrane region to perform its function (Akira et al., 2006). All grouper TLR1 subfamily members (EcTLR1b, EcTLR2s and EcTLR14) have this typical structure with the exception of EcTLR1a, in which no trans-membrane region was predicted (SFig. 1) (Wei et al., 2011). Wang et al. (2013) also indicated that no trans-membrane region was found in TLR1 of rainbow trout, spotted green pufferfish and Japanese flounder. Whether the lack of a trans-membrane region affects its localization and even the function of TLR1 needs further study. The LRR domain contains several LRR motifs, which adopt a horseshoe shape (Jin and Lee, 2008). Except for TLR14/18 (21 LRRs), the other TLR1 subfamily members typically have 20 LRR motifs (Matsushima et al., 2007). Using the method described by Matsushima et al. (2007), we found EcTLR1a and EcTLR2s have 20 LRR motifs, but EcTLR1b has one additional LRR at the N-terminus (Table 1 and SFig. 1AeD). The additional LRR sequence is “GGIRDLSHHNLRSVPSNLPND”, which is a bacterial type LRR, as in Japanese puffer TLR1. In zebrafish, carp and rohu TLR2, 21 LRR motifs were identified and the last LRR motif may interfere with the capping of the LRR-CT (Matsushima et al., 2007; Ribeiro et al., 2010; Samanta et al., 2012). In Japanese flounder TLR2, however, only 19 LRRs were found, so that there is no LRR corresponding to LRR7 in species with 20 LRRs. EcTLR14 has 21 LRR motifs, which number was conserved among reported piscine TLR14/18s including those of Japanese puffer, zebrafish, channel catfish and Atlantic salmon (Table 1 and SFig. 1E) (Lee et al., 2014; Matsushima et al., 2007; Quiniou et al., 2013). The N-terminus and C-terminus LRR domains (LRR-NT and LRRCT) are capped by two cysteine clusters in most TLRs, which mostly contain two and four cysteine residues, respectively, and form disulfide bonds (Matsushima et al., 2005). EcTLR2s both share a similar Cx2Cx4-5CxC motif, but EcTLR2a has one more amino acid than EcTLR2b between the second and third cysteine (Table 1). The cysteine motif model was observed in other piscine TLR2s, but is different from mammalian TLR2s (Cx5C) (Quiniou et al., 2013). A Cx10C type LRR-NT is conserved among EcTLR14 and other piscine TLR14/18s (Table 1). Similar to mammalian and other piscine TLR1s, EcTLR1s also have no LRR-NT. EcTLR1s, EcTLR2s and EcTLR14 share a similar LRR-CT motif CxCx22-31Cx20C, but the amino acid numbers are different between the second and the third cysteine (Table 1) (Matsushima et al., 2007). The first two cysteines are sited in the last LRR. In conclusion, the LRR numbers and LRR-NT and LRR-CT motifs are highly conserved in TLR14/18, compared to other TLR1 subfamily members, among fish species. The TIR domain is essential for signaling, and three highly conserved motifs, namely boxes 1, 2 and 3 were identified in this domain (Slack et al., 2000; Xu et al., 2000). Box1/2 is proposed to be involved in signaling transduction, while box3 mainly participates in directing the localization of receptors (Slack et al., 2000; Xu et al., 2000). These boxes are conserved in the cytoplasmic regions of the grouper and other piscine TLR1s, TLR2s, and TLR14/18s with the exception of EcTLR1a in box2, where the first conserved amino acid arginine is replaced by another basic amino acid lysine (Fig. 1). However, the important amino acid residue proline in box2 is conserved in all piscine and human TLR1 subfamily members. The TIR domains of human TLR1 and TLR2 are composed of five central
Table 1 Sequence characteristics of deduced EcTLR1s, EcTLR2s and EcTLR14 proteins. TLRs
Length (aa)
N-glycosylation sites
Molecular weight (kDa)
LRR
LRRNT
LRRTC
TLR1a TLR1b TLR2a TLR2b TLR14
801 827 821 797 879
6 6 5 3 2
90.1 93.3 92.9 90.6 100.3
20 21 20 20 21
None None Cx2Cx5CxC Cx2Cx4CxC Cx10C
CxCx31Cx20C CxCx22Cx20C CxCx26Cx20C CxCx25Cx20C CxCx25Cx20C
Fig. 1. Multiple sequence alignment of EcTLR1s (A), EcTLR2s (B) and EcTLR14 (C) TIR domain with that of other vertebrate species. Three highly conserved boxes were marked by red boxes, respectively. Numbers represented the amino acid positions of EcTLR1b, EcTLR2b or EcTLR14. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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parallel b-sheets, which are surrounded by five a-helices on both sides (Xu et al., 2000). By homology modeling, we found that grouper TLR1s, TLR2b and TLR14 TIR domains all have 3D models that are similar to those of human TLR1 (1fyv.1.A) and TLR2 (1fyw.1.A), with five b-sheets surrounded by several a-helices (Fig. 2). However, in the grouper TLR2a TIR domain, a total of seven b-sheets were observed, and the additional two b-sheets are localized between the first a-helix and the second b-sheet in human and other TLR2 TIR domains. Whether this difference in structure is affected by lower sequence identity between human TLR2 and grouper TLR2a is unclear. 3.2. Identity and phylogenetic analysis In groupers, two types of TLR1 or TLR2 were identified by Wei et al. (2011) and our group (in this study), and sequence alignment suggested that the identity of TLR1s or TLR2s are very low (Fig. 3). As shown in STable 2, EcTLR1s share only 32% sequence identity with each other, and the TIR domain has 52% identity. However, EcTLR1b displays 52%e79% sequence identity with that of other fish, and the TIR domain shared high sequence identity (74%e 92%). EcTLR1b demonstrated equal sequence identity (full-length about 30% and TIR domain about 50%) with Homo sapiens TLR1/6/ 10, which is the same as the result obtained in Japanese puffer (Oshiumi et al., 2003). EcTLR2b shares 40%e45% sequence identity with EcTLR2a and human and other piscine TLR2s, and the TIR domain displays higher sequence identity (59%e66%) (STable 2).
