Developmental and Comparative Immunology 79 (2018) 128e136 Contents lists available at ScienceDirect Developmental and Comparative Immunology journal homepage: www.elsevier.com/locate/dci A conserved Toll-like receptor-to-NF-kB signaling pathway in the endangered coral Orbicella faveolata Leah M. Williams a, Lauren E. Fuess b, Joseph J. Brennan a, Katelyn M. Mansfield a, Erick Salas-Rodriguez a, Julianne Welsh a, Jake Awtry c, Sarah Banic c, Cecilia Chacko c, Aarthia Chezian c, Donovan Dowers c, Felicia Estrada c, Yu-Hsuan Hsieh c, Jiawen Kang c, Wanwen Li c, Zoe Malchiodi c, John Malinowski c, Sean Matuszak c, Thomas McTigue IV c, David Mueller c, Brian Nguyen c, Michelle Nguyen c, Phuong Nguyen c, Sinead Nguyen c, Ndidi Njoku c, Khusbu Patel c, William Pellegrini c, Tessa Pliakas c, Deena Qadir c, Emma Ryan c, Alex Schiffer c, Amber Thiel c, Sarah A. Yunes c, Kathryn E. Spilios c, * Jorge H. Pinzon C b, Laura D. Mydlarz b, Thomas D. Gilmore a, a Department of Biology, Boston University, Boston, MA 02215, USA b Department of Biology, University of Texas at Arlington, Arlington, TX 76019, USA c Molecular Biology Laboratory (BB522), Program in Biochemistry & Molecular Biology, Boston University, Boston, MA 02215, USA article info abstract Article history: Herein, we characterize the Toll-like receptor (TLR)-to-NF-kB innate immune pathway of Orbicella Received 16 September 2017 faveolata (Of), which is an ecologically important, disease-susceptible, reef-building coral. As compared Received in revised form to human TLRs, the intracellular TIR domain of Of-TLR is most similar to TLR4, and it can interact in vitro 23 October 2017 with the human TLR4 adapter MYD88. Treatment of O. faveolata tissue with lipopolysaccharide, a ligand Accepted 24 October 2017 for mammalian TLR4, resulted in gene expression changes consistent with NF-kB pathway mobilization. Available online 26 October 2017 Biochemical and cell-based assays revealed that Of-NF-kB resembles the mammalian non-canonical NF- kB protein p100 in that C-terminal truncation results in translocation of Of-NF-kB to the nucleus and Keywords: k NF-kappaB increases its DNA-binding and transcriptional activation activities. Moreover, human I B kinase (IKK) and Toll-like receptor Of-IKK can both phosphorylate conserved residues in Of-NF-kB in vitro and induce C-terminal processing Evolution of Of-NF-kB in vivo. These results are the first characterization of TLR-to-NF-kB signaling proteins in an Coral endangered coral, and suggest that these corals have conserved innate immune pathways. Innate immunity © 2017 Elsevier Ltd. All rights reserved. Orbicella faveolata 1. Introduction innate immune effector molecules such as cytokines and anti- microbial peptides (Akira et al., 2006; Kawai and Akira, 2007). The Toll-like receptor (TLR)-to-NF-kB signaling pathway is a TLR-to-NF-kB pathways have been intensively studied for their prominent innate immune pathway in higher metazoans. Specific roles in immunity in many model systems from flies to humans pathogen-associated molecular patterns (PAMPs) are detected by (Aderem and Ulevitch, 2000; Kawai and Akira, 2007; Minakhina membrane-bound TLRs, which then initiate intracellular signaling and Steward, 2006; Silverman and Maniatis, 2001; Vasselon and cascades. One main TLR pathway leads to activation of transcription Detmers, 2002). Recently, genome and transcriptome sequencing factor NF-kB to induce changes in the expression of genes encoding 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 Abbreviations: IKK, IkappaB kinase; LRR, leucine-rich repeat domain; Nv, in these basal organisms are not well understood (Bosch et al., Nematostella vectensis; NF-kB, nuclear factor kappa B; Of, Orbicella faveolata; TIR, 2009). Toll/interleukin-1 receptor; TLR, Toll-like Receptor; TM, transmembrane. In mammals, TLRs are single-pass transmembrane proteins with * Corresponding author. Biology Department, Boston University, 5 Cummington Mall, Boston, MA 02215, USA. an N-terminal extracellular leucine-rich repeat (LRR) domain, a E-mail address: [email protected] (T.D. Gilmore). central transmembrane (TM) domain, and a C-terminal https://doi.org/10.1016/j.dci.2017.10.016 0145-305X/© 2017 Elsevier Ltd. All rights reserved. L.M. Williams et al. / Developmental and Comparative Immunology 79 (2018) 128e136 129 intracellular Toll/interleukin-1 receptor (TIR) domain. Ligand Guldberg et al., 2007; Hughes et al., 2017; Weis, 2008). Bleaching recognition by the LRR domain promotes engagement of the often causes coral death, but in some cases, corals can recover from intracellular TIR domain with other TIR domain-containing adapter a bleaching event and re-establish a symbiotic relationship with proteins, which initiates downstream signaling cascades (Kawai Symbiodinium. Nevertheless, recovered corals often show increased and Akira, 2007). These adapter proteins include myeloid differ- susceptibility to microbial diseases such as yellow band disease, entiation