The Toll-like receptor (TLR)-to-NF-kB signaling pathway is a prominent innate immune pathway in higher metazoans. Specific pathogen-associated molecular patterns (PAMPs) are detected by membrane-bound TLRs, which then initiate intracellular signaling cascades. One main TLR pathway leads to activation of transcription factor NF-kB to induce changes in the expression of genes encoding innate immune effector molecules such as cytokines and antimicrobial peptides (Akira et al., 2006;Kawai and Akira, 2007). TLR-to-NF-kB pathways have been intensively studied for their roles in immunity in many model systems from flies to humans (Aderem and Ulevitch, 2000;Kawai and Akira, 2007;Minakhina and Steward, 2006;Silverman and Maniatis, 2001;Vasselon and Detmers, 2002). Recently, genome and transcriptome sequencing has revealed that many basal metazoans and some pre-metazoans also have homologs of the TLR-to-NF-kB pathway (Gilmore and Wolenski, 2012). However, the biological roles of TLR and NF-kB in these basal organisms are not well understood (Bosch et al., 2009).
In mammals, TLRs are single-pass transmembrane proteins with an N-terminal extracellular leucine-rich repeat (LRR) domain, a central transmembrane (TM) domain, and a C-terminal Abbreviations: IKK, IkappaB kinase; LRR, leucine-rich repeat domain; Nv, Nematostella vectensis; NF-kB, nuclear factor kappa B; Of, Orbicella faveolata; TIR, Toll/interleukin-1 receptor; TLR, Toll-like Receptor; TM, transmembrane.
intracellular Toll/interleukin-1 receptor (TIR) domain. Ligand recognition by the LRR domain promotes engagement of the intracellular TIR domain with other TIR domain-containing adapter proteins, which initiates downstream signaling cascades (Kawai and Akira, 2007). These adapter proteins include myeloid differentiation primary response protein 88 (MYD88), TIR domaincontaining adapter protein (TIRAP/MAL), TIR domain-containing adapter inducing IFNb (TRIF), and Trif-related adapter protein (TRAM) (Aderem and Ulevitch, 2000;Kawai and Akira, 2007). The number of TLRs varies widely among organisms, with ten TLRs in humans, 13 in mice, nine in fruit flies, and over 200 in sea urchins (Buckley and Rast, 2012;Valanne et al., 2011;Vasselon and Detmers, 2002). In more basal organisms, such as sponges and cnidarians (which include hydras, jellyfish, sea anemones, and corals), there are two types of TLR-like proteins. Most often, these basal TLR-like proteins contain only a TM domain and a TIR domain; however, some basal animals have genes encoding full-length TLRs with LRR, TM, and TIR domains, similar to mammalian TLRs. Within the phylum Cnidaria, there are animals having no complete TLRs (Aiptasia pallida, Hydra vulgaris), ones having one complete TLR (Nematostella vectensis), and ones having multiple complete TLR and TLR-like proteins (Acropora digitifera) (Miller et al., 2007;Poole and Weis, 2014;Shinzato et al., 2011).
In insects and mammals, the NF-kB superfamily comprises multiple related transcription factors that bind distinct DNA sequences known as kB sites (Hayden and Ghosh, 2004). All NF-kB proteins have a conserved N-terminal domain of approximately 300 amino acids called the Rel Homology Domain (RHD). The RHD has residues important for DNA binding, dimerization, and nuclear localization (Gilmore, 2006). The NF-kB superfamily can be divided into two subclasses: the NF-kB proteins (e.g., human p52/p100 and p50/p105, and Drosophila Relish) and the Rel proteins (e.g., human p65/RelA, c-Rel, and RelB, and Drosophila Dorsal and Dif) (Gilmore, 2006). Based on DNA binding-site preference and sequence similarity, the NF-kB RHDs are more related to each other than to the RHDs of Rel proteins (Finnerty and Gilmore, 2015;Siggers et al., 2011). Where characterized, basal organisms (e.g., cnidarians, sponges, pre-metazoans) have single NF-kB family proteins, which are phylogenetically most similar to the vertebrate and insect NF-kB subclass (Finnerty and Gilmore, 2015).
