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Characterization and expression profiling of NOD-like receptor C3 (NLRC3) in mucosal tissues of turbot (Scophthalmus maximus L.) following bacterial challenge
Accepted Manuscript Characterization and expression profiling of NOD-like receptor C3 (NLRC3) in mucosal tissues of turbot (Scophthalmus maximus L.) following bacterial challenge Zhumei Hou, Zhi Ye, Dongdong Zhang, Chengbin Gao, Baofeng Su, Lin Song, Fenghua Tan, Huanhuan Song, Yu Wang, Chao Li PII:
S1050-4648(17)30248-6
DOI:
10.1016/j.fsi.2017.05.014
Reference:
YFSIM 4567
To appear in:
Fish and Shellfish Immunology
Received Date: 10 January 2017 Revised Date:
30 April 2017
Accepted Date: 2 May 2017
Please cite this article as: Hou Z, Ye Z, Zhang D, Gao C, Su B, Song L, Tan F, Song H, Wang Y, Li C, Characterization and expression profiling of NOD-like receptor C3 (NLRC3) in mucosal tissues of turbot (Scophthalmus maximus L.) following bacterial challenge, Fish and Shellfish Immunology (2017), doi: 10.1016/j.fsi.2017.05.014. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Characterization and expression profiling of NOD-like receptor C3 (NLRC3) in mucosal tissues of Turbot (Scophthalmus maximus L.) following bacterial challenge
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Zhumei Houa,1, Zhi Ye b,1, Dongdong Zhang b, Chengbin Gaoa, Baofeng Sud,e, Lin Songa, Fenghua Tanc, Huanhuan Songa, Yu Wanga, Chao Lia*
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Marine Science and Engineering College, Qingdao Agricultural University, Qingdao 266109, China
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School of Fisheries, Aquaculture, and Aquatic Sciences, Auburn University, Auburn, AL 36849, USA
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266109, China
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Fisheries Research Institute, Chinese Academy of Fishery Sciences, Harbin 150070, China
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Heilongjiang Fisheries Research Institute, Chinese Academy of Fishery Sciences, Harbin 150070,
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School of international education and exchange, Qingdao Agricultural University, Qingdao
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National and Local Joint Engineering Laboratory of Freshwater Fish Breeding, Heilongjiang
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Ministry of Agriculture Key Laboratory of Freshwater Aquatic Biotechnology and Breeding,
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Both of these authors contributed equally to this work.
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Corresponding author: Chao Li; email: [email protected]
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Abstract: The mucosal surfaces are important for teleost as they are directly and continuously
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exposed to pathogen-rich aquatic environments. Scrutinization and recognition of the attached
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pathogens is the first crucial step of mucosal immunity initiation. Nucleotide oligomerization
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domain (NOD)-like receptors (NLRs) are a large group of intracellular pathogen recognition
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receptors (PRRs) which play key roles in pathogen recognition and subsequent immune signaling
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pathways activation. In this study, we identified two NLRC3 genes (NLRC3a and NLRC3b), a
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subfamily of NLRs from turbot, and profiled their expression patterns in mucosal tissues
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following bacterial challenge. NLRC3a transcript contains an open reading frame (ORF) of
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3,405 bp that encodes a putative peptide of 1,134 amino acids. While NLRC3b has an ORF of
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3,114 bp encoding 1,037 amino acids. A caspase recruitment domain (CARD) at N-terminus
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characterized turbot NLRC3a, while NLRC3b seems to be unique to teleost, containing a fish
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specific NACHT associated (FISNA) domain and an extra B30.2 (PRY/SPRY) domain at C-
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terminus. In addition, NLRC3a and NLRC3b were detected in all the examined tissues, with the
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highest expression levels in kidney and blood, respectively. After bacteria challenge, expression
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levels of turbot NLRC3 genes were strongly induced in intestine rather than in skin and gill,
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while NLRC3a had relatively higher expression level than that of NLRC3b. Taken together,
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NLRC3 genes found in this study were the first NLR members identified in turbot. The different
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expression signatures of NLRC3a and NLRC3b in mucosal tissues following two bacterial
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infections indicated they probably have important roles in early response to bacterial infection in
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the first line of host defense system.
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Keywords: Turbot, NLRC3, Pathogen recognition receptors, Mucosal immunity
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1. Introduction
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Mucosal immune system contains a unique array of innate and adaptive immune cells and molecules that together constitutes the first line of host defense against pathogens’ intrusion. The
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mucosal surfaces serve as dynamic interfaces driving a variety of critical physiological
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processes[1]. For teleost fish, mucosal immune system is of great importance as their mucosal
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surfaces are directly exposed to and continuously colonized by complex and diverse microbial
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communities which bombard mucosal epithelial barriers, thus posing additional challenges to
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fish mucosal immunity [2,3]. The first critical step of mucosal immune response is to scrutinize
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and recognize the attached potential pathogens, and then to initiate the host immunity in a rapid
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and accurate manner. In this respect, pathogen recognition receptors (PRRs) which can
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distinguish the microbial unique and conserved molecular signatures, so-called pathogen-
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associated molecular patterns (PAMPs), are key factors of mucosal surfaces for pathogen
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recognition and subsequent immune signaling pathways activation [4]. Nevertheless, our
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understanding of teleost PRRs as well as their associated activities in early mucosal immune
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defense against pathogen invasion is still limited.
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PRRs discovered so far are classified into three major families according to their domain
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properties: the Toll-like receptors (TLRs), nucleotide-binding oligomerization domain (NOD)-
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like receptors (NLRs; alternatively called nucleotide-binding domain and leucine-rich repeat
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containing receptors), and retinoic acid-inducible gene (RIG-1)-like receptors (RLRs) [5].
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Among these, NLRs are a large group of intracellular sensor proteins which are capable of
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recognizing intracellular PAMPs or damage/danger associated molecular patterns (DAMPs) and
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regulating the antimicrobial immune responses [6,7]. The absence of signal peptides and lack of
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transmembrane domains indicated the cytoplasmic location of NLRs [8,9]. The NLRs have three
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characterized and structural domains: a variable N-terminal effector domain responsible for
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triggering downstream signaling pathways through protein-protein interaction, a central
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nucleotide binding oligomerization (NACHT) domain for self-regulation and oligomerization,
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and a leucine-rich repeat (LRR) C-terminus required for ligand binding specificity [10,11].
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Ligand binding contributes to the oligomerization of the NACHT domain, and thereafter forms
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an active signaling platform which allows the effector domains to bind downstream signaling
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molecules and to trigger immune cascades [12]. Among the three domains, the N-terminus is
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widely diverse across the species. They typically can be either a caspase recruitment domain
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(CARD) or pyrin domain (PYD) or in a fewer cases, a baculovirus inhibitor of apoptosis repeat
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(BIR) domain or some other less defined (X) domains [9,12].
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Recently, NLRs are classified into NLRA/CIITA, NLRB/NAIP, NLRC, NLRP and NLRX1 subfamilies according to their domain structures and phylogenetic relationship using standard
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nomenclature [9,13]. NLRC is one of the biggest and most important groups, including
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NLRC1/NOD1, NLRC2/NOD2, NLRC3/NOD3, NLRC4/IPAF, and NLRC5 [9,13]. Among
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these, NLRC1 and NLRC2 with one or two N-terminal CARDs are two widely studied groups of
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NLRCs that augment pro-inflammatory pathways through the activation of caspase-1 or NF-κB
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signaling pathways [7]. Conversely, NLRC3 has been reported to negatively regulate
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inflammation and emerged as a negative regulator of innate immune signaling by interfering in
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TLR or STING signaling pathways [14,15]. For example, zebrafish (Danio rerio) NLRC3-like
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receptor was reported to negatively regulate inflammation and macrophage activation [16].
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However, other studies indicated that the negative regulatory role of NLRC3 may be ruled out in
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some teleost because they play a significant role as PRRs in pathogen recognition [17,18]. Until
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now, NLRC3 genes have been documented in several teleost species, such as zebrafish [19],
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Japanese flounder (Paralichthys olivaceus) [18,20], channel catfish (Ictalurus punctatus)
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[8,20,21] and Asian seabass (Lates calcarifer) [17]. In those studies, NLRC3 can be induced by
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bacterial infection or ligand (such as LPS), demonstrating their important role in the innate
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immune response in teleost.