EcTLR14 full-length or TIR domains share 59%e81% or 86%e95% amino acids identity, respectively, with other reported piscine TLR14/18s (STable 2). To analyze the evolutionary relationships of these receptors, a phylogenetic tree was constructed using fish, bird, mouse and human TLR1 subfamily proteins (Fig. 4). The results indicated that the tree is clustered into three groups: one group contains bird, mouse and human TLR1/6/10 and piscine TLR1, another group has TLR2 from different species including EcTLR2s, and the last group contains piscine TLR14 and TLR18. The above results indicated that EcTLR1a and EcTLR1b are evolutionarily different, and the former is more closely related to mammalian TLR1/6/10, while the latter is more closely related to piscine TLR1. Similarly, EcTLR2a is evolutionarily closer to piscine TLR2 than EcTLR2b. Therefore, the lower identity between EcTLR1a and EcTLR1b, or EcTLR2a and EcTLR2b, obtained above can be explained by the different degrees of evolution of each receptor. 3.3. Tissue distribution of EcTLRs in healthy groupers In the present study, a total of seven tissues were used to detect the expression profiles of the three TLRs. qPCR results demonstrated that expression of all TLRs can be detected in the tissues tested, but the expression profiles differ. Relatively high expression of both EcTLR1b and EcTLR2b was observed in the spleen, followed by the gills or skin, and weak expression was detected in the thymus and brain (Fig. 5). However, EcTLR14 transcript levels were
Fig. 2. The 3D model of EcTLR1a (A), EcTLR1b (B), EcTLR2a (C), EcTLR2b (D) and EcTLR14 (D) TIR domain. The model of EcTLR1s TIR domain was performed using human TLR1 TIR domain (1fyv.1.A) as template, while EcTLR2s and EcTLR14 using human TLR2 TIR domain (1fyw.1.A) as template.
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Fig. 3. Sequence alignment of EcTLR1b and EcTLR2b (A), or EcTLR2a and EcTLR2b (B). The highly conserved boxes are marked in red. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 4. Phylogenetic analysis of TLR1 subfamily proteins. The neighbor-joining tree was constructed using Mega 5.04 software and numbers on the lines indicate the percentages of bootstrap values after 1000 replicates. Mus musculus TLR1: AAG37302; M. musculus TLR6: BAA78632; Homo sapiens TLR1: AAY85640; H. sapiens TLR6: BAA78631; H. sapiens TLR10: AAY78491; Gallus gallusTLR1: BAD67422; Maylandia zebra TLR1: XP_004553798; Haplochromis burtoni TLR1: XP_005933874; Oreochromis niloticus TLR1: XP_005460413; Neolamprologus brichardi TLR1: XP_006799802; Stegastes partitus TLR1: XP_008288611; Oryzias latipes TLR1: XP_004083162; Lepisosteus oculatus TLR1: XP_006633625; Danio rerio TLR18: AAI62732; Ctenopharyngodon idella TLR18: AIB55030; Ictalurus punctatus TLR18: AEI59674; Salmo salar TLR18: CDK60413; Takifugu rubripes TLR14: AAW69369; Paralichthys olivaceus TLR14: BAJ78225; H. sapiens TLR2: AAH33756; G. gallus TLR2: BAB16113; M. musculus TLR2: EDL15415; M. zebra TLR2: XP_004571641; O. niloticus TLR2: XP_005460222; Poecilia formosa TLR2: XP_007568121; Oncorhynchus mykiss TLR2: CCK73195; I. punctatus TLR2: AEI59663; D. rerio TLR2: NP_997977; C. idella TLR2: ACT68333; Carassius carassius TLR2: AGO57934; Cyprinus carpio TLR2: ACP20793; Cirrhinus mrigala TLR2: AHI59129; Labeo rohita TLR2: ADQ74644.
notably detected in the head, kidney, skin and trunk kidney but were low in the brain and thymus, similar to EcTLR1b and EcTLR2b (Fig. 5). The expression profiles of EcTLR1s and EcTLR2s were similar to each other except that relatively higher transcript levels of EcTLR1a and EcTLR2b were found in the head kidney and skin, respectively (Wei et al., 2011). Nevertheless, the highest expression level of these receptors is found mainly in the immune organs, which is in line with the expression of grouper TLR21 and TLR22 (Ding et al., 2012; Li et al., 2012). The basic expression of TLR1 subfamily members has been broadly determined in several teleost fish, and most of these genes displayed a constitutive tissues distribution, but each demonstrated a unique expression pattern. (1) Wu et al. and Zhang et al. have reported that TLR1, in spotted green pufferfish and channel catfish, was ubiquitously expressed in various tissues, but a low expression
level was observed in pufferfish (Wu et al., 2008; Zhang et al., 2013). In large yellow croaker and rainbow trout, TLR1 was notably expressed in the spleen, which is similar to EcTLR1s (Palti et al., 2010; Wang et al., 2013). (2) In large yellow croaker, two TLR2 homologues were identified but, unlike EcTLR2s, the basic expression pattern of both TLR2s was inconsistent with the highest TLR2a expressed in the blood, while the highest TLR2b expression was seen in the intestine (Ao et al., 2016). Catfish TLR2 expression was detected at high levels in the liver, brain and gills, whereas only low expression was observed in the skin and muscle (Baoprasertkul et al., 2007). However, rohu TLR2 was predominantly expressed in the spleen followed by the intestine and gills (Samanta et al., 2012). (3) Grass carp TLR18 was predominantly expressed in the spleen, gills and heart, and weak expression was observed in the skin, liver and brain (Huang et al., 2015). The salmon TLR18
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very weakly in the heart and muscle in Japanese flounder (Hwang et al., 2010). Ubiquitous expression of TLRs in fish species suggests their important immune surveillance role, while the unique tissue expression patterns may imply their specific function characteristics.
3.4. Expression of EcTLRs post C. irritans infection
Fig. 5. Constitutive expression of EcTLR1b, EcTLR2b and EcTLR14 in fish tissues was determined using real-time PCR. The mRNA expression levels were normalized to bactin transcripts, and the data are expressed as mean ± SE (N ¼ 3).
transcript was broadly detected in the muscle and liver, but expression was very weak in the head kidney (Lee et al., 2014). However, TLR14 was abundantly expressed in the head kidney, but
In mammals, the MyD88-dependent signaling pathway plays a crucial role in host defense against parasite infection, and parasite components such as GPI, profilin-like protein and genomic DNA can be recognized by TLR2, TLR4, TLR9, TLR11 or TLR12 (McDonald et al., 2013; Ropert et al., 2008; Tarleton, 2007; Yarovinsky, 2014). Further studies have demonstrated that individual TLR deficiencies have less notable effects than simple MyD88 deficiency on the infection intensity of a parasite in vivo, and multiple TLRs are required for optimal resistance (Andrade et al., 2013; SchamberReis et al., 2013). To date, however, the role of piscine TLRs in the host immune response against parasite infection has not been thoroughly studied. To analyze the possible involvement of TLR1 subfamily members in grouper anti-C. irritans infection, herein, we detected the expression profiles of these TLRs after infection with the parasite, and the results are shown in Fig. 6. The expression of EcTLR1s in the gills showed a similar trend, firstly increasing and reaching a peak at day 1, and then decreasing to normal levels at day 3 (Fig. 6A and B). In the spleen, however, no
Fig. 6. Expression of EcTLR1a (A), EcTLR1b (B), EcTLR2a (C) and EcTLR14 (D) in the gills and spleen after infection with C. irritans was detected using real-time PCR at various time points. mRNA expression levels were firstly normalized to the transcripts of b-actin, and then the change fold of test group's gene expression to its control at the same time point was calculated. The data are shown as mean ± SE (N ¼ 3). Significant differences in gene expression between the control and C. irritans-infected groups at each time point are indicated with* (P < 0.05).