primary response protein 88 (MYD88), TIR domain- black band disease, and plague (Kushmaro et al., 1996; Pinzon et al., containing adapter protein (TIRAP/MAL), TIR domain-containing 2015). adapter inducing IFNb (TRIF), and Trif-related adapter protein Recent reports of transcriptional changes in immune-related (TRAM) (Aderem and Ulevitch, 2000; Kawai and Akira, 2007). The molecules in bleached and pathogen-infected corals have sug- number of TLRs varies widely among organisms, with ten TLRs in gested that the cnidarian innate immune system plays a role in humans, 13 in mice, nine in fruit flies, and over 200 in sea urchins coral diseases (Anderson et al., 2016; Fuess et al., 2016, 2017; Pinzon (Buckley and Rast, 2012; Valanne et al., 2011; Vasselon and et al., 2015; Zhou et al., 2017). To gain deeper insights into molec- Detmers, 2002). In more basal organisms, such as sponges and ular processes important for coral immunity and health, we have cnidarians (which include hydras, jellyfish, sea anemones, and had an ongoing interest in characterizing cnidarian homologs of corals), there are two types of TLR-like proteins. Most often, these mammalian immunoregulatory molecules and pathways basal TLR-like proteins contain only a TM domain and a TIR domain; (Wolenski et al., 2011, 2013). In this report, we have used phylo- however, some basal animals have genes encoding full-length TLRs genetic, biochemical, and cell-based assays to characterize the with LRR, TM, and TIR domains, similar to mammalian TLRs. Within structure, activity, and regulation of TLR and NF-kB proteins from the phylum Cnidaria, there are animals having no complete TLRs O. faveolata. We also show that lipopolysaccharide (LPS) treatment (Aiptasia pallida, Hydra vulgaris), ones having one complete TLR of O. faveolata tissue can induce a gene expression profile consistent (Nematostella vectensis), and ones having multiple complete TLR with induction of the NF-kB pathway. These results represent the and TLR-like proteins (Acropora digitifera)(Miller et al., 2007; Poole first characterization of proteins in the conserved immunoregula- and Weis, 2014; Shinzato et al., 2011). tory TLR-to-NF-kB pathway of a critically endangered coral. In insects and mammals, the NF-kB superfamily comprises multiple related transcription factors that bind distinct DNA se- 2. Materials and methods quences known as kB sites (Hayden and Ghosh, 2004). All NF-kB proteins have a conserved N-terminal domain of approximately 2.1. Phylogenetic analyses 300 amino acids called the Rel Homology Domain (RHD). The RHD has residues important for DNA binding, dimerization, and nuclear For comparative analysis of Toll-like receptors, the predicted TIR localization (Gilmore, 2006). The NF-kB superfamily can be divided domains of Orbicella faveolata (Of) TLR, Nematostella vectensis (Nv) into two subclasses: the NF-kB proteins (e.g., human p52/p100 and TLR, Drosophila melanogaster (Dm) Toll, and an Amphimedon p50/p105, and Drosophila Relish) and the Rel proteins (e.g., human queenslandica (Aq) TLR-like protein (Gauthier et al., 2010) were p65/RelA, c-Rel, and RelB, and Drosophila Dorsal and Dif) (Gilmore, analyzed along with the ten human TLR proteins. The TIR domains 2006). Based on DNA binding-site preference and sequence simi- of Of-TLR, Nv-TLR, and Aq-TLR were identified through MEME larity, the NF-kB RHDs are more related to each other than to the analysis (Bailey et al., 2006), and sequences were trimmed to RHDs of Rel proteins (Finnerty and Gilmore, 2015; Siggers et al., contain only the TIR domains based on motif prediction and known 2011). Where characterized, basal organisms (e.g., cnidarians, human TIR domains (Supplemental Table 1). Human TLR and Dm- sponges, pre-metazoans) have single NF-kB family proteins, which Toll amino acid sequences, along with their annotated TIR do- are phylogenetically most similar to the vertebrate and insect NF- mains, were obtained from the UniProt database. Clustal Omega kB subclass (Finnerty and Gilmore, 2015). (Sievers et al., 2011) was then used to align the trimmed and culled In addition to the RHD, members of the NF-kB subclass have a C- TIR sequence dataset. The tree was rooted with the Aq-TLR, and terminal inhibitory domain consisting primarily of a series of phylogenetic comparison was performed using neighbor-joining Ankyrin (ANK) repeats, which must be removed by proteolysis to analysis bootstrapped 1000 times using PAUP* (Swofford, 2001). activate the DNA-binding activity of the transcription factor For phylogenetic analysis of the NF-kB and Rel proteins, the RHD (Hayden and Ghosh, 2004; Sun, 2011). For example, the vertebrate sequences of O. faveolata (Of) NF-kB (from NCBI) Aiptasia pallida NF- NF-kB proteins p100 and p105 are processed to p52 and p50, kB (NCBI), Actinia tenebrosa NF-kB (NCBI), N. vectensis NF-kB (Uni- respectively, which then translocate from the cytoplasm to the Prot), D. melanogaster Relish, Dorsal, and Dif (UniProt), and Homo nucleus.