In addition to the RHD, members of the NF-kB subclass have a Cterminal inhibitory domain consisting primarily of a series of Ankyrin (ANK) repeats, which must be removed by proteolysis to activate the DNA-binding activity of the transcription factor (Hayden and Ghosh, 2004;Sun, 2011). For example, the vertebrate NF-kB proteins p100 and p105 are processed to p52 and p50, respectively, which then translocate from the cytoplasm to the nucleus. In the mammalian non-canonical NF-kB signaling pathway, IkB kinase (IKK)-mediated phosphorylation of p100 at three serine residues located C-terminal to the ANK repeats promotes its processing to p52 by the proteasome (Sun, 2011). On the other hand, the Drosophila melanogaster NF-kB protein Relish is activated by a discrete caspase-mediated proteolytic event that removes the C-terminal ANK repeat domain (St€ oven et al., 2003;Valanne et al., 2011). Overall, it is not known how NF-kB proteins are regulated and activated in organisms basal to Drosophila.
The mountainous star coral Orbicella faveolata (Of), previously known as Montastraea faveolata, is an endangered reef-building coral found in the Caribbean Sea and the Gulf of Mexico. O. faveolata forms a symbiotic relationship with a dinoflagellate of the genus Symbiodinium (Davy et al., 2012;Steele et al., 2011). Like most reef-building corals, O. faveolata is susceptible to an environmentally induced loss of symbiosis, commonly referred to as "bleaching" because this process gives the coral tissue a white appearance (Brown, 1997;Gleason and Wellington, 1993;Hoegh-Guldberg et al., 2007;Hughes et al., 2017;Weis, 2008). Bleaching often causes coral death, but in some cases, corals can recover from a bleaching event and re-establish a symbiotic relationship with Symbiodinium. Nevertheless, recovered corals often show increased susceptibility to microbial diseases such as yellow band disease, black band disease, and plague (Kushmaro et al., 1996;Pinz on et al., 2015).
Recent reports of transcriptional changes in immune-related molecules in bleached and pathogen-infected corals have suggested that the cnidarian innate immune system plays a role in coral diseases (Anderson et al., 2016;Fuess et al., 2016Fuess et al., , 2017;;Pinz on et al., 2015;Zhou et al., 2017). To gain deeper insights into molecular processes important for coral immunity and health, we have had an ongoing interest in characterizing cnidarian homologs of mammalian immunoregulatory molecules and pathways (Wolenski et al., 2011(Wolenski et al., , 2013)). In this report, we have used phylogenetic, biochemical, and cell-based assays to characterize the structure, activity, and regulation of TLR and NF-kB proteins from O. faveolata. We also show that lipopolysaccharide (LPS) treatment of O. faveolata tissue can induce a gene expression profile consistent with induction of the NF-kB pathway. These results represent the first characterization of proteins in the conserved immunoregulatory TLR-to-NF-kB pathway of a critically endangered coral.
For comparative analysis of Toll-like receptors, the predicted TIR domains of Orbicella faveolata (Of) TLR, Nematostella vectensis (Nv) TLR, Drosophila melanogaster (Dm) Toll, and an Amphimedon queenslandica (Aq) TLR-like protein (Gauthier et al., 2010) were analyzed along with the ten human TLR proteins. The TIR domains of Of-TLR, Nv-TLR, and Aq-TLR were identified through MEME analysis (Bailey et al., 2006), and sequences were trimmed to contain only the TIR domains based on motif prediction and known human TIR domains (Supplemental Table 1). Human TLR and Dm-Toll amino acid sequences, along with their annotated TIR domains, were obtained from the UniProt database. Clustal Omega (Sievers et al., 2011) was then used to align the trimmed and culled TIR sequence dataset. The tree was rooted with the Aq-TLR, and phylogenetic comparison was performed using neighbor-joining analysis bootstrapped 1000 times using PAUP* (Swofford, 2001).
For phylogenetic analysis of the NF-kB and Rel proteins, the RHD sequences of O. faveolata (Of) NF-kB (from NCBI) Aiptasia pallida NF-kB (NCBI), Actinia tenebrosa NF-kB (NCBI), N. vectensis NF-kB (Uni-Prot), D. melanogaster Relish, Dorsal, and Dif (UniProt), and Homo sapiens p100, p105, RelA, RelB, and c-Rel (UniProt) were obtained. The tree was rooted with an NF-kB-like protein from Capsaspora owczarzaki (NCBI). As described above, conserved motifs from MEME analysis (Bailey et al., 2006) were truncated based on motif predictions (see Supplemental Table 2), were aligned by Clustal Omega (Sievers et al., 2011), and were inputted into PAUP* (Swofford, 2001) for maximum likelihood analysis bootstrapped 1000 times.