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Turbot (Scophthalmus maximus) is one of the most important cultured marine fishes in
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China. Its commercial production has been significantly hindered due to widespread disease
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outbreaks caused by a number of pathogens, including Vibrio anguillarum, Streptococcus iniae
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and Edwardsiella tarda. Up to date, many innate immune components as well as their associated
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activities during the infection have been characterized in turbot, including stomatin-like protein 2
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[22], chemokines [23,24], MyD88 [25], lysozyme [26], and particularly some PRRs like TLRs
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[27,28] and peptidoglycan recognition protein (PGRP) [29]. The expression patterns of these
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PPRs in mucosal tissues (skin, gill and intestine) following bacterial infection indicated their
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important roles during early recognition of invading pathogen [30]. However, characterization of
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another important category of PRRs, NLRs especially NLRC3 and their associated immune
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activities in mucosal tissues are still lacking. In this regard, we sought here to identify NLRC3
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members in turbot, conducted phylogenetic analysis, and profiled their expression patterns in
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mucosal barriers following bacterial infection in the early time points.
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2. Materials and methods
2.1. Sequence identification and analysis
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By means of NLRC3 protein sequences from other species as queries, turbot NLRC3 genes were identified from turbot transcriptome databases generated by our group [31], using
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tBLASTn program with a cutoff E-value of 1e-10. The retrieved nucleotide sequences were
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translated by the ORF Finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). The predicted amino
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acid sequences were further verified by BLASTP (http://blast.ncbi.nlm.nih.gov/Blast.cgi) against
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NCBI non-redundant protein database. The conserved domains and signal peptides were
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identified using simple modular architecture research tool (SMART; http://smart.embl-
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heidelberg.de/). The theoretical pI, molecular mass and N-glycosylation sites were analyzed
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using online ExPASy server [32]. The percentages of similarity and identity of NLRC amino
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acids with other species were calculated by MatGAT program [33].
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2.2. Phylogenetic analysis
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Amino acids sequences of NLRC3 from turbot and other organisms were selected to construct the phylogenetic tree, including human (Homo sapiens; NP_849172.2), mouse
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(Mus musculus; NP_001074749.1), chicken (Gallus gallus; XP_015150161.1), green sea turtle
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(Chelonia mydas; XP_007054520.1), western clawed frog (Xenopus tropicalis;
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XP_002932504.1), zebrafish (D. rerio; XP_009295904.1), Atlantic salmon (Salmo salar;
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XP_014049925.1), fugu (Takifugu rubripes; XP_003961491.2 and XP_003961491.2), half-
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smooth tongue sole (Cynoglossus semilaevis; XP_008312409.1 and XP_008312409.1), large
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yellow croaker (Larimichthys crocea; XP_003438651.1), medaka (Oryzias latipes;
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XP_004080575.1 and XP_004080575.1) and Nile tilapia (Oreochromis niloticus;
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XP_004080575.1 and XP_003438651.1). Amino acid sequence alignments of the NLRC3
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proteins were conducted using the ClustalW2 multiple alignment program [34]. Phylogenetic and
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molecular evolutionary analyses were performed using MEGA (Molecular Evolutionary
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Genetics Analysis) package (version 6.0) with the neighbor-joining method [35]. Poisson
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correction was selected for substitution model, gaps were removed by complete deletion and
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1,000 bootstrapping replications were used to evaluate the reliability of the neighbor-joining
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trees.
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2.3. Bacteria challenge and sample collection
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In order to investigate the immune roles of NLRC3 genes against bacterial infection, the Gram-negative bacteria V. anguillarum and the Gram-positive bacteria S. iniae were selected to
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conduct the bath challenge, respectively. Turbot fingerlings (approximately 15.6 g in mass and
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5.5 cm in length) were obtained from the turbot hatchery (Haiyang, Shandong, China) and
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acclimated in aquariums with aerated flow-through water (27 ± 0.5 oC) for one week in the
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laboratory prior to challenge. The bacteria isolate of V. anguillarum and S. iniae were provided
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by the disease lab of Qingdao Agricultural University. A pre-challenge was conducted and the
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bacteria were re-isolated from symptomatic fish and biochemically confirmed before cultured.
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V. anguillarum and S. iniae were inoculated in LB broth respectively and cultured in a
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shaker (180 rpm) at 28 oC overnight. The concentration of the final culture was measured by
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streak plate serial ten-fold dilutions of the culture and recorded as colony forming units per
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milliliter (CFU/mL). Fish were challenged by 2 h immersion of V. anguillarum and S. iniae at a
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final concentration of 5×107 CFU/mL and 5×106 CFU/mL, respectively. Control fish followed an
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identical procedure but were in sterilized media without bacteria.
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2.4. Sample collection and total RNA extraction
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Turbot tissues including brain, skin, gill, intestine, head kidney, liver, spleen and blood
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were collected from healthy individuals. While only mucosal tissues (skin, gill and intestine)
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were collected after challenge of V. anguillarum at 2 h, 6 h and 24 h and S. iniae at 2 h, 4 h, 8 h
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and 12 h, respectively. At each time point, 15 fish were randomly collected and divided into 3
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replicate pools (5 fish each). Fish were euthanized with tricaine methanesulfonate (MS-222) at
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200 mg/L (buffered with sodium bicarbonate). Approximately equal amounts of tissue from each
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fish within the replicates were collected, flash-frozen in liquid nitrogen and stored at -80 °C until
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RNA isolation. Prior to RNA extraction, samples were removed from the -80 °C freezer and
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ground to a fine powder with sterilized mortar and pestle in the presence of liquid nitrogen. Total
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RNA was extracted from tissues using the Trizol® Reagent (Invitrogen, USA) following
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manufacturer’s instructions. The RNA integrity and the genomic DNA contamination were
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monitored by electrophoresis with 1% agarose gel. The purity and concentration of RNA were
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measured on a nanodrop 2000 (Thermo Electron North America LLC, FL). All extracted
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samples had an A260/280 ratio greater than 1.8, and were diluted to 250 ng/µl.
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2.5. QPCR analysis
Quantitative real-time PCR (QPCR) was used to examine NLRC3 mRNA expression in
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different tissues at different time points post bacteria challenge. Primers for QPCR were
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designed using Primer 3 software based on the sequences of turbot NLRC3 transcripts. Turbot
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18S rRNA gene was used as an internal control for normalization of the expression levels. The
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PCR products were sequenced to confirm the specificity of these primers. First strand cDNA
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(500 ng RNA per 10 µl reaction) was synthesized by PrimeScript RT reagent Kit (Takara, Dalian,
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China) and QPCR was performed on a CFX96 real-time PCR Detection System (Bio-Rad
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Laboratories, Hercules, CA) following the manufacturer’s instructions. The PCR reaction
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mixture was denatured at 95 °C for 30 s and then subjected to 40 cycles of 95 °C for 5 s, 58 °C
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for 5 s and followed by dissociation curve analysis, 5 s at 65 oC, then up to 95 °C at a rate of
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0.1 °C/s increment, to verify the specificity of the amplicons. Results were expressed relative to
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the expression levels of 18S rRNA in each sample using the Relative Expression Software Tool
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(REST) [36] to capture significance at the level of P < 0.05. The Ct values of liver was set as the
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control to determine the gene expression patterns in healthy turbot tissues. A no-template control
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was run on all plates.
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3. Results and discussion
3.1 Identification of turbot NLRC3 genes
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Two turbot NLRC3 transcripts NLRC3a (KY437762) and NLRC3b (KY437763) were
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captured from the turbot transcriptomic database [31]. NLRC3a transcript has a full-length of
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5,471 bp, containing a 3,405 bp open reading frame (ORF) that encodes a putative peptide of
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1,134 amino acids with an estimated molecular mass of 125.03 kDa and a theoretical pI of 8.35.
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Similarly, the NLRC3b is 3,700 bp long and contains a 3,114 bp ORF that encodes 1,037 amino
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acids with an estimated molecular mass of 113.96 kDa and a theoretical pI of 7.66. The domain
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analysis and protein sequence alignment of the turbot NLRC3 with other species showed that all
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NLRC3 sequences have the characteristic NACHT domain and LRR domain, but the N-terminus
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is variable and in some cases, additional C-terminus exist (Fig. 1 and Fig. 2). Sequence identity and similarity of NLRC3 genes were analyzed. Turbot NLRC3a and
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NLRC3b amino acid sequences showed very low identity (24.1%) to each other (Table 1).