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significant change was observed during the course of this experiment. In the gills, the EcTLR2a transcript was mainly downregulated and a significant up-regulation was detected only at day 1 (Fig. 6C). However, the expression of EcTLR2a was mainly upregulated in the spleen, and a significant increase in its expression was observed at 6 h and day 2 post infection. The changes in EcTLR2a expression were similar to those of EcTLR2b, as its expression decreased in the infection sites (skin and gills), while significantly increasing in the systemic immune organs (spleen and head kidney) (Li et al., 2011). Unlike EcTLR1s and EcTLR2s, where the expression pattern is different in the gills and spleen, the EcTLR14 transcript level was noticeably increased in both the gills and spleen during the course of parasite infection (Fig. 6D). The peak expression of EcTLR14 was reached at day 1 (about 10-fold) and 6 h (about 7-fold) in the gills and spleen, respectively, post C. irritans challenge. Nevertheless, the expression of four TLRs was significantly up-regulated at day 1 in the gills, but at day 2 in the spleen, suggesting that the host immune response was triggered more quickly at the local infection site than in the systemic immune organs. TLR1 and TLR2 expression could also be significantly induced in channel catfish and naked carp post challenge with another ciliate parasite, Ichthyophthirius multifiliis, but the TLR18 transcript level had not obviously changed (Tong et al., 2015; Zhao et al., 2013). These findings implied that the immune response mechanisms of salt and fresh water fish against the two parasites may be different. In addition to the present study, the expression of TLR1 subfamily genes was also detected after stimulation by other pathogens. Wei et al. (2011) has indicated that EcTLR1a expression can be up-regulated in the spleen and head kidney post LPS, poly (I:C) or Vibrio alginolyticus stimulation. Similar results were also observed in salmon and zebrafish TLR1 after infection with Piscirickettsia salmonisor Mycobacterium marinum, respectively, and pufferfish TLR1 post LPS stimulation (Meijer et al., 2004; Salazar et al., 2015; Wu et al., 2008). In LYCK cells, however, although LPS could significantly induce TLR1 expression at later time points, PGN or poly (I:C) had no obvious effect on expression (Wang et al., 2013). Similarly, trout TLR1 expression was not affected by treatment with mammalian TLR agonists in anterior kidney leukocytes (Palti et al., 2010). As observed in EcTLR1a, LPS, poly (I:C) or V. alginolyticus can up-regulate the expression of EcTLR2a (Wei et al., 2011). In large yellow croaker, the expressions of TLR2a and TLR2b could be substantially increased post Vibrio parahaemolyticus, LPS and polyI:C, or inactivated trivalent bacterial vaccine stimulation (Ao et al., 2016; Fan et al., 2015). Besides, mrigal TLR2 expression could also be induced following PGN, Streptococcus uberis or Aeromonas hydrophila treatment (Basu et al., 2012). In Japanese flounder, Hwang et al. (2010) demonstrated that TLR14 expression was induced by viral hemorrhagic septicemia virus or bacterial (Streptococcus iniae and Edwardsiella tarda) challenge. Huang et al. (2015) also indicated that the TLR18 transcript level could been substantially increased by either grass carp reovirus or A. hydrophila infection in immune-relevant tissues or by flagellin, LPS or poly (I:C) stimulation in CIK cells. However, salmon TLR18 expression was significantly down-regulated in the primary head kidney cell post IFNg, type I IFN or IL-1b stimulation (Lee et al., 2014). These studies suggested that TLR1 subfamily members may participate in fish anti-pathogen infection, but further study is needed to identify the specific ligand recognized by these receptors. In conclusion, in the present study, three more TLR1 subfamily members, including EcTLR1b, EcTLR2b and EcTLR14, were identified in groupers, and these TLRs displayed conserved TLR structure. Sequence alignment indicated that EcTLR1s or EcTLR2s share relatively low sequence identity with each other, and are characterized by differing evolutionary status. EcTLR14 exhibited high
sequence identity with that of other fish, and clustered into the same branch with other piscine TLR14/18s. The expressions of these three TLRs were determined in healthy and C. irritans-infected groupers, and the results implied that this parasite could induce different immune responses of these TLRs in the gills and spleen. Further work should focus on studying the functional similarity/ disparity of EcTLR1s and EcTLR2s. Acknowledgments This work was funded by the National Natural Science Foundation of China (31302208 and 31272681) and the Marine Fisheries Science and Technology Promotion Project of Guangdong Province (A201501B11). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.dci.2016.03.028. References Akira, S., Uematsu, S., Takeuchi, O., 2006. Pathogen recognition and innate immunity. Cell 124, 783e801. Andrade, W.A., Souza, M.d.C., Martinez, E.R., Nagpal, K., Dutra, M.S., Melo, M.B., Bartholomeu, D.C., Ghosh, S., Golenbock, D.T., Gazzinelli, R.T., 2013. Combined action of nucleic acid-sensing toll-like receptors (TLRs) and TLR11/TLR12 heterodimers imparts resistance to Toxoplasma gondii in mice. Cell Host Microbe 13, 42e53. Ao, J., Mu, Y., Wang, K., Sun, M., Wang, X., Chen, X., 2016. Identification and characterization of a novel Toll-like receptor 2 homologue in the large yellow croaker Larimichthys crocea. Fish. Shellfish Immunol. 48, 221e227. Aravalli, R.N., Hu, S., Rowen, T.N., Palmquist, J.M., Lokensgard, J.R., 2005. TLR2mediated proinflammatory cytokine and chemokine production by microglial cells in response to herpes simplex virus. J. Immunol. 175, 4189e4193. Baoprasertkul, P., Peatman, E., Abernathy, J., Liu, Z., 2007. Structural characterisation and expression analysis of toll-like receptor 2 gene from catfish. Fish. Shellfish Immunol. 22, 418e426. Basu, M., Swain, B., Sahoo, B.R., Maiti, N.K., Samanta, M., 2012. Induction of toll-like receptor (TLR) 2, and MyD88-dependent TLR- signaling in response to ligand stimulation and bacterial infections in the Indian major carp, mrigal (Cirrhinus mrigala). Mol. Biol. Rep. 39, 6015e6028. Brietzke, A., Arnemo, M., Gjoen, T., Rebl, H., Korytar, T., Goldammer, T., Rebl, A., Seyfert, H.M., 2016. Structurally diverse genes encode Tlr2 in rainbow trout: the conserved receptor cannot be stimulated by classical ligands to activate NF-kB in vitro. Dev. Comp. Immunol. 54, 75e88. Dan, X.M., Li, A.X., Lin, X.T., Teng, N., Zhu, X.Q., 2006. A standardized method to propagate Cryptocaryon irritans on a susceptible host pompano Trachinotus ovatus. Aquaculture 258, 127e133. Ding, X., Lu, D.Q., Hou, Q.H., Li, S.S., Liu, X.C., Zhang, Y., Lin, H.R., 2012. Orangespotted grouper (Epinephelus coioides) toll-like receptor 22: molecular characterization, expression pattern and pertinent signaling pathways. Fish. Shellfish Immunol. 33, 494e503. Fan, Z.J., Jia, Q.J., Yao, C.L., 2015. Characterization and expression analysis of Toll-like receptor 2 gene in large yellow croaker, Larimichthys crocea. Fish. Shellfish Immunol. 44, 129e137. Farhat, K., Riekenberg, S., Heine, H., Debarry, J., Lang, R., Mages, J., BuwittBeckmann, U., Roschmann, K., Jung, G., Wiesmuller, K.H., Ulmer, A.J., 2008. Heterodimerization of TLR2 with TLR1 or TLR6 expands the ligand spectrum but does not lead to differential signaling. J. Leukoc. Biol. 83, 692e701. Govindaraj, R.G., Manavalan, B., Lee, G., Choi, S., 2010. Molecular modeling-based evaluation of hTLR10 and identification of potential ligands in Toll-like receptor signaling. PLoS One 5, e12713. Huang, W.J., Shen, Y., Xu, X.Y., Hu, M.Y., Li, J.L., 2015. Identification and characterization of the TLR18 gene in grass carp (Ctenopharyngodon idella). Fish. Shellfish Immunol. 47, 681e688. Hwang, S.D., Kondo, H., Hirono, I., Aoki, T., 2010. Molecular cloning and characterization of Toll-like receptor 14 in Japanese flounder, Paralichthys olivaceus. Fish. Shellfish Immunol. 30, 425e429. Jin, M.S., Lee, J.O., 2008. Structures of the toll-like receptor family and its ligand complexes. Immunity 29, 182e191. Kawai, T., Akira, S., 2011. Toll-like receptors and their crosstalk with other innate receptors in infection and immunity. Immunity 34, 637e650. Krishnegowda, G., Hajjar, A.M., Zhu, J., Douglass, E.J., Uematsu, S., Akira, S., Woods, A.S., Gowda, D.C., 2005. Induction of proinflammatory responses in macrophages by the glycosylphosphatidylinositols of plasmodium falciparum: cell signaling receptors, glycosylphosphatidylinositol (GPI) structural requirement, and regulation of GPI activity. J. Biol. Chem. 280, 8606e8616.
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Short communication
Identification and characterization of three TLR1 subfamily members from the orange-spotted grouper, Epinephelus coioides Yan-Wei Li a, b, Dong-Dong Xu c, Xia Li b, Ze-Quan Mo a, Xiao-Chun Luo c, An-Xing Li b, *, Xue-Ming Dan a, ** a b c
College of Animal Science, South China Agricultural University, Guangzhou 510642, Guangdong Province, PR China State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, Guangdong Province, PR China School of Bioscience and Biotechnology, South China University of Technology, Guangzhou 510006, Guangdong Province, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 March 2016 Received in revised form 27 March 2016 Accepted 27 March 2016 Available online 30 March 2016
Toll-like receptors (TLRs), which play important roles in host defense against pathogen infection, are the most intensively studied pattern recognition receptors (PRRs). In this study, we identified three novel TLR1 subfamily members, including TLR1 (EcTLR1b), TLR2 (EcTLR2b) and TLR14 (EcTLR14), from the orange-spotted grouper (Epinephelus coioides). EcTLR1b and EcTLR2b displayed low sequence identity with the previously reported grouper TLR1 (EcTLR1a) and TLR2 (EcTLR2a), respectively. The open reading frames (ORFs) of EcTLR1b, EcTLR2b and EcTLR14 contain 2484 bp, 2394 bp and 2640 bp, which encode the corresponding 827 amino acids (aa), 797 aa and 879 aa, respectively. All three TLRs have leucine-rich repeat (LRR) domains (including an LRR-NT (except for EcTLR1b), several LRR motifs and an LRR-CT), a trans-membrane region and a Toll/interleukin-1 receptor (TIR) domain. The TIR domains of the three TLRs exhibited conserved boxes, namely box1, box2 and box3, and their 3D models were similar to those of human TLR1 or TLR2. Sequence alignment demonstrated that the TIR domains of the three TLRs shared higher sequence identity with those of other species than the full-length receptors. Phylogenetic analysis indicated that EcTLR1s and EcTLR2s are characterized by their differing evolutionary status, whereas EcTLR14 was found to be in the same group as other piscine TLR14/18s. The three TLRs were ubiquitously expressed in seven tested tissues of healthy groupers, although their expression profiles were different. Post Cryptocaryon irritans infection, TLR1s expression was up-regulated in the gills. The expression of TLR2b was mainly increased in the spleen, but decreased in the gills, which was similar to the expression pattern of TLR2a post C. irritans infection. Unlike EcTLR1b and EcTLR2b, however, the grouper TLR14 transcript was substantially induced in both tissues post challenge. These findings may be helpful in understanding the innate immune mechanism of host anti-parasite infection. Crown Copyright © 2016 Published by Elsevier Ltd. All rights reserved.
Keywords: Epinephelus coioides Toll-like receptor Innate immune Cryptocaryon irritans
1. Introduction Toll-like receptor (TLR) is a crucial pattern recognition receptor (PRR) that recognizes specific molecules that are conserved among microbes, called pathogen associated molecular patterns (PAMPs), to trigger the host innate immune response (Kawai and Akira,
* Corresponding author. State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, 135 Xingang West Street, Haizhu District, Guangzhou 510275, Guangdong Province, PR China. ** Corresponding author. College of Animal Science, South China Agricultural University, 483 Wushan Street, Tianhe District, Guangzhou 510642, PR China. E-mail addresses: [email protected] (A.-X. Li), [email protected] (X.-M. Dan). http://dx.doi.org/10.1016/j.dci.2016.03.028 0145-305X/Crown Copyright © 2016 Published by Elsevier Ltd. All rights reserved.
2011). TLRs are type I trans-membrane glycoproteins that contain two typical domains, namely an N-terminal leucine-rich repeat (LRR) domain and a C-terminal Toll/IL-1R homology (TIR) domain, and the two domains are linked by a single trans-membrane helix (Sasai and Yamamoto, 2013; Takeuchi and Akira, 2010). The LRR domain binds specifically the conserved ligand(s) and the TIR domain is responsible for signal transduction. To date, 10 and 12 TLRs have been found in humans and mice, respectively, and these TLRs are categorized into six subfamilies according to their sequence identities and function (Roach et al., 2005). The TLR1 subfamily, including TLR1, TLR2, TLR6 and TLR10, localizes on the cell surface, and senses pathogen associated molecular patterns (PAMPs) mainly on the surfaces or membranes of microbes (Akira et al., 2006). In mammals, TLR2 recognizes a broad range of
Y.-W. Li et al. / Developmental and Comparative Immunology 61 (2016) 180e189
PAMPs, including lipoteichoic acid (LTA), peptidoglycans (PGN), lipoarabinomannan, zymosan, glycosylphosphatidylinositols (GPI) and glycoproteins from bacteria, yeasts, parasites or viruses (Aravalli et al., 2005; Krishnegowda et al., 2005; Sato et al., 2003; Schwandner et al., 1999; Underhill et al., 1999). In addition, TLR2 can specifically recognize triacylated or diacylated lipopeptides via heterodimerization with TLR1 or TLR6, and this heterodimerization is indispensible in expanding the ligand spectrum of TLR2 but not for differential signaling (Farhat et al., 2008). To date, however, the ligand responsible for the activation of TLR10 has not been absolutely identified (Govindaraj et al., 2010). Upon interaction with ligands, TLR1 subfamily members directly recruit adaptor protein MyD88 via their TIR domain to trigger the MyD88-dependent signaling pathway and subsequently regulate the expression of pro-inflammatory cytokines (Takeuchi and Akira, 2010). In fish, more than 20 TLRs have been discovered, and several of these TLRs are common to both mammals and fish. However, the TLR repertoire in fish is more diverse than that in mammals (Rebl et al., 2010; Takano et al., 2010). For the TLR1 subfamily, counterparts of mammalian TLR1 and TLR2 have been found in fish, and piscine TLR1 displays equally high sequence identity to mammalian TLR1, TLR6 and TLR10. However, thus far, no TLR6 or TLR10 sequence has been identified in any fish species. Ribeiro et al. (2010) have reported that, using p38 MAPK phosphorylation as the output, common carp TLR2 can be activated by LTA and PGN from Staphylococcus aureus, as well as synthetic triacylated lipopeptide (Pam3CSK4), but found no response to the diacylated lipopeptide (MALP-2). This may be due to the lack of TLR6 in fish, which prevents the heterodimerization of TLR2 with TLR6 that is required to sense MALP-2 (Pietretti and Wiegertjes, 2014). However, Brietzke et al. (2016) recently demonstrated that classical ligands of mammalian TLR2 cannot activate rainbow trout TLR2 to induce the activation of NF-kB. In addition to TLR1 and TLR2, a nonmammalian receptor TLR14 (sometimes called TLR18) has been discovered that is a new TLR1 subfamily member unique to fish. Huang et al. (2015) found that overexpression of grass carp TLR18 increased NF-kB activation, induced the expression of IL-8, IFN-1 and TNF-a and, more importantly, reduced the Aeromonas hydrophila infection in CIK cells. However, the ligand specifically recognized by TLR18 is so far unclear. In recent years, TLRs have been reported in many teleost fish (Palti, 2011). However, to date, only six TLRs, including TLR1, TLR2, TLR3, TLR9, TLR21 and TLR22 (Ding et al., 2012; Li et al., 2012; Qiao et al., 2012; Wei et al., 2011), have been identified and characterized from the orange-spotted grouper (Epinephelus coioides), which is one of the more important mari-cultured species reared in South China. In the present study, three TLR1 subfamily members, including a novel TLR1 and TLR2 and a non-mammalian TLR14, were identified. The structure conservation and phylogenetic status of these receptors were analyzed, and subsequently the expressions of these TLRs in healthy groupers and Cryptocaryon irritans-infected fish were determined. C. irritans is a protozoan ectoparasite, which can infect most farmed marine fish and causes heavy economic losses in South China. The results obtained here contribute to an understanding of the innate immune mechanism of host defense against infection by this parasite. 2. Materials and methods 2.1. Screening of cDNA and ORF amplification Five EST sequences were identified from a transcriptome database of the grouper, and they each shared high sequence identity with one of the piscine TLR1 subfamily members (unpublished data). Among them, two ESTs shared 100% sequence identity with
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grouper TLR1 and TLR2 reported by Wei et al. (2011), respectively, and are here referred to as EcTLR1a and EcTLR2a. However, two other ESTs displayed sequence identity with other piscine TLR1s or TLR2s, and we refer to them as EcTLR1b and EcTLR2b. A 408 bp EcTLR2b has been amplified in our lab, and the expression level of this receptor in C. irritans-infected grouper tissues has been determined (Li et al., 2011). The last EST sequence with high sequence identity with other piscine TLR14/18 is referred to as EcTLR14. All three novel TLRs contain complete open reading frames (ORFs). Subsequently, primers of TLR1bFLF/R, TLR2bFLF/R and TLR14FLF/R (STable 1) were designed to amplify the ORFs of EcTLR1b, EcTLR2b and EcTLR14, respectively, with PrimeSTAR® max DNA polymerase (Takara, Japan). After insertion into the pEASYblunt simple cloning vector (TransGen Biotech, China) and transformation into Escherichia coli Trans1-T1 phage resistant chemically competent cells (TransGen Biotech), positive clones were identified using colony PCR and sequenced by the Life Technologies Corporation (Guangdong, China). 2.2. Bioinformatic analysis Sequence alignment was constructed using NCBI BLAST (http:// blast.ncbi.nlm.nih.gov/Blast.cgi) or Clustal Omega software (http:// www.ebi.ac.uk/Tools/msa/clustalo/). ORFs were identified using the NCBI ORF Finder tool (http://www.ncbi.nlm.nih.gov/gorf/gorf. html). N-glycosylation sites were predicted using NetNGlyc 1.0 Server (http://www.cbs.dtu.dk/services/NetNGlyc/). The theoretical Mw (molecular weight) was computed with the Compute pI/Mw tool (http://web.expasy.org/compute_pi/). Protein structure analysis was performed using SMART software (http://smart.emblheidelberg.de/), and the numbers of LRRs were manually identified through the method described by Matsushima et al. (2007). A three-dimensional (3D) model of the TIR domain was constructed using SWISS-MODLE software (http://swissmodel.expasy.org/ interactive). The phylogenetic tree was constructed using MEGA 5.04 software. 2.3. Fish and sampling Orange-spotted groupers, weighing 12.3 ± 3.3 g, were purchased from the Marine Fisheries Development Center of Guangdong Province, China, where no disease outbreak had occurred during the course of breeding. Fish were then acclimated for 2 weeks at 25 C in a flow-through water system and fed daily with a commercial grouper feed. C. irritans used in the present study was propagated using pompano Trachinotus ovatus as the host following the methods described by Dan et al. (2006). For the challenge, 80 groupers were randomly divided into two groups, a parasiteinfected group and an untreated control group. The former was exposed to C. irritans at a dose of 4800 theronts per fish as previously described, and the latter was treated the same as the infection group with the exception of parasite infection (Li et al., 2014). Samples of gill and spleen tissue were collected from both groups at 6 h, 12 h, 1 d, 2 d and 3 d post challenge, and were used as the template to analyze the expression profiles of the target genes after parasite infection. In addition, samples of skin, gill, thymus, brain, spleen, head kidney and trunk kidney were isolated from healthy groupers to detect the tissue distribution of the target genes. All samples were immediately frozen in liquid nitrogen after collection and then stored at 80 C until RNA isolation. 2.4. RNA isolation and cDNA synthesis Total RNA was isolated from the tissues sampled in Section 2.3 using Trizol Reagent (Invitrogen, USA) following the manual's