Expression plasmids for HA-tagged human IKKb, FLAG-Nv-NF-kB, FLAG-Hu-IKKb, and the empty pcDNA-FLAG vector have been described previously (Starczynowski et al., 2007;Wolenski et al., 2011). cDNAs encoding human cell codon-optimized versions of Of-NF-kB, Of-IKK, and Of-TLR were synthesized (GenScript) (Supplemental Figs. 1,2,and 3,respectively). These cDNAs and PCRgenerated Of-NF-kB truncation mutants (Of-RHD and Of-Cterm) were subcloned into pcDNA-FLAG or the yeast GAL4-fusion vector pGBT9. Details about primers and plasmid constructions are included in Supplemental Material (Supplemental Tables 3 and4).
DF-1 chicken fibroblasts and human HEK 293 or 293T cells were grown in Dulbecco's modified Eagle's Medium (Invitrogen) supplemented with 10% fetal bovine serum (Biologos), 50 units/ml penicillin, and 50 mg/ml streptomycin as described previously (Wolenski et al., 2011). Transfection of cells with expression plasmids was performed using polyethylenimine (PEI) (Polysciences, Inc.) essentially as described previously (Sullivan et al., 2009;Wolenski et al., 2011). Briefly, on the day of transfection, cells were incubated with plasmid DNA and PEI at a DNA:PEI ratio of 1:6. Media was changed 24 h post-transfection, and whole-cell lysates were prepared 24 h later in AT Lysis Buffer (20 mM HEPES, pH 7.9, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 20% wt/vol glycerol, 1% [wt/ vol] Triton X-100, 20 mM NaF, 1 mM Na 4 P 2 O 7 $10H 2 O, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 mg/ml leupeptin, 1 mg/ml pepstatin A, 10 mg/ml aprotinin). If cells were used for immunofluorescence, they were passaged onto glass coverslips on the day prior to fixation.
GST and GST-TIR domain fusion proteins were expressed in BL21 and BL21 pLyse bacterial cells, respectively, and were purified from extracts using glutathione beads as described previously (Garbati et al., 2010). To assess the amounts and sizes of the purified GST proteins 1% of GST and 10% of the GST samples were electrophoresed on a 10% SDS-polyacrylamide gel, which was then stained with Coomassie blue (Bio-Rad). The remaining portions of the GST proteins on beads were incubated with extracts from 293 cells expressing FLAG-MYD88. The beads were then washed four times with cold PBS, and were boiled in 2Â SDS sample buffer to release proteins from the beads. Proteins were electrophoresed on a 7.5% SDS-polyacrylamide gel, and the gel was subjected to anti-FLAG Western blotting as described elsewhere (Wolenski et al., 2011) and below.
Western blotting for FLAG-and HA-tagged proteins was performed essentially as described previously (Wolenski et al., 2011), using a rabbit anti-FLAG (1:1000, Cell Signaling Technology) or anti-HA (1:500, Cell Signaling Technology) antisera. Briefly, samples were subjected to SDS-PAGE, proteins were transferred to nitrocellulose, incubated with primary antibody, and then with anti-rabbit horseradish peroxidase-linked secondary antibody (1:5000, Cell Signaling Technology). Immunoreactive proteins were detected with SuperSignal West Dura Extended Duration Substrate (Pierce). EMSAs were performed using a 32 P-labeled kB-site probe (GGGAATTCCC, see Supplemental Table 4) and 293 whole-cell extracts, as described previously (Wolenski et al., 2011). Yeast GAL4site LacZ and 293 cell kB-site luciferase reporter gene assays were performed as described previously (Wolenski et al., 2011). Transfection and indirect immunofluorescence of DF-1 cells were performed essentially as described previously, and fixed cells were probed with rabbit anti-FLAG primary antiserum (1:50, Cell Signaling Technology) (Wolenski et al., 2011).