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Turbot NLRC3a showed high identity (more than 80%) to the selected teleost fish NLRC3a
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sequences, including Nile tilapia, fugu, medaka and half-smooth tongue sole, but had much
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lower identity (45.7~51.3%) to NLRC3 from zebrafish and higher vertebrates like human, mouse,
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chick, turtle and frog. Similarly, NLRC3b showed higher identity (58.1~72.7%) to NLRC3b of
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fugu, Atlantic salmon, medaka, large yellow croaker, half-smooth tongue sole and Nile tilapia,
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and its identity was lower to zebrafish and some higher vertebrates (less than 30%) (Table 1).
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In addition to low identity between the two NLRC3 sequences of turbot, they also have
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different domain compositions, implying they may have different immune response mechanisms.
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Alignment of NLRC3a with sequences of Nile tilapia, fugu, medaka and half-smooth tongue sole,
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as well as NLRC3 sequences of zebrafish, human , mouse, chick, turtle and frog revealed highly
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conserved motifs, one NACHT domain and 14 LLRs (Fig.1). Except human and mouse, all of
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them have one N-terminal CARD (Fig.1), which has been found in a wide array of proteins that
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are typically associated with inflammation and apoptosis [37–39].
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Teleost specific NLRC3 were detected including turbot NLRC3b and other teleost NLRC3b
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in fugu, Atlantic salmon, medaka, large yellow croaker, half-smooth tongue sole because they
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have a typically specific NACHT associated (FISNA) domain rather than CARD at N-terminus,
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and an extra highly conserved B30.2 (PRY/SPRY) domain at C-terminus (Fig. 2). Exceptionally,
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tilapia NLRC3b lacked B30.2 domain and zebrafish has no FISNA and B30.2 domains. However,
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NLRC3 sequences of zebrafish and other higher vertebrates showed some similarities to FISNA
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and B30.2 (Fig. 2). The FISNA has been reported to locate immediately upstream of the NACHT
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domain of fish NLR family, but its function is uncharacterized [40]. The C-terminal B30.2
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domain contains two single Serine-Proline-Arginine-Tyrosine (SPRY) and Proline-Arginine-
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Tyrosine (PRY) regions, which are implicated in innate immunity [41] and interact directly with
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caspase-1 to modulate IL-1β production [42]. This B30.2 domain has also been found in some
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other immune related proteins, such as teleost-specific tripartite motif (Trim) proteins that
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involve in virus infection [43].
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In addition, both of the predicted turbot NLRC3a and NLRC3b proteins lack the signal
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peptides and transmembrane domain, indicating their cytoplasmic location and functioning as the
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intracellular receptor, which was consistent with the characterized of NLRs in other species [9].
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3.2 Phylogenetic analyses
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In order to further confirm the identities of turbot NLRC3 genes (NLRC3a and NLRC3b) and clarify the phylogenetic relationship of NLRC3 among species, a neighbor-joining
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phylogenetic tree was constructed using MEGA6 with the NLRC3 amino acid sequences from
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selected vertebrate species (Fig.3). As shown in Fig.3, all NLRC3 sequences were clustered into
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two major distinct groups. In the first major cluster, fish NLRC3 and higher vertebrates NLRC3
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sequences were separated into two subclades with high bootstrapping values (100 match score).
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Turbot NLRC3a shared a common ancestor with clade consisted of O. niloticus and O.
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latipeswith high similarity match score of 81, then clustered with T. rubripes, and then fell into
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the clade with C. semilaevis with 100 match score (Fig. 3).
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Fish specific NLRC3b containing extra B30.2 and FISNA domains were clustered together, forming the second independent major clade. Turbot NLRC3b also showed the closest
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relationship to O. niloticus like that of NLRC3a but with lower match score of 50, and then
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clustered with L. crocea (match score 86), and then fell into the clade with C. semilaevis with
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match score 46 (Fig. 2).
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Taken together, the phylogenetic tree analysis was almost consistent with their phylogenetic relationships. Collectively, the phylogenetic analysis confirmed the identification of NLRC3a
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and NLRC3b, and NLRC3a showed homology to their counterparts identified in other species,
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while NLRC3b seemed to be unique to teleost with relatively lower match scores among species.
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3.3. Basal tissue expression of two turbot NLRC3 genes
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QPCR analyses was performed to determine the basal expression levels of these two turbot
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NLRC3 genes in different tissues, including liver, skin, spleen, gill, intestine, blood, head kidney
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and brain. NLRC3a and NLRC3b were both expressed in almost all examined tissues, but with
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varied expression levels (Fig. 4). Turbot NLRC3a showed the strongest expression level in head
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kidney and gill (> 25 fold), followed by spleen (~17.4 fold), intestine (~16.2 fold), blood (~12.9
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fold), skin (~9.4 fold), brain (~2.0 fold), and the lowest expression level in liver (Fig. 4). While
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NLRC3b had much higher expression in blood, spleen and head kidney (> 60 fold), modest in
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gill (~21.8 fold), and lower expression level in skin, intestine and brain (Fig. 2).
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In channel catfish, NLRC3a also had higher expression level in immune related tissues such
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as gill, head kidney, intestine and spleen, but lower expression level in skin and brain [8].
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Similarly, Asian seabass NLRC3a, had higher expression level in gill and intestine, modest
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expression level in liver, and much lower expression level in skin and brain [17]. In miiuy
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croaker (Miichthys miiuy), NLRC3a also expressed weakly in skin but higher in gill, intestine
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and blood [44]. However, the information about the expression patterns of teleost specific
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NLRC3b was much limited. A Japanese flounder NLRC transcript (GenBank acc. No JF271924)
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that contains B30.2 domain and lacks FISNA domain, had higher expression levels in gill and
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kidney [18]. While another poNLRC3 gene in Japanese flounder with similar domain
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compositions as turbot NLRC3b, had relatively abundant expression in hepatopancreas and
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lower expression level in blood, which was opposite to that of turbot NLRC3b in the current
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study [20] Some other NLRC genes, such as one of the most studied NOD1, had the highest
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expression level in spleen of goldfish (Carassius auratus L.) [45], and was highly expressed in
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spleen and liver of Rainbow trout (Oncorhynchus mykiss) [46]. Additionally, NLRC5 gene was
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highly expressed in the head kidney, spleen, and hindgut of Atlantic salmon [47] and in the gill,
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intestine and spleen of healthy Japanese flounder [48]. Taken together, fish NLRC genes were
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mainly distributed in mucosal tissues of gill, intestine and/or other immune functional tissues
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such as kidney, liver and spleen, indicating their important roles for fish immunity. The different
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expression patterns between turbot NLRC3a and NLRC3b genes suggest the different roles of
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turbot NLRC3 genes might play. However, further studies are needed to characterize the detailed
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roles of NLRC3 in mucosal immune response after infection in teleost.
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3.4. Expression profiles of turbot NLRC3 genes following bacterial challenge
Expression profiles of NLRC3 in mucosal tissues of teleost fish, particularly after bacterial infection are scarce. Following V. anguillarum challenge, turbot NLRC3a showed different
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expression patterns in different tissues (Fig.5A). In general, turbot NLRC3a had relatively higher
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expression level in intestine than that of in gill and skin. As shown in Fig. 5A, the expression of
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NLRC3a in intestine was greatly up-regulated for 84.4 fold at 12 h and 74.2 fold at 24 h,
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respectively, but moderate induced at earlier time points (P < 0.05). The expression of turbot
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NLRC3a was also significantly induced in gill at all early time points but was still in a very low
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level, 4.4~8.9 fold. While in skin, a significant induction was only observed at 24 h post-
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infection with 5.5 fold (Fig. 5A).
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Turbot NLRC3b also had different expression patterns in different mucosal tissues
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following V. anguillarum challenge (Fig. 5B). In detail, the expression of NLRC3b was
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significantly induced at all time points in intestine and the highest expression was around 18~19
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fold at 12 h and 24 h. While the expression of NLRC3b in skin and gill was not significantly
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upregulated until 24 h post infection but still less than 5 fold. Obviously, expression of turbot
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NLRC3a and NLRC3b both were strongly induced in intestine rather than in skin and gill, and
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particularly at 12 h and 24 h post-infection. It has been reported that the turbot intestine may be a
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portal entry for the fish pathogen V. anguillarum [49] and turbot intestinal tract can serve as an
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enrichment site for V. anguillarum [50]. Additionally, NLRC3a had relative higher expression
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than NLRC3b in the gill and intestine, indicating NLRC3a may be more responsive to V.