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protocols and then dissolved in 20 mL RNase-free water. The quality of the extracted RNA was determined by agarose gel electrophoresis and using an OD260/280 test. After treatment with RNase-free DNase I (ThermoFisher Scientific, USA) at 37 C for 30 min, 1 mg total RNA was used as the template to synthesize the first-strand cDNA by treating with ReverTra Ace-a reverse transcriptase (Toyobo, Japan) at 42 C for 60 min. The cDNA was then stored at 20 C until use. 2.5. Expression analysis Real-time PCR was used to determine the tissue expression profiles of EcTLR1b, EcTLR2b and EcTLR14 in healthy fish, and the temporal expressions of EcTLR1a, EcTLR1b, EcTLR2a and EcTLR14 post C. irritans infection. Primers are listed in STable 1 and b-actin was used as the internal control. Real-time PCR was performed with Maxima SYBR Green qPCR Master Mix (2) on a Roche LightCycler 480 Realtime PCR Detection System (Roche). The PCR cycling conditions were as follows: one cycle of 95 C for 10 min, followed by 40 cycles of 95 C for 15 s, 57 C for 30 s and 72 C for 30 s. The specificities of the PCR products were confirmed by melting curve analysis and sequencing. Each sample was amplified in triplicate. The mRNA expression of each target gene relative to the reference gene was calculated using the 2△△Ct method (Livak and Schmittgen, 2001). Data were analyzed using SPSS software and results are expressed as the mean ± standard error (SE). The significance of differences was determined by Duncan's test, and the level of statistically significant difference was set at P < 0.05. 3. Results and discussion 3.1. Sequence and domain analysis The ORFs of EcTLR1b, EcTLR2b and EcTLR14 were submitted to the NCBI GenBank under accession nos. KM282519, KM282521 and KM282520, respectively. The ORFs of EcTLR1b, EcTLR2b and EcTLR14 contained 2484 bp, 2394 bp and 2640 bp, which encode the corresponding 827 amino acids (aa), 797 aa and 879 aa, respectively (Table 1 and SFig. 1). The calculated Mws were about 90 kDa for TLR1s and TLR2s and 100 kDa for EcTLR14 (Table 1) (Wei et al., 2011). Weber et al. (2004) indicated that N-glycosylation is required for TLR function in humans, and each TLR has its unique conserved N-glycosylation sites. In this study, several putative Nglycosylation sites were identified from the LRR domains of EcTLR1a (at positions 73, 105, 122, 152, 376 and 429), EcTLR1b (at positions 62, 116, 233, 268, 300, 349 and 567), EcTLR2a (at positions 189, 218, 246, 294 and 400), EcTLR2b (at positions 39, 225 and 439) and EcTLR14 (at positions 96 and 576), and the amounts and positions of N-glycosylation sites between TLR1s or TLR2s were different (SFig. 1). However, these N-glycosylation sites are predicted by software, and whether they really contain N-linked oligosaccharides at these sites is unclear. TLR has a characteristic structure with an LRR domain, a short trans-membrane region and a TIR domain, which is normally located on the cell surface or in intracellular compartments such as
endosomes, using its trans-membrane region to perform its function (Akira et al., 2006). All grouper TLR1 subfamily members (EcTLR1b, EcTLR2s and EcTLR14) have this typical structure with the exception of EcTLR1a, in which no trans-membrane region was predicted (SFig. 1) (Wei et al., 2011). Wang et al. (2013) also indicated that no trans-membrane region was found in TLR1 of rainbow trout, spotted green pufferfish and Japanese flounder. Whether the lack of a trans-membrane region affects its localization and even the function of TLR1 needs further study. The LRR domain contains several LRR motifs, which adopt a horseshoe shape (Jin and Lee, 2008). Except for TLR14/18 (21 LRRs), the other TLR1 subfamily members typically have 20 LRR motifs (Matsushima et al., 2007). Using the method described by Matsushima et al. (2007), we found EcTLR1a and EcTLR2s have 20 LRR motifs, but EcTLR1b has one additional LRR at the N-terminus (Table 1 and SFig. 1AeD). The additional LRR sequence is “GGIRDLSHHNLRSVPSNLPND”, which is a bacterial type LRR, as in Japanese puffer TLR1. In zebrafish, carp and rohu TLR2, 21 LRR motifs were identified and the last LRR motif may interfere with the capping of the LRR-CT (Matsushima et al., 2007; Ribeiro et al., 2010; Samanta et al., 2012). In Japanese flounder TLR2, however, only 19 LRRs were found, so that there is no LRR corresponding to LRR7 in species with 20 LRRs. EcTLR14 has 21 LRR motifs, which number was conserved among reported piscine TLR14/18s including those of Japanese puffer, zebrafish, channel catfish and Atlantic salmon (Table 1 and SFig. 1E) (Lee et al., 2014; Matsushima et al., 2007; Quiniou et al., 2013). The N-terminus and C-terminus LRR domains (LRR-NT and LRRCT) are capped by two cysteine clusters in most TLRs, which mostly contain two and four cysteine residues, respectively, and form disulfide bonds (Matsushima et al., 2005). EcTLR2s both share a similar Cx2Cx4-5CxC motif, but EcTLR2a has one more amino acid than EcTLR2b between the second and third cysteine (Table 1). The cysteine motif model was observed in other piscine TLR2s, but is different from mammalian TLR2s (Cx5C) (Quiniou et al., 2013). A Cx10C type LRR-NT is conserved among EcTLR14 and other piscine TLR14/18s (Table 1). Similar to mammalian and other piscine TLR1s, EcTLR1s also have no LRR-NT. EcTLR1s, EcTLR2s and EcTLR14 share a similar LRR-CT motif CxCx22-31Cx20C, but the amino acid numbers are different between the second and the third cysteine (Table 1) (Matsushima et al., 2007). The first two cysteines are sited in the last LRR. In conclusion, the LRR numbers and LRR-NT and LRR-CT motifs are highly conserved in TLR14/18, compared to other TLR1 subfamily members, among fish species. The TIR domain is essential for signaling, and three highly conserved motifs, namely boxes 1, 2 and 3 were identified in this domain (Slack et al., 2000; Xu et al., 2000). Box1/2 is proposed to be involved in signaling transduction, while box3 mainly participates in directing the localization of receptors (Slack et al., 2000; Xu et al., 2000). These boxes are conserved in the cytoplasmic regions of the grouper and other piscine TLR1s, TLR2s, and TLR14/18s with the exception of EcTLR1a in box2, where the first conserved amino acid arginine is replaced by another basic amino acid lysine (Fig. 1). However, the important amino acid residue proline in box2 is conserved in all piscine and human TLR1 subfamily members. The TIR domains of human TLR1 and TLR2 are composed of five central
Table 1 Sequence characteristics of deduced EcTLR1s, EcTLR2s and EcTLR14 proteins. TLRs
Length (aa)
N-glycosylation sites
Molecular weight (kDa)
LRR
LRRNT
LRRTC
TLR1a TLR1b TLR2a TLR2b TLR14
801 827 821 797 879
6 6 5 3 2
90.1 93.3 92.9 90.6 100.3
20 21 20 20 21
None None Cx2Cx5CxC Cx2Cx4CxC Cx10C
CxCx31Cx20C CxCx22Cx20C CxCx26Cx20C CxCx25Cx20C CxCx25Cx20C
Fig. 1. Multiple sequence alignment of EcTLR1s (A), EcTLR2s (B) and EcTLR14 (C) TIR domain with that of other vertebrate species. Three highly conserved boxes were marked by red boxes, respectively. Numbers represented the amino acid positions of EcTLR1b, EcTLR2b or EcTLR14. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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parallel b-sheets, which are surrounded by five a-helices on both sides (Xu et al., 2000). By homology modeling, we found that grouper TLR1s, TLR2b and TLR14 TIR domains all have 3D models that are similar to those of human TLR1 (1fyv.1.A) and TLR2 (1fyw.1.A), with five b-sheets surrounded by several a-helices (Fig. 2). However, in the grouper TLR2a TIR domain, a total of seven b-sheets were observed, and the additional two b-sheets are localized between the first a-helix and the second b-sheet in human and other TLR2 TIR domains. Whether this difference in structure is affected by lower sequence identity between human TLR2 and grouper TLR2a is unclear. 3.2. Identity and phylogenetic analysis In groupers, two types of TLR1 or TLR2 were identified by Wei et al. (2011) and our group (in this study), and sequence alignment suggested that the identity of TLR1s or TLR2s are very low (Fig. 3). As shown in STable 2, EcTLR1s share only 32% sequence identity with each other, and the TIR domain has 52% identity. However, EcTLR1b displays 52%e79% sequence identity with that of other fish, and the TIR domain shared high sequence identity (74%e 92%). EcTLR1b demonstrated equal sequence identity (full-length about 30% and TIR domain about 50%) with Homo sapiens TLR1/6/ 10, which is the same as the result obtained in Japanese puffer (Oshiumi et al., 2003). EcTLR2b shares 40%e45% sequence identity with EcTLR2a and human and other piscine TLR2s, and the TIR domain displays higher sequence identity (59%e66%) (STable 2).
EcTLR14 full-length or TIR domains share 59%e81% or 86%e95% amino acids identity, respectively, with other reported piscine TLR14/18s (STable 2). To analyze the evolutionary relationships of these receptors, a phylogenetic tree was constructed using fish, bird, mouse and human TLR1 subfamily proteins (Fig. 4). The results indicated that the tree is clustered into three groups: one group contains bird, mouse and human TLR1/6/10 and piscine TLR1, another group has TLR2 from different species including EcTLR2s, and the last group contains piscine TLR14 and TLR18. The above results indicated that EcTLR1a and EcTLR1b are evolutionarily different, and the former is more closely related to mammalian TLR1/6/10, while the latter is more closely related to piscine TLR1. Similarly, EcTLR2a is evolutionarily closer to piscine TLR2 than EcTLR2b. Therefore, the lower identity between EcTLR1a and EcTLR1b, or EcTLR2a and EcTLR2b, obtained above can be explained by the different degrees of evolution of each receptor. 3.3. Tissue distribution of EcTLRs in healthy groupers In the present study, a total of seven tissues were used to detect the expression profiles of the three TLRs. qPCR results demonstrated that expression of all TLRs can be detected in the tissues tested, but the expression profiles differ. Relatively high expression of both EcTLR1b and EcTLR2b was observed in the spleen, followed by the gills or skin, and weak expression was detected in the thymus and brain (Fig. 5). However, EcTLR14 transcript levels were
Fig. 2. The 3D model of EcTLR1a (A), EcTLR1b (B), EcTLR2a (C), EcTLR2b (D) and EcTLR14 (D) TIR domain. The model of EcTLR1s TIR domain was performed using human TLR1 TIR domain (1fyv.1.A) as template, while EcTLR2s and EcTLR14 using human TLR2 TIR domain (1fyw.1.A) as template.
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Fig. 3. Sequence alignment of EcTLR1b and EcTLR2b (A), or EcTLR2a and EcTLR2b (B). The highly conserved boxes are marked in red. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 4. Phylogenetic analysis of TLR1 subfamily proteins. The neighbor-joining tree was constructed using Mega 5.04 software and numbers on the lines indicate the percentages of bootstrap values after 1000 replicates. Mus musculus TLR1: AAG37302; M. musculus TLR6: BAA78632; Homo sapiens TLR1: AAY85640; H. sapiens TLR6: BAA78631; H. sapiens TLR10: AAY78491; Gallus gallusTLR1: BAD67422; Maylandia zebra TLR1: XP_004553798; Haplochromis burtoni TLR1: XP_005933874; Oreochromis niloticus TLR1: XP_005460413; Neolamprologus brichardi TLR1: XP_006799802; Stegastes partitus TLR1: XP_008288611; Oryzias latipes TLR1: XP_004083162; Lepisosteus oculatus TLR1: XP_006633625; Danio rerio TLR18: AAI62732; Ctenopharyngodon idella TLR18: AIB55030; Ictalurus punctatus TLR18: AEI59674; Salmo salar TLR18: CDK60413; Takifugu rubripes TLR14: AAW69369; Paralichthys olivaceus TLR14: BAJ78225; H. sapiens TLR2: AAH33756; G. gallus TLR2: BAB16113; M. musculus TLR2: EDL15415; M. zebra TLR2: XP_004571641; O. niloticus TLR2: XP_005460222; Poecilia formosa TLR2: XP_007568121; Oncorhynchus mykiss TLR2: CCK73195; I. punctatus TLR2: AEI59663; D. rerio TLR2: NP_997977; C. idella TLR2: ACT68333; Carassius carassius TLR2: AGO57934; Cyprinus carpio TLR2: ACP20793; Cirrhinus mrigala TLR2: AHI59129; Labeo rohita TLR2: ADQ74644.
notably detected in the head, kidney, skin and trunk kidney but were low in the brain and thymus, similar to EcTLR1b and EcTLR2b (Fig. 5). The expression profiles of EcTLR1s and EcTLR2s were similar to each other except that relatively higher transcript levels of EcTLR1a and EcTLR2b were found in the head kidney and skin, respectively (Wei et al., 2011). Nevertheless, the highest expression level of these receptors is found mainly in the immune organs, which is in line with the expression of grouper TLR21 and TLR22 (Ding et al., 2012; Li et al., 2012). The basic expression of TLR1 subfamily members has been broadly determined in several teleost fish, and most of these genes displayed a constitutive tissues distribution, but each demonstrated a unique expression pattern. (1) Wu et al. and Zhang et al. have reported that TLR1, in spotted green pufferfish and channel catfish, was ubiquitously expressed in various tissues, but a low expression
level was observed in pufferfish (Wu et al., 2008; Zhang et al., 2013). In large yellow croaker and rainbow trout, TLR1 was notably expressed in the spleen, which is similar to EcTLR1s (Palti et al., 2010; Wang et al., 2013). (2) In large yellow croaker, two TLR2 homologues were identified but, unlike EcTLR2s, the basic expression pattern of both TLR2s was inconsistent with the highest TLR2a expressed in the blood, while the highest TLR2b expression was seen in the intestine (Ao et al., 2016). Catfish TLR2 expression was detected at high levels in the liver, brain and gills, whereas only low expression was observed in the skin and muscle (Baoprasertkul et al., 2007). However, rohu TLR2 was predominantly expressed in the spleen followed by the intestine and gills (Samanta et al., 2012). (3) Grass carp TLR18 was predominantly expressed in the spleen, gills and heart, and weak expression was observed in the skin, liver and brain (Huang et al., 2015). The salmon TLR18
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very weakly in the heart and muscle in Japanese flounder (Hwang et al., 2010). Ubiquitous expression of TLRs in fish species suggests their important immune surveillance role, while the unique tissue expression patterns may imply their specific function characteristics.
3.4. Expression of EcTLRs post C. irritans infection
Fig. 5. Constitutive expression of EcTLR1b, EcTLR2b and EcTLR14 in fish tissues was determined using real-time PCR. The mRNA expression levels were normalized to bactin transcripts, and the data are expressed as mean ± SE (N ¼ 3).
transcript was broadly detected in the muscle and liver, but expression was very weak in the head kidney (Lee et al., 2014). However, TLR14 was abundantly expressed in the head kidney, but
In mammals, the MyD88-dependent signaling pathway plays a crucial role in host defense against parasite infection, and parasite components such as GPI, profilin-like protein and genomic DNA can be recognized by TLR2, TLR4, TLR9, TLR11 or TLR12 (McDonald et al., 2013; Ropert et al., 2008; Tarleton, 2007; Yarovinsky, 2014). Further studies have demonstrated that individual TLR deficiencies have less notable effects than simple MyD88 deficiency on the infection intensity of a parasite in vivo, and multiple TLRs are required for optimal resistance (Andrade et al., 2013; SchamberReis et al., 2013). To date, however, the role of piscine TLRs in the host immune response against parasite infection has not been thoroughly studied. To analyze the possible involvement of TLR1 subfamily members in grouper anti-C. irritans infection, herein, we detected the expression profiles of these TLRs after infection with the parasite, and the results are shown in Fig. 6. The expression of EcTLR1s in the gills showed a similar trend, firstly increasing and reaching a peak at day 1, and then decreasing to normal levels at day 3 (Fig. 6A and B). In the spleen, however, no
Fig. 6. Expression of EcTLR1a (A), EcTLR1b (B), EcTLR2a (C) and EcTLR14 (D) in the gills and spleen after infection with C. irritans was detected using real-time PCR at various time points. mRNA expression levels were firstly normalized to the transcripts of b-actin, and then the change fold of test group's gene expression to its control at the same time point was calculated. The data are shown as mean ± SE (N ¼ 3). Significant differences in gene expression between the control and C. irritans-infected groups at each time point are indicated with* (P < 0.05).
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significant change was observed during the course of this experiment. In the gills, the EcTLR2a transcript was mainly downregulated and a significant up-regulation was detected only at day 1 (Fig. 6C). However, the expression of EcTLR2a was mainly upregulated in the spleen, and a significant increase in its expression was observed at 6 h and day 2 post infection. The changes in EcTLR2a expression were similar to those of EcTLR2b, as its expression decreased in the infection sites (skin and gills), while significantly increasing in the systemic immune organs (spleen and head kidney) (Li et al., 2011). Unlike EcTLR1s and EcTLR2s, where the expression pattern is different in the gills and spleen, the EcTLR14 transcript level was noticeably increased in both the gills and spleen during the course of parasite infection (Fig. 6D). The peak expression of EcTLR14 was reached at day 1 (about 10-fold) and 6 h (about 7-fold) in the gills and spleen, respectively, post C. irritans challenge. Nevertheless, the expression of four TLRs was significantly up-regulated at day 1 in the gills, but at day 2 in the spleen, suggesting that the host immune response was triggered more quickly at the local infection site than in the systemic immune organs. TLR1 and TLR2 expression could also be significantly induced in channel catfish and naked carp post challenge with another ciliate parasite, Ichthyophthirius multifiliis, but the TLR18 transcript level had not obviously changed (Tong et al., 2015; Zhao et al., 2013). These findings implied that the immune response mechanisms of salt and fresh water fish against the two parasites may be different. In addition to the present study, the expression of TLR1 subfamily genes was also detected after stimulation by other pathogens. Wei et al. (2011) has indicated that EcTLR1a expression can be up-regulated in the spleen and head kidney post LPS, poly (I:C) or Vibrio alginolyticus stimulation. Similar results were also observed in salmon and zebrafish TLR1 after infection with Piscirickettsia salmonisor Mycobacterium marinum, respectively, and pufferfish TLR1 post LPS stimulation (Meijer et al., 2004; Salazar et al., 2015; Wu et al., 2008). In LYCK cells, however, although LPS could significantly induce TLR1 expression at later time points, PGN or poly (I:C) had no obvious effect on expression (Wang et al., 2013). Similarly, trout TLR1 expression was not affected by treatment with mammalian TLR agonists in anterior kidney leukocytes (Palti et al., 2010). As observed in EcTLR1a, LPS, poly (I:C) or V. alginolyticus can up-regulate the expression of EcTLR2a (Wei et al., 2011). In large yellow croaker, the expressions of TLR2a and TLR2b could be substantially increased post Vibrio parahaemolyticus, LPS and polyI:C, or inactivated trivalent bacterial vaccine stimulation (Ao et al., 2016; Fan et al., 2015). Besides, mrigal TLR2 expression could also be induced following PGN, Streptococcus uberis or Aeromonas hydrophila treatment (Basu et al., 2012). In Japanese flounder, Hwang et al. (2010) demonstrated that TLR14 expression was induced by viral hemorrhagic septicemia virus or bacterial (Streptococcus iniae and Edwardsiella tarda) challenge. Huang et al. (2015) also indicated that the TLR18 transcript level could been substantially increased by either grass carp reovirus or A. hydrophila infection in immune-relevant tissues or by flagellin, LPS or poly (I:C) stimulation in CIK cells. However, salmon TLR18 expression was significantly down-regulated in the primary head kidney cell post IFNg, type I IFN or IL-1b stimulation (Lee et al., 2014). These studies suggested that TLR1 subfamily members may participate in fish anti-pathogen infection, but further study is needed to identify the specific ligand recognized by these receptors. In conclusion, in the present study, three more TLR1 subfamily members, including EcTLR1b, EcTLR2b and EcTLR14, were identified in groupers, and these TLRs displayed conserved TLR structure. Sequence alignment indicated that EcTLR1s or EcTLR2s share relatively low sequence identity with each other, and are characterized by differing evolutionary status. EcTLR14 exhibited high
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