In vitro kinase assays were performed as described previously (Wolenski et al., 2011). Briefly, human 293T cells were transfected with pcDNA-FLAG-IKKb and pcDNA-FLAG-Of-IKK constructs, lysed two days later, and the kinases were immunoprecipitated with anti-FLAG beads (Sigma). These immunoprecipitates were then incubated with approximately 4 mg of GST alone, GST-Of-NF-kB or GST-Of-NF-kB-3X-Ala C-terminal peptides and 5 mCi [g-32 P]ATP (Perkin Elmer) in kinase reaction buffer (25 mM Tris-HCl, pH 7.5, 20 mM b-glycerophosphate, 10 mM NaF, 10 mM MgCl 2 , 2 mM DTT, 500 mM Na 3 VO 4 , 50 mM ATP) for 30 min at 30 C. Samples were then boiled in 2Â SDS sample buffer and electrophoresed on a 10% SDSpolyacrylamide gel. The 32 P-labeled GST-Of-NF-kB peptides were detected by phosphorimaging. As a control for protein input, 4 mg of GST alone, GST-Of-NF-kB or GST-Of-NF-kB-3X-Ala was electrophoresed on a 10% SDS-polyacrylamide gel, and proteins were detected by staining with Coomassie blue (Bio-Rad).
The collection and maintenance of O. faveolata fragments have been described previously (Fuess et al., 2017). Collected O. faveolata were exposed to lipopolysaccharide (LPS) from Escherichia coli 0127:B8 (Sigma) at a final concentration of 10 mg/ml for 30 min, and samples were then aerated with seawater for 4 h before being removed and frozen in liquid nitrogen. Samples were lysed in a 50 mM Tris-base, pH 7.8, with 0.05 mM dithiothreitol, and then RNA was extracted, made into cDNA, and sequenced. Ingenuity Pathway Analysis was performed as described elsewhere (Fuess et al., 2017).
To determine whether proteins in the vertebrate TLR-to-NF-kB innate immune pathway (Silverman and Maniatis, 2001) are present in the coral O. faveolata, we scanned transcriptomic data (Pinz on et al., 2015). Homologous transcripts of many of the receptor and downstream signaling components of the mammalian TLR pathway were present in O. faveolata (Fig. 1). Nevertheless, in many cases, there were reduced numbers of signaling components in each family (Supplemental Table 5). For example, there are five NF-kB/Rel proteins in humans, but there was a single NF-kB-like protein in O. faveolata. Similarly, there are ten complete TLRs in humans, but in O. faveolata there was only a single predicted fulllength TLR and four TLR-like proteins containing only TM and TIR domains.
From transcriptomic analysis, the single full-length Of-TLR is predicted to contain bona fide LRR, TM, and TIR domains. By phylogenetic analysis using the neighbor-joining method, the TIR domain of Of-TLR was most closely related to the TIR domains of the single characterized N. vectensis (Nv) TLR (Brennan et al., 2017) and to the D. melanogaster Toll protein. Among the human TLRs, Of-TLR appeared to be most similar to the TIR domain of human TLR4 (Fig. 2A).
Since the TIR domain of human TLR4 interacts with the intracellular adapter protein MYD88 as an early step in signaling to NF-kB( O' Neill et al., 2003), we sought to determine whether the TIR domain of Of-TLR (Of-TIR) could also interact with human MYD88. We incubated bacterially expressed GST-Of-TIR with 293 cell extracts overexpressing FLAG-MYD88, and performed a pull-down assay (Fig. 2B). As shown in Fig. 2C, MYD88 was detected by Western blotting in pull-down fractions containing GST-Of-TIR, but MYD88 was not seen with GST alone. As a positive control, we showed that MYD88 also interacted with GST-Nv-TIR, which we have recently shown can interact with human MYD88 (Brennan et al., 2017). Thus, the phylogenetic similarity of Of-TLR and human TLR4 proteins was reinforced functionally by Of-TLR's ability to interact with the human TLR4 adapter protein MYD88.
Having established that Of-TLR had similarities to human TLR4, we sought to determine whether O. faveolata tissue would elicit a response to gram-negative lipopolysaccharide (LPS), an activator of the mammalian TLR4-to-NF-kB pathway. Therefore, we treated O. faveolata tissue for 30 min with LPS, and followed that treatment with a 4-h washout period with seawater. RNA was then extracted from treated tissues and subjected to RNA-seq analysis. Ingenuity Pathway Analysis (IPA) of the RNA-seq data identified 39 pathways in O. faveolata that were significantly activated (as compared to control tissue), and 13 of these pathways had numerical z-scores. Of these 13 pathways, NF-kB signaling was the most significantly activated pathway (IPA: z-score ¼ 1.8, p ¼ 0.03) (Fuess et al., 2017). A total of 16 genes in the NF-kB signaling pathway were expressed in our samples, resulting in a 9.3 E À02 pathway ratio (Table 1). Among the NF-kB pathway components activated by LPS treatment were the following: TLR1-and 2-like proteins; members of the tumor necrosis factor receptor associated factor family (TRAF2, 3, 5), which are proteins involved with regulation of the pathway; kinases including RIPK1, MAP3K3, and PIK3R4; and the adapter protein MYD88. Nearly all genes in the NF-kB signaling pathway were up-or down-regulated in a manner that was consistent with activation of the pathway as it known in mammalian systems (Table 1).