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anguillarum infection.
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In case of S. iniae infection, the expression of NLRC3a was initially induced as early as 2 h
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with 16.9 fold, increased to the highest value at 4 h with 72.7 fold, but there was a reduction at 8
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h (31.6 h) and then recovered to 52.8 fold at 12 h post infection (Fig. 6A, P < 0.05). The
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expression of NLRC3a was significantly upregulated in skin and gill 4 h post-infection but kept
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at a low level afterwards (3.1~6.9 fold, P < 0.05, Fig. 6A). The magnitude of expression of
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NLRC3b after S. iniae infection were similar to that of NLRC3b after V. anguillarum infection
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as shown in Fig. 6B and Fig. 5B. After infection of 2 h, NLRC3b had significant upregulation in
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intestine at 2 h (4.3 fold),4 h (18.3 fold), decreased at 8 h (8.3 fold) and then rose again at 12 h
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(12.2 fold). While the expression of NLRC3b was not significantly expressed in skin and gill
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until 4 h post-infection, and had the highest expression at 12 h for 5.3 fold in skin and at 8 h for
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3.6 fold in gill, respectively (Fig. 6B). Here, NLRC3a and NLRC3b showed similar expression
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patterns to the infection of S. iniae, both induced as early as 2 h post infection and stronger in the
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intestine compared to that of in skin and gill, but the former had a stronger response. Our
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previous studies also revealed that several other immune-related genes were significantly
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induced in the intestine of turbot following S. iniae infection [26–29,51]. All these results
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indicated the intestine probably serves as an important entry of S. iniae.
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When compared with other fish species, Asian seabass NLRC3a was also significantly
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induced by Gram-negative V. alginolyticus infection in intestine at early time points [17]. While
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significant downregulation of NLRC3 genes were observed in the intestine of channel catfish
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after E. ictalurid infection [21]. When Asian seabass challenged by less virulent Gram-positive S.
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aureus, NLRC3a was significantly induced in the intestine but with low expression level (less
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than 4 fold) [17]. Additionally, a Japanese flounder NLRC that clustered into NLRC3 group, was
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upregulated as early as 3 h post S. iniae infection and 6 h post E. tarda stimulation [18]. Another
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flounder poNLRC3 gene with similar domain composition to turbot NLRC3b was significantly
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induced after 24 h post E. tarda challenge [20]. Therefore, in addition to domain structure, the
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species variation and different pathogenesis may also contribute to the differences of gene
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expression [28].
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These observations suggested NLRC3 may have important roles in mucosal immune response during Gram-negative and Gram-positive bacterial infection, however, further research
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maybe needed to discover whether NLRC3 activate or inhibit the innate immunity. Some reports
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showed that NLRC3 negatively regulated pro-inflammatory genes in mammals [14,15], however,
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the expression of IL-1β gene was positively regulated by two Japanese flounder NRLC3 genes,
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respectively [18,20].
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In conclusion, two members of NLRC3 genes were identified from turbot, the domain
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structures of their predicted proteins were characterized and their expression patterns were
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profiled following two different bacterial challenges in mucosal tissues. NLRC3a with a N-
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terminal CARD domain (or lack) and long LRRs showed high homology to their counterparts
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identified in other species, while NLRC3b possessing an uncharacterized N-terminal domain and
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an additional B30.2 (PRY/SPRY) domain at C-terminus appeared to be unique in teleost fish.
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The significant mucosal expression signatures of NLRC3 genes, particularly in the intestine,
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indicated their important roles in immediate response to both Gram-negative and Gram-positive
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bacteria in the first line of host defense system. NLRC3 genes found in this study were the first
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NLR members identified in turbot and their characterization will expand our knowledge of NLRs
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in mucosal immunity. Furthermore, understanding of the mechanism of host-bacteria interactions
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in turbot may provide insight into the development of effective disease prevention strategies and
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benefit the turbot industry.
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Acknowledgments
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This study was supported by the Advanced Talents Foundation of QAU grant (Grant No.: 6631114337), the national natural science foundation of China (Grant No.: 31602193).
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Table1. Similarities (%) and identity (%) of turbot NLRC3a and NLRC3b sequences with other
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species (MatGAT analysis).
Half -smooth tongue sole NLRC3a Fugu NLRC3a
Medaka NLRC3a Nile tilapia NLRC3a
Atlantic salmon NLRC3b Half-smooth tongue sole NLRC3b
Medaka NLRC3b
537 538 539 540 541 542 543 544
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Nile tilapia NLRC3b 535
25.6 24.8 25.2 24.8 24.3 23.1
81.1 80.3
43.7 43.3
25.6 24.7
92.2 93.7 45.4
82.7 86.5 24.8
42.3 44.2 66.2
23.8 24.5 60.1
40.0 42.5 43.8 44.3 40.7
22.3 23.7 24.5 23.1 23.7
72.8 78.7 85.1 79.2 69.3
61.7 63.9 72.7 64.6 58.1
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Fugu NLRC3b Large yellow croaker NLRC3b
90.7 89.5
45.8 44.4 44.5 45.9 43.7 41.5
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Western clawed frog NLRC3 Zebrafish NLRC3
24.1 48.1 47.7 50.5 51.0 45.7 51.3
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43.1 67.6 67.8 72.0 70.5 64.8 73.1
Turbot NLRC3b Similarity Identity
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Turbot NLRC3a Similarity Identity
Species Turbot NLRC3a Turbot NLRC3b Human NLRC3 Mouse NLRC3 Chicken NLRC3
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Fig. 1. Alignment of the deduced amino acid sequences of turbot NLRC3a with other species.
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Dashes represent amino acid deletions. Asterisks indicated identical amino acids; colons
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indicated similar amino acids and empty spaces represented absence or low level of similarities.
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Fig. 2. Alignment of the deduced amino acid sequences of turbot NLRC3b with other species.
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Dashes represent amino acid deletions. Asterisks indicated identical amino acids; colons
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indicated similar amino acids and empty spaces represented absence or low level of similarities.
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Fig. 3. Phylogenetic tree for the turbot NLRC3 genes. The phylogenetic tree was constructed
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based on the amino acid sequences of NLRC3 from fish and other vertebrate species using the
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neighbor-joining method in MEGA 6. Gaps were removed by complete deletion, the
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phylogenetic tree was evaluated with 10,000 bootstrap replications, and the bootstrapping values
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were indicated by numbers at the nodes. Dark solid circles indicated the newly characterized
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turbot NLRC3 genes (NLRC3a and NLRC3b).
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Fig. 4. Basal gene expression of the NLRC3 genes (NLRC3a and NLRC3b) in different turbot
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tissues. Expression levels were calibrated against tissue of liver which had the lowest expression
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level, and 18S rRNA was used as the reference gene. The Ct values of liver was set as the control
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to determine the gene expression patterns in healthy turbot tissues. HK was the abbreviation for
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head kidney.
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Fig. 5. Real-time qPCR analysis for NLRC3 expression levels following Vibrio anguillarum
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infection. The NLRC3 expression was measured in the mucosal tissues including skin, gill, and
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intestine at the time points of 2 h, 6 h, 12 h, and 24 h post-infection. Fold change was calculated
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by the change in expression at a given time point relative to the untreated control and normalized
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by change in the 18S rRNA reference gene. The results were presented as mean ± SE of fold
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changes and * indicated statistical significance at P < 0.05. (A) Relative gene expression level of
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NLRC3a following V. anguillarum infection in the mucosal tissues at different time points; (B)
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Relative gene expression level of NLRC3b following V. anguillarum infection in the mucosal
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tissues at different time points
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Fig. 6. Real-time qPCR analysis for NLRC3 expression in the mucosal tissues of turbot
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following Streptococcus iniae infection at the time points of 2 h, 4 h, 8 h and 12 h. Fold change
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was calculated by the change in expression at a given time point relative to the untreated control
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and normalized by changes in the 18S rRNA housekeeping gene. The results were presented as
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mean ± SE of fold changes and the * indicated statistical significance at P < 0.05. (A) Relative
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gene expression level of NLRC3a following S. iniae infection in the mucosal tissues at different
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time points; (B) Relative gene expression level of NLRC3b following S. iniae infection in the
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mucosal tissues at different time points.