Because IPA predicted that LPS could activate the NF-kB pathway in O. faveolata, we sought to characterize NF-kB-like proteins in O. faveolata. By scanning an O. faveolata transcriptomic database (Pinz on et al., 2015), we identified transcripts encoding a single NF-kB-like protein, but no Rel-like protein. The amino acid sequence of the Of-NF-kB protein (Supplemental Fig. 1) indicated that it had an overall domain structure that was most similar to mammalian NF-kB proteins, i.e., with an N-terminal RHD, followed by a glycine-rich region, and then a series of ANK repeats (Fig. 3A). A maximum-likelihood analysis of Of-NF-kB also indicated that it was most similar to other invertebrate NF-kB proteins, most notably those from sea anemones (N. vectensis, Aiptasia sp, and A. tenebrosa)( Fig. 3B). Moreover, Of-NF-kB clustered with NF-kB proteins from D. melanogaster (Relish) and humans (p100 and so that the amounts of GST proteins analyzed in the pull-downs were more similar, one-half of the sample from the GST alone pull-down was loaded on the gel used in the Western blot.). p105), and was distinct from the Rel proteins. This result is similar to the clustering of Nv-NF-kB with NF-kB proteins, but distinct from Rel proteins, as reported by Sullivan et al. (2007).
The mammalian NF-kB proteins p100 and p105 require removal of their C-terminal ANK repeat domain in order to become active transcription factors. Due to the structural similarity of Of-NF-kB to human NF-kB p100 and p105, we sought to determine whether Cterminal truncation of Of-NF-kB would unveil active transcription factor properties. Therefore, we created expression vectors for FLAG-tagged codon-optimized versions of full-length Of-NF-kB, an N-terminal form (Of-RHD), and a protein containing only the Cterminal glycine-rich and ANK repeat domain (Of-Cterm) (Fig. 4A). As a control, we used an expression vector for the 440 amino acid N. vectensis NF-kB protein (Nv-NF-kB), which we have characterized previously as an active NF-kB protein (Wolenski et al., 2011). Each of the Of-NF-kB constructs directed the expression of an appropriately sized protein when transfected into HEK 293 cells (Fig. 4B).
To determine the subcellular localization of the Of-NF-kB proteins, we transfected the plasmids into chicken fibroblasts and later performed anti-FLAG indirect immunofluorescence. We found that full-length Of-NF-kB and Of-Cterm were present in the cytoplasm of transfected chicken fibroblasts, whereas Of-RHD and the control Nv-NF-kB protein were both located in the nucleus of these cells (Fig. 4C).
We next analyzed the DNA-binding and transactivation properties of the full-length and truncated Of-NF-kB proteins. To measure DNA-binding activity, 293 cells were transfected with the Of-NF-kB expression plasmids, and extracts were analyzed for DNAbinding activity by an electrophoretic mobility shift assay (EMSA) using a kB-site probe that we have previously shown can be avidly bound by Nv-NF-kB ( Wolenski et al., 2011). Lysates from cells transfected with expression plasmids for Of-RHD and the positive control Nv-NF-kB both contained proteins that strongly bound to the kB-site probe (Fig. 4D). In contrast, extracts from cells transfected with the empty vector control, Of-NF-kB or Of-Cterm showed only low or background kB-site DNA-binding activity (Fig. 4D). In addition, Of-RHD and Nv-NF-kB both activated transcription of an NF-kB-responsive luciferase reporter plasmid when overexpressed in 293 cells as compared to empty vector controltransfected cells (Fig. 4E). As expected, full-length Of-NF-kB and O. faveolata tissue was exposed to E. coli LPS for 30 min and then returned to seawater for 4 h. RNA-seq was performed to identify transcripts whose expression was affected by this treatment, and Ingenuity Pathway Analysis was then used to identify pathways whose gene expression patterns were significantly changed by LPS treatment. The "NF-kB pathway" was found to be the most significantly activated pathway overall. Listed are 16 O. faveolata genes with homologs in the NF-kB pathway whose expression was affected by LPS treatment. Average Log2-Fold Change is indicated as well as associated error, encoded protein function from IPA analysis (Function), and consistency with pathway activation (Activation Status). The complete RNA-seq dataset for differently expressed genes can be found in Fuess et al. (2017). Of-Cterm did not activate the reporter above vector control levels (Fig. 4E). Taken together, these results showed that removal of Cterminal ANK repeat domain sequences of Of-NF-kB enabled the protein to enter the nucleus, bind to DNA, and activate transcription in vertebrate cells.
To further demonstrate that Of-NF-kB was a functional transcriptional activator, we assessed its ability to activate transcription in yeast, which do not have endogenous NF-kB proteins. To accomplish this, we fused the Of-RHD to the GAL4 DNA-binding domain (GAL4-Of-RHD) and expressed this protein in yeast cells, which contain an integrated GAL4-site LacZ reporter locus. In these cells, GAL4-Of-RHD activated transcription of the GAL4-site reporter substantially (~135-fold) above control (GAL4 alone) levels. However, the GAL4-Cterm fusion protein did not activate transcription of the reporter in yeast (Fig. 4F). As we have shown previously (Alshanbayeva et al., 2015;Wolenski et al., 2011), a GAL4-Nv-NF-kB fusion protein strongly (~240-fold) activated transcription in yeast as compared to the GAL4 alone control. Therefore, the ability of the Of-RHD sequences to activate transcription appeared to be conserved from yeast to human cells, and appeared to be an intrinsic property of sequences within the N-terminal half of the protein.
Upon activation, the human NF-kB p100 protein is converted to its active p52 form by proteasomal processing of C-terminal sequences up to the glycine-rich region (Sun, 2011). This processing is initiated by IKK-dependent phosphorylation of a cluster of three serine residues located C-terminal to the ANK repeats of p100 (Sun, 2011). The Of-NF-kB protein contains three serine residues with similar spacing and flanking sequences to the IKK target serines in human p100 and in an NF-kB protein from the sea anemone A. pallida (Fig. 5A). In an in vitro kinase assay (Fig. 5B), a constitutively active form of human IKKb and the predicted Of-IKK protein could each phosphorylate a bacterially expressed GST-fusion peptide containing the C-terminal serine residues of Of-NF-kB (amino acids 843-874); however, neither IKK phosphorylated the analogous GST-fusion protein containing alanine substitutions for the three serine residues or GST alone (Fig. 5B).
To determine whether IKK-dependent phosphorylation could induce processing of Of-NF-kB, we transfected human 293T cells with expression vectors for FLAG-Of-NF-kB and either FLAG-Of-IKK or HA-Hu-IKKb, which we have recently shown can induce processing of the sea anemone A. pallida NF-kB protein in similar experiments (Mansfield et al., 2017). As shown in Fig. 5C, cotransfection of 293T cells with FLAG-Of-NF-kB and either FLAG-Of-IKK or HA-Hu-IKKb resulted in the appearance of an Of-NF-kB
The data presented in this paper suggest that there are highly conserved elements of a TLR-to-NF-kB innate immune pathway in the endangered coral O. faveolata. That is, we show that the intracellular TIR domain of the single Of-TLR protein is most similar by phylogenetic analysis to human TLR4, and that the TIR domain of Of-TLR can directly interact with human MYD88 (Fig. 2), which is an essential adapter protein for human TLR4-based signaling to NF-kB. Moreover, the single Of-NF-kB protein has sequence similarity, an overall structural organization, and transactivation properties that are more like human NF-kB proteins than human Rel proteins (Figs. 3 and4). Furthermore, the Of-NF-kB protein can be processed in response to overexpression of human and O. faveolata IKK proteins in a manner consistent with non-canonical pathway processing of NF-kB p100 in human cells (Fig. 5). Finally, we show that a mammalian ligand of TLR4-to-NF-kB activation, namely gramnegative bacterial LPS, activates a gene expression profile in isolated O. faveolata tissue that is consistent with overall activation of NF-kB pathway genes (Table 1).
To date, a handful of studies have identified TLR-to-NF-kB pathway components in environmentally stressed cnidarians. Anderson et al. (2016) and Pinz on et al. ( 2015) identified transcripts encoding NF-kB signaling components in their transcriptomes from bleached O. faveolata. In the coral Acropora palmata, transcripts for a putative tumor necrosis factor receptor-associated factor 3 (TRAF3) homolog and NF-kB were shown to be upregulated when coral were exposed to increased water temperature (DeSalvo et al., 2010). The upregulation of NF-kB pathway components at the transcriptional level has also recently been described in the sea anemone A. pallida following treatment with heat or chemicals that induce bleaching (Mansfield et al., 2017). However, the precise mechanisms by which these conserved signal transduction pathways elicit their responses, the gene targets regulated by NF-kB in these organisms, and their biological roles in these cnidarians remain largely unknown.
Treatment of O. faveolata tissue with LPS results in significant differential expression of genes in a manner indicating activation of the NF-kB pathway. These results are consistent with other studies that have reported upregulation of NF-kB pathway transcripts as a response to pathogen-induced stress in cnidarians. For example, Franzenburg et al. (2012) demonstrated that microbial infection of Hydra elicits a TLR-like pathway response and bacterial flagellin can activate TLR-to-NF-kB signaling when the Hydra TLRs are expressed in mammalian cells (Bosch et al., 2009). Similarly, Brennan et al. (2017) have shown that a coral bacterial pathogen can activate the N. vectensis TLR. However, our results are the first to show that NF-kB pathway components in an endangered coral are upregulated in response to treatment with a known mammalian inducer of NF-kB, i.e., LPS (Table 1). Whether this activation of NF-kB components by LPS occurs through the Of-TLR that we characterize here is not known.
Although our results suggest a link between LPS treatment and activation of NF-kB in O. faveolata, it is important to note the differences between our findings and the LPS-induced rapid activation of NF-kB that is usually studied in mammalian systems. That is, our results show that the levels of mRNAs encoding several NF-kB pathway components are upregulated approximately 4 h after treatment of O. faveolata tissue with LPS. In contrast, LPS-induced activation of NF-kB in mammalian systems is usually studied as the rapid nuclear localization of a latent cytoplasmic NF-kB complex approximately 10e30 min following treatment with LPS. Thus, we have not directly shown that LPS treatment activates the NF-kB protein in a conventional sense.
Our results suggest that the TLR-to-NF-kB pathway in O. faveolata has characteristics of both non-canonical and canonical NF-kB signaling in mammals. On the one hand, the Of-NF-kB pathway is like the mammalian non-canonical pathway in that 1) the overall organization of Of-NF-kB is similar to the mammalian NF-kB protein p100, 2) C-terminal truncation leads to nuclear localization, enhanced DNA binding, and enhanced transactivation in vertebrate cells, 3) IKKs can induce partial processing of overexpressed Of-NF-kB in human cells, 4) sites of IKK-dependent serine phosphorylation in p100 are conserved in Of-NF-kB, and 5) these serine residues are required for processing of Of-NF-kB in human cells. On the other hand, human IKKb, which is normally associated with canonical NF-kB signaling in mammalian cells, is as active as, if not even more active than, Of-IKK towards Of-NF-kB in our in vitro phosphorylation and human cell induction experiments. Moreover, TLR4 generally activates the canonical NF-kB pathway in mammalian cells. Furthermore, although the TIR domain of the Of-TLR has properties of human TLR4, the extracellular LRR domain of Of-TLR is more similar to the Drosophila Toll protein (a multiple cysteine cluster TLR) than to TLR4 (a single cysteine cluster TLR), similar to what is seen with Nv-TLR (Brennan et al., 2017). Finally, the clustering of the TIR domain of Of-TLR with human TLR4 is not seen with other methods of phylogenetic analysis (e.g., maximum-likelihood and maximum parsimony [data not shown]). Thus, the evolutionary processes by which cnidarian TLR and TLR-like proteins, as well as a single cnidarian IKK, diversified into the multiple member higher metazoan TLR and IKK families vis-a-vis canonical vs. non-canonical signaling to NF-kB are not obvious.
Coral bleaching and coral pathogen disease will be persistent problems as ocean temperatures continue to rise as a result of climate change. Defining the biological processes and molecular pathways that are altered in bleached and diseased corals will likely be critical for determining how these organisms respond and survive. Moreover, given the broad impact that the understanding of immune pathways has had on human disease diagnosis and therapy, our data will no doubt contribute to the understanding of diseases in environmentally sensitive organisms as well. Thus, our characterization of key conserved immune regulatory molecules, namely TLR and NF-kB, in an endangered coral is an important contribution to molecular ecology and comparative immunology.