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NLRC3a and NLRC3b were identified in turbot. Turbot NLRC3a is homologous to their counterparts in other vertebrates. NLRC3b appears to be unique to teleost.
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NLRC3a and NLRC3b were ubiquitously expressed in turbot tissues.
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Expression of NLRC3a and NLRC3b were significantly induced after bacterial challenge.
S1050-4648(17)30248-6
DOI:
10.1016/j.fsi.2017.05.014
Reference:
YFSIM 4567
To appear in:
Fish and Shellfish Immunology
Received Date: 10 January 2017 Revised Date:
30 April 2017
Accepted Date: 2 May 2017
Please cite this article as: Hou Z, Ye Z, Zhang D, Gao C, Su B, Song L, Tan F, Song H, Wang Y, Li C, Characterization and expression profiling of NOD-like receptor C3 (NLRC3) in mucosal tissues of turbot (Scophthalmus maximus L.) following bacterial challenge, Fish and Shellfish Immunology (2017), doi: 10.1016/j.fsi.2017.05.014. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Characterization and expression profiling of NOD-like receptor C3 (NLRC3) in mucosal tissues of Turbot (Scophthalmus maximus L.) following bacterial challenge
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Zhumei Houa,1, Zhi Ye b,1, Dongdong Zhang b, Chengbin Gaoa, Baofeng Sud,e, Lin Songa, Fenghua Tanc, Huanhuan Songa, Yu Wanga, Chao Lia*
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Marine Science and Engineering College, Qingdao Agricultural University, Qingdao 266109, China
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School of Fisheries, Aquaculture, and Aquatic Sciences, Auburn University, Auburn, AL 36849, USA
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266109, China
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Fisheries Research Institute, Chinese Academy of Fishery Sciences, Harbin 150070, China
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Heilongjiang Fisheries Research Institute, Chinese Academy of Fishery Sciences, Harbin 150070,
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China
School of international education and exchange, Qingdao Agricultural University, Qingdao
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National and Local Joint Engineering Laboratory of Freshwater Fish Breeding, Heilongjiang
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Ministry of Agriculture Key Laboratory of Freshwater Aquatic Biotechnology and Breeding,
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Both of these authors contributed equally to this work.
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*
Corresponding author: Chao Li; email: [email protected]
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Abstract: The mucosal surfaces are important for teleost as they are directly and continuously
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exposed to pathogen-rich aquatic environments. Scrutinization and recognition of the attached
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pathogens is the first crucial step of mucosal immunity initiation. Nucleotide oligomerization
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domain (NOD)-like receptors (NLRs) are a large group of intracellular pathogen recognition
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receptors (PRRs) which play key roles in pathogen recognition and subsequent immune signaling
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pathways activation. In this study, we identified two NLRC3 genes (NLRC3a and NLRC3b), a
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subfamily of NLRs from turbot, and profiled their expression patterns in mucosal tissues
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following bacterial challenge. NLRC3a transcript contains an open reading frame (ORF) of
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3,405 bp that encodes a putative peptide of 1,134 amino acids. While NLRC3b has an ORF of
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3,114 bp encoding 1,037 amino acids. A caspase recruitment domain (CARD) at N-terminus
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characterized turbot NLRC3a, while NLRC3b seems to be unique to teleost, containing a fish
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specific NACHT associated (FISNA) domain and an extra B30.2 (PRY/SPRY) domain at C-
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terminus. In addition, NLRC3a and NLRC3b were detected in all the examined tissues, with the
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highest expression levels in kidney and blood, respectively. After bacteria challenge, expression
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levels of turbot NLRC3 genes were strongly induced in intestine rather than in skin and gill,
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while NLRC3a had relatively higher expression level than that of NLRC3b. Taken together,
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NLRC3 genes found in this study were the first NLR members identified in turbot. The different
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expression signatures of NLRC3a and NLRC3b in mucosal tissues following two bacterial
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infections indicated they probably have important roles in early response to bacterial infection in
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the first line of host defense system.
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Keywords: Turbot, NLRC3, Pathogen recognition receptors, Mucosal immunity
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1. Introduction
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Mucosal immune system contains a unique array of innate and adaptive immune cells and molecules that together constitutes the first line of host defense against pathogens’ intrusion. The
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mucosal surfaces serve as dynamic interfaces driving a variety of critical physiological
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processes[1]. For teleost fish, mucosal immune system is of great importance as their mucosal
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surfaces are directly exposed to and continuously colonized by complex and diverse microbial
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communities which bombard mucosal epithelial barriers, thus posing additional challenges to
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fish mucosal immunity [2,3]. The first critical step of mucosal immune response is to scrutinize
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and recognize the attached potential pathogens, and then to initiate the host immunity in a rapid
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and accurate manner. In this respect, pathogen recognition receptors (PRRs) which can
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distinguish the microbial unique and conserved molecular signatures, so-called pathogen-
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associated molecular patterns (PAMPs), are key factors of mucosal surfaces for pathogen
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recognition and subsequent immune signaling pathways activation [4]. Nevertheless, our
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understanding of teleost PRRs as well as their associated activities in early mucosal immune
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defense against pathogen invasion is still limited.
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PRRs discovered so far are classified into three major families according to their domain
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properties: the Toll-like receptors (TLRs), nucleotide-binding oligomerization domain (NOD)-
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like receptors (NLRs; alternatively called nucleotide-binding domain and leucine-rich repeat
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containing receptors), and retinoic acid-inducible gene (RIG-1)-like receptors (RLRs) [5].
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Among these, NLRs are a large group of intracellular sensor proteins which are capable of
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recognizing intracellular PAMPs or damage/danger associated molecular patterns (DAMPs) and
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regulating the antimicrobial immune responses [6,7]. The absence of signal peptides and lack of
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transmembrane domains indicated the cytoplasmic location of NLRs [8,9]. The NLRs have three
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characterized and structural domains: a variable N-terminal effector domain responsible for
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triggering downstream signaling pathways through protein-protein interaction, a central
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nucleotide binding oligomerization (NACHT) domain for self-regulation and oligomerization,
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and a leucine-rich repeat (LRR) C-terminus required for ligand binding specificity [10,11].
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Ligand binding contributes to the oligomerization of the NACHT domain, and thereafter forms
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an active signaling platform which allows the effector domains to bind downstream signaling
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molecules and to trigger immune cascades [12]. Among the three domains, the N-terminus is
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widely diverse across the species. They typically can be either a caspase recruitment domain
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(CARD) or pyrin domain (PYD) or in a fewer cases, a baculovirus inhibitor of apoptosis repeat
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(BIR) domain or some other less defined (X) domains [9,12].
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Recently, NLRs are classified into NLRA/CIITA, NLRB/NAIP, NLRC, NLRP and NLRX1 subfamilies according to their domain structures and phylogenetic relationship using standard
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nomenclature [9,13]. NLRC is one of the biggest and most important groups, including
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NLRC1/NOD1, NLRC2/NOD2, NLRC3/NOD3, NLRC4/IPAF, and NLRC5 [9,13]. Among
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these, NLRC1 and NLRC2 with one or two N-terminal CARDs are two widely studied groups of
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NLRCs that augment pro-inflammatory pathways through the activation of caspase-1 or NF-κB
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signaling pathways [7]. Conversely, NLRC3 has been reported to negatively regulate
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inflammation and emerged as a negative regulator of innate immune signaling by interfering in
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TLR or STING signaling pathways [14,15]. For example, zebrafish (Danio rerio) NLRC3-like
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receptor was reported to negatively regulate inflammation and macrophage activation [16].
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However, other studies indicated that the negative regulatory role of NLRC3 may be ruled out in
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some teleost because they play a significant role as PRRs in pathogen recognition [17,18]. Until
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now, NLRC3 genes have been documented in several teleost species, such as zebrafish [19],
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Japanese flounder (Paralichthys olivaceus) [18,20], channel catfish (Ictalurus punctatus)
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[8,20,21] and Asian seabass (Lates calcarifer) [17]. In those studies, NLRC3 can be induced by
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bacterial infection or ligand (such as LPS), demonstrating their important role in the innate
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immune response in teleost.
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Turbot (Scophthalmus maximus) is one of the most important cultured marine fishes in
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China. Its commercial production has been significantly hindered due to widespread disease
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outbreaks caused by a number of pathogens, including Vibrio anguillarum, Streptococcus iniae
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and Edwardsiella tarda. Up to date, many innate immune components as well as their associated
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activities during the infection have been characterized in turbot, including stomatin-like protein 2
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[22], chemokines [23,24], MyD88 [25], lysozyme [26], and particularly some PRRs like TLRs
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[27,28] and peptidoglycan recognition protein (PGRP) [29]. The expression patterns of these
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PPRs in mucosal tissues (skin, gill and intestine) following bacterial infection indicated their
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important roles during early recognition of invading pathogen [30]. However, characterization of
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another important category of PRRs, NLRs especially NLRC3 and their associated immune
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activities in mucosal tissues are still lacking. In this regard, we sought here to identify NLRC3
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members in turbot, conducted phylogenetic analysis, and profiled their expression patterns in
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mucosal barriers following bacterial infection in the early time points.
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2. Materials and methods
2.1. Sequence identification and analysis
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By means of NLRC3 protein sequences from other species as queries, turbot NLRC3 genes were identified from turbot transcriptome databases generated by our group [31], using
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tBLASTn program with a cutoff E-value of 1e-10. The retrieved nucleotide sequences were
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translated by the ORF Finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). The predicted amino
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acid sequences were further verified by BLASTP (http://blast.ncbi.nlm.nih.gov/Blast.cgi) against
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NCBI non-redundant protein database. The conserved domains and signal peptides were
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identified using simple modular architecture research tool (SMART; http://smart.embl-
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heidelberg.de/). The theoretical pI, molecular mass and N-glycosylation sites were analyzed
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using online ExPASy server [32]. The percentages of similarity and identity of NLRC amino
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acids with other species were calculated by MatGAT program [33].
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2.2. Phylogenetic analysis
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Amino acids sequences of NLRC3 from turbot and other organisms were selected to construct the phylogenetic tree, including human (Homo sapiens; NP_849172.2), mouse
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(Mus musculus; NP_001074749.1), chicken (Gallus gallus; XP_015150161.1), green sea turtle
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(Chelonia mydas; XP_007054520.1), western clawed frog (Xenopus tropicalis;
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XP_002932504.1), zebrafish (D. rerio; XP_009295904.1), Atlantic salmon (Salmo salar;
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XP_014049925.1), fugu (Takifugu rubripes; XP_003961491.2 and XP_003961491.2), half-
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smooth tongue sole (Cynoglossus semilaevis; XP_008312409.1 and XP_008312409.1), large
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yellow croaker (Larimichthys crocea; XP_003438651.1), medaka (Oryzias latipes;
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XP_004080575.1 and XP_004080575.1) and Nile tilapia (Oreochromis niloticus;
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XP_004080575.1 and XP_003438651.1). Amino acid sequence alignments of the NLRC3
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proteins were conducted using the ClustalW2 multiple alignment program [34]. Phylogenetic and
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molecular evolutionary analyses were performed using MEGA (Molecular Evolutionary
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Genetics Analysis) package (version 6.0) with the neighbor-joining method [35]. Poisson
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correction was selected for substitution model, gaps were removed by complete deletion and
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1,000 bootstrapping replications were used to evaluate the reliability of the neighbor-joining
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trees.
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2.3. Bacteria challenge and sample collection
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In order to investigate the immune roles of NLRC3 genes against bacterial infection, the Gram-negative bacteria V. anguillarum and the Gram-positive bacteria S. iniae were selected to
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conduct the bath challenge, respectively. Turbot fingerlings (approximately 15.6 g in mass and
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5.5 cm in length) were obtained from the turbot hatchery (Haiyang, Shandong, China) and
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acclimated in aquariums with aerated flow-through water (27 ± 0.5 oC) for one week in the
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laboratory prior to challenge. The bacteria isolate of V. anguillarum and S. iniae were provided
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by the disease lab of Qingdao Agricultural University. A pre-challenge was conducted and the
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bacteria were re-isolated from symptomatic fish and biochemically confirmed before cultured.
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V. anguillarum and S. iniae were inoculated in LB broth respectively and cultured in a
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shaker (180 rpm) at 28 oC overnight. The concentration of the final culture was measured by
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streak plate serial ten-fold dilutions of the culture and recorded as colony forming units per
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milliliter (CFU/mL). Fish were challenged by 2 h immersion of V. anguillarum and S. iniae at a
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final concentration of 5×107 CFU/mL and 5×106 CFU/mL, respectively. Control fish followed an
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identical procedure but were in sterilized media without bacteria.
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2.4. Sample collection and total RNA extraction
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Turbot tissues including brain, skin, gill, intestine, head kidney, liver, spleen and blood
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were collected from healthy individuals. While only mucosal tissues (skin, gill and intestine)
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were collected after challenge of V. anguillarum at 2 h, 6 h and 24 h and S. iniae at 2 h, 4 h, 8 h
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and 12 h, respectively. At each time point, 15 fish were randomly collected and divided into 3
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replicate pools (5 fish each). Fish were euthanized with tricaine methanesulfonate (MS-222) at
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200 mg/L (buffered with sodium bicarbonate). Approximately equal amounts of tissue from each
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fish within the replicates were collected, flash-frozen in liquid nitrogen and stored at -80 °C until
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RNA isolation. Prior to RNA extraction, samples were removed from the -80 °C freezer and
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ground to a fine powder with sterilized mortar and pestle in the presence of liquid nitrogen. Total
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RNA was extracted from tissues using the Trizol® Reagent (Invitrogen, USA) following
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manufacturer’s instructions. The RNA integrity and the genomic DNA contamination were
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monitored by electrophoresis with 1% agarose gel. The purity and concentration of RNA were
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measured on a nanodrop 2000 (Thermo Electron North America LLC, FL). All extracted
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samples had an A260/280 ratio greater than 1.8, and were diluted to 250 ng/µl.
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2.5. QPCR analysis
Quantitative real-time PCR (QPCR) was used to examine NLRC3 mRNA expression in
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different tissues at different time points post bacteria challenge. Primers for QPCR were
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designed using Primer 3 software based on the sequences of turbot NLRC3 transcripts. Turbot
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18S rRNA gene was used as an internal control for normalization of the expression levels. The
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PCR products were sequenced to confirm the specificity of these primers. First strand cDNA
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(500 ng RNA per 10 µl reaction) was synthesized by PrimeScript RT reagent Kit (Takara, Dalian,
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China) and QPCR was performed on a CFX96 real-time PCR Detection System (Bio-Rad
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Laboratories, Hercules, CA) following the manufacturer’s instructions. The PCR reaction
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mixture was denatured at 95 °C for 30 s and then subjected to 40 cycles of 95 °C for 5 s, 58 °C
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for 5 s and followed by dissociation curve analysis, 5 s at 65 oC, then up to 95 °C at a rate of
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0.1 °C/s increment, to verify the specificity of the amplicons. Results were expressed relative to
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the expression levels of 18S rRNA in each sample using the Relative Expression Software Tool
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(REST) [36] to capture significance at the level of P < 0.05. The Ct values of liver was set as the
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control to determine the gene expression patterns in healthy turbot tissues. A no-template control
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was run on all plates.
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3. Results and discussion
3.1 Identification of turbot NLRC3 genes
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Two turbot NLRC3 transcripts NLRC3a (KY437762) and NLRC3b (KY437763) were
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captured from the turbot transcriptomic database [31]. NLRC3a transcript has a full-length of
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5,471 bp, containing a 3,405 bp open reading frame (ORF) that encodes a putative peptide of
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1,134 amino acids with an estimated molecular mass of 125.03 kDa and a theoretical pI of 8.35.
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Similarly, the NLRC3b is 3,700 bp long and contains a 3,114 bp ORF that encodes 1,037 amino
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acids with an estimated molecular mass of 113.96 kDa and a theoretical pI of 7.66. The domain
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analysis and protein sequence alignment of the turbot NLRC3 with other species showed that all
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NLRC3 sequences have the characteristic NACHT domain and LRR domain, but the N-terminus
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is variable and in some cases, additional C-terminus exist (Fig. 1 and Fig. 2). Sequence identity and similarity of NLRC3 genes were analyzed. Turbot NLRC3a and
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NLRC3b amino acid sequences showed very low identity (24.1%) to each other (Table 1).
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Turbot NLRC3a showed high identity (more than 80%) to the selected teleost fish NLRC3a
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sequences, including Nile tilapia, fugu, medaka and half-smooth tongue sole, but had much
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lower identity (45.7~51.3%) to NLRC3 from zebrafish and higher vertebrates like human, mouse,
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chick, turtle and frog. Similarly, NLRC3b showed higher identity (58.1~72.7%) to NLRC3b of
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fugu, Atlantic salmon, medaka, large yellow croaker, half-smooth tongue sole and Nile tilapia,
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and its identity was lower to zebrafish and some higher vertebrates (less than 30%) (Table 1).
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In addition to low identity between the two NLRC3 sequences of turbot, they also have
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different domain compositions, implying they may have different immune response mechanisms.
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Alignment of NLRC3a with sequences of Nile tilapia, fugu, medaka and half-smooth tongue sole,
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as well as NLRC3 sequences of zebrafish, human , mouse, chick, turtle and frog revealed highly
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conserved motifs, one NACHT domain and 14 LLRs (Fig.1). Except human and mouse, all of
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them have one N-terminal CARD (Fig.1), which has been found in a wide array of proteins that
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are typically associated with inflammation and apoptosis [37–39].
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Teleost specific NLRC3 were detected including turbot NLRC3b and other teleost NLRC3b
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in fugu, Atlantic salmon, medaka, large yellow croaker, half-smooth tongue sole because they
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have a typically specific NACHT associated (FISNA) domain rather than CARD at N-terminus,
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and an extra highly conserved B30.2 (PRY/SPRY) domain at C-terminus (Fig. 2). Exceptionally,
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tilapia NLRC3b lacked B30.2 domain and zebrafish has no FISNA and B30.2 domains. However,
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NLRC3 sequences of zebrafish and other higher vertebrates showed some similarities to FISNA
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and B30.2 (Fig. 2). The FISNA has been reported to locate immediately upstream of the NACHT
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domain of fish NLR family, but its function is uncharacterized [40]. The C-terminal B30.2
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domain contains two single Serine-Proline-Arginine-Tyrosine (SPRY) and Proline-Arginine-
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Tyrosine (PRY) regions, which are implicated in innate immunity [41] and interact directly with
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caspase-1 to modulate IL-1β production [42]. This B30.2 domain has also been found in some
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other immune related proteins, such as teleost-specific tripartite motif (Trim) proteins that
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involve in virus infection [43].
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In addition, both of the predicted turbot NLRC3a and NLRC3b proteins lack the signal
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peptides and transmembrane domain, indicating their cytoplasmic location and functioning as the
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intracellular receptor, which was consistent with the characterized of NLRs in other species [9].
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3.2 Phylogenetic analyses
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In order to further confirm the identities of turbot NLRC3 genes (NLRC3a and NLRC3b) and clarify the phylogenetic relationship of NLRC3 among species, a neighbor-joining
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phylogenetic tree was constructed using MEGA6 with the NLRC3 amino acid sequences from
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selected vertebrate species (Fig.3). As shown in Fig.3, all NLRC3 sequences were clustered into
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two major distinct groups. In the first major cluster, fish NLRC3 and higher vertebrates NLRC3
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sequences were separated into two subclades with high bootstrapping values (100 match score).
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Turbot NLRC3a shared a common ancestor with clade consisted of O. niloticus and O.
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latipeswith high similarity match score of 81, then clustered with T. rubripes, and then fell into
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the clade with C. semilaevis with 100 match score (Fig. 3).
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Fish specific NLRC3b containing extra B30.2 and FISNA domains were clustered together, forming the second independent major clade. Turbot NLRC3b also showed the closest
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relationship to O. niloticus like that of NLRC3a but with lower match score of 50, and then
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clustered with L. crocea (match score 86), and then fell into the clade with C. semilaevis with
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match score 46 (Fig. 2).
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Taken together, the phylogenetic tree analysis was almost consistent with their phylogenetic relationships. Collectively, the phylogenetic analysis confirmed the identification of NLRC3a
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and NLRC3b, and NLRC3a showed homology to their counterparts identified in other species,
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while NLRC3b seemed to be unique to teleost with relatively lower match scores among species.
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3.3. Basal tissue expression of two turbot NLRC3 genes
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QPCR analyses was performed to determine the basal expression levels of these two turbot
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NLRC3 genes in different tissues, including liver, skin, spleen, gill, intestine, blood, head kidney
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and brain. NLRC3a and NLRC3b were both expressed in almost all examined tissues, but with
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varied expression levels (Fig. 4). Turbot NLRC3a showed the strongest expression level in head
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kidney and gill (> 25 fold), followed by spleen (~17.4 fold), intestine (~16.2 fold), blood (~12.9
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fold), skin (~9.4 fold), brain (~2.0 fold), and the lowest expression level in liver (Fig. 4). While
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NLRC3b had much higher expression in blood, spleen and head kidney (> 60 fold), modest in
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gill (~21.8 fold), and lower expression level in skin, intestine and brain (Fig. 2).
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In channel catfish, NLRC3a also had higher expression level in immune related tissues such
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as gill, head kidney, intestine and spleen, but lower expression level in skin and brain [8].
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Similarly, Asian seabass NLRC3a, had higher expression level in gill and intestine, modest
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expression level in liver, and much lower expression level in skin and brain [17]. In miiuy
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croaker (Miichthys miiuy), NLRC3a also expressed weakly in skin but higher in gill, intestine
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and blood [44]. However, the information about the expression patterns of teleost specific
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NLRC3b was much limited. A Japanese flounder NLRC transcript (GenBank acc. No JF271924)
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that contains B30.2 domain and lacks FISNA domain, had higher expression levels in gill and
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kidney [18]. While another poNLRC3 gene in Japanese flounder with similar domain
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compositions as turbot NLRC3b, had relatively abundant expression in hepatopancreas and
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lower expression level in blood, which was opposite to that of turbot NLRC3b in the current
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study [20] Some other NLRC genes, such as one of the most studied NOD1, had the highest
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expression level in spleen of goldfish (Carassius auratus L.) [45], and was highly expressed in
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spleen and liver of Rainbow trout (Oncorhynchus mykiss) [46]. Additionally, NLRC5 gene was
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highly expressed in the head kidney, spleen, and hindgut of Atlantic salmon [47] and in the gill,
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intestine and spleen of healthy Japanese flounder [48]. Taken together, fish NLRC genes were
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mainly distributed in mucosal tissues of gill, intestine and/or other immune functional tissues
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such as kidney, liver and spleen, indicating their important roles for fish immunity. The different
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expression patterns between turbot NLRC3a and NLRC3b genes suggest the different roles of
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turbot NLRC3 genes might play. However, further studies are needed to characterize the detailed
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roles of NLRC3 in mucosal immune response after infection in teleost.
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3.4. Expression profiles of turbot NLRC3 genes following bacterial challenge
Expression profiles of NLRC3 in mucosal tissues of teleost fish, particularly after bacterial infection are scarce. Following V. anguillarum challenge, turbot NLRC3a showed different
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expression patterns in different tissues (Fig.5A). In general, turbot NLRC3a had relatively higher
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expression level in intestine than that of in gill and skin. As shown in Fig. 5A, the expression of
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NLRC3a in intestine was greatly up-regulated for 84.4 fold at 12 h and 74.2 fold at 24 h,
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respectively, but moderate induced at earlier time points (P < 0.05). The expression of turbot
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NLRC3a was also significantly induced in gill at all early time points but was still in a very low
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level, 4.4~8.9 fold. While in skin, a significant induction was only observed at 24 h post-
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infection with 5.5 fold (Fig. 5A).
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Turbot NLRC3b also had different expression patterns in different mucosal tissues
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following V. anguillarum challenge (Fig. 5B). In detail, the expression of NLRC3b was
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significantly induced at all time points in intestine and the highest expression was around 18~19
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fold at 12 h and 24 h. While the expression of NLRC3b in skin and gill was not significantly
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upregulated until 24 h post infection but still less than 5 fold. Obviously, expression of turbot
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NLRC3a and NLRC3b both were strongly induced in intestine rather than in skin and gill, and
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particularly at 12 h and 24 h post-infection. It has been reported that the turbot intestine may be a
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portal entry for the fish pathogen V. anguillarum [49] and turbot intestinal tract can serve as an
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enrichment site for V. anguillarum [50]. Additionally, NLRC3a had relative higher expression
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than NLRC3b in the gill and intestine, indicating NLRC3a may be more responsive to V.
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anguillarum infection.
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In case of S. iniae infection, the expression of NLRC3a was initially induced as early as 2 h
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with 16.9 fold, increased to the highest value at 4 h with 72.7 fold, but there was a reduction at 8
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h (31.6 h) and then recovered to 52.8 fold at 12 h post infection (Fig. 6A, P < 0.05). The
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expression of NLRC3a was significantly upregulated in skin and gill 4 h post-infection but kept
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at a low level afterwards (3.1~6.9 fold, P < 0.05, Fig. 6A). The magnitude of expression of
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NLRC3b after S. iniae infection were similar to that of NLRC3b after V. anguillarum infection
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as shown in Fig. 6B and Fig. 5B. After infection of 2 h, NLRC3b had significant upregulation in
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intestine at 2 h (4.3 fold),4 h (18.3 fold), decreased at 8 h (8.3 fold) and then rose again at 12 h
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(12.2 fold). While the expression of NLRC3b was not significantly expressed in skin and gill
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until 4 h post-infection, and had the highest expression at 12 h for 5.3 fold in skin and at 8 h for
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3.6 fold in gill, respectively (Fig. 6B). Here, NLRC3a and NLRC3b showed similar expression
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patterns to the infection of S. iniae, both induced as early as 2 h post infection and stronger in the
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intestine compared to that of in skin and gill, but the former had a stronger response. Our
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previous studies also revealed that several other immune-related genes were significantly
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induced in the intestine of turbot following S. iniae infection [26–29,51]. All these results
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indicated the intestine probably serves as an important entry of S. iniae.
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When compared with other fish species, Asian seabass NLRC3a was also significantly
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induced by Gram-negative V. alginolyticus infection in intestine at early time points [17]. While
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significant downregulation of NLRC3 genes were observed in the intestine of channel catfish
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after E. ictalurid infection [21]. When Asian seabass challenged by less virulent Gram-positive S.
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aureus, NLRC3a was significantly induced in the intestine but with low expression level (less
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than 4 fold) [17]. Additionally, a Japanese flounder NLRC that clustered into NLRC3 group, was
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upregulated as early as 3 h post S. iniae infection and 6 h post E. tarda stimulation [18]. Another
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flounder poNLRC3 gene with similar domain composition to turbot NLRC3b was significantly
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induced after 24 h post E. tarda challenge [20]. Therefore, in addition to domain structure, the
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species variation and different pathogenesis may also contribute to the differences of gene
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expression [28].
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These observations suggested NLRC3 may have important roles in mucosal immune response during Gram-negative and Gram-positive bacterial infection, however, further research
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maybe needed to discover whether NLRC3 activate or inhibit the innate immunity. Some reports
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showed that NLRC3 negatively regulated pro-inflammatory genes in mammals [14,15], however,
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the expression of IL-1β gene was positively regulated by two Japanese flounder NRLC3 genes,
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respectively [18,20].
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In conclusion, two members of NLRC3 genes were identified from turbot, the domain
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structures of their predicted proteins were characterized and their expression patterns were
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profiled following two different bacterial challenges in mucosal tissues. NLRC3a with a N-
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terminal CARD domain (or lack) and long LRRs showed high homology to their counterparts
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identified in other species, while NLRC3b possessing an uncharacterized N-terminal domain and
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an additional B30.2 (PRY/SPRY) domain at C-terminus appeared to be unique in teleost fish.
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The significant mucosal expression signatures of NLRC3 genes, particularly in the intestine,
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indicated their important roles in immediate response to both Gram-negative and Gram-positive
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bacteria in the first line of host defense system. NLRC3 genes found in this study were the first
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NLR members identified in turbot and their characterization will expand our knowledge of NLRs
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in mucosal immunity. Furthermore, understanding of the mechanism of host-bacteria interactions
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in turbot may provide insight into the development of effective disease prevention strategies and
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benefit the turbot industry.
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Acknowledgments
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This study was supported by the Advanced Talents Foundation of QAU grant (Grant No.: 6631114337), the national natural science foundation of China (Grant No.: 31602193).
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[30] S. Akira, S. Uematsu, O. Takeuchi, Pathogen recognition and innate immunity, Cell. 124 (2006) 783–801. [31] C. Gao, Q. Fu, B. Su, S. Zhou, F. Liu, L. Song, M. Zhang, Y. Ren, X. Dong, F. Tan, C. Li, Transcriptomic profiling revealed the signatures of intestinal barrier alteration and pathogen entry in turbot (Scophthalmus maximus) following Vibrio anguillarum challenge, Dev. Comp. Immunol. 65 (2016) 159–168. [32] E. Gasteiger, C. Hoogland, A. Gattiker, S. ’everine Duvaud, M. Wilkins, R. Appel, A. Bairoch, Protein Identification and Analysis Tools on the ExPASy Server, in: J. Walker (Ed.), Proteomics Protoc. Handb., Humana Press, 2005: pp. 571–607. [33] J.J. Campanella, L. Bitincka, J. Smalley, MatGAT: An application that generates similarity/identity matrices using protein or DNA sequences, BMC Bioinformatics. 4 (2003) 29. [34] M.A. Larkin, G. Blackshields, N.P. Brown, R. Chenna, P.A. McGettigan, et al., Clustal W and Clustal X version 2.0, Bioinforma. Oxf. Engl. 23 (2007) 2947–2948. [35] K. Tamura, G. Stecher, D. Peterson, A. Filipski, S. Kumar, MEGA6: Molecular Evolutionary Genetics Analysis version 6.0, Mol. Biol. Evol. 30 (2013) 2725–2729. [36] M.W. Pfaffl, G.W. Horgan, L. Dempfle, Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR, Nucleic Acids Res. 30 (2002) e36. [37] L. Bouchier-Hayes, S.J. Martin, CARD games in apoptosis and immunity, EMBO Rep. 3 (2002) 616–621. [38] W.-P. Kao, C.-Y. Yang, T.-W. Su, Y.-T. Wang, Y.-C. Lo, S.-C. Lin, The versatile roles of CARDs in regulating apoptosis, inflammation, and NF-κB signaling, Apoptosis Int. J. Program. Cell Death. 20 (2015) 174–195. [39] H.H. Park, Y.-C. Lo, S.-C. Lin, L. Wang, J.K. Yang, H. Wu, The death domain superfamily in intracellular signaling of apoptosis and inflammation, Annu. Rev. Immunol. 25 (2007) 561–586. [40] C. Stein, M. Caccamo, G. Laird, M. Leptin, Conservation and divergence of gene families encoding components of innate immune response systems in zebrafish, Genome Biol. 8 (2007) R251. [41] A.A. D’Cruz, J.J. Babon, R.S. Norton, N.A. Nicola, S.E. Nicholson, Structure and function of the SPRY/B30.2 domain proteins involved in innate immunity, Protein Sci. Publ. Protein Soc. 22 (2013) 1–10. [42] J.J. Chae, G. Wood, S.L. Masters, K. Richard, G. Park, B.J. Smith, D.L. Kastner, The B30.2 domain of pyrin, the familial Mediterranean fever protein, interacts directly with caspase-1 to modulate IL-1β production, Proc. Natl. Acad. Sci. 103 (2006) 9982–9987. [43] L.M. van der Aa, J.-P. Levraud, M. Yahmi, E. Lauret, V. Briolat, P. Herbomel, A. Benmansour, P. Boudinot, A large new subset of TRIM genes highly diversified by duplication and positive selection in teleost fish, BMC Biol. 7 (2009) 7. [44] J. Li, L. Kong, Y. Gao, C. Wu, T. Xu, Characterization of NLR-A subfamily members in miiuy croaker and comparative genomics revealed NLRX1 underwent duplication and lose in actinopterygii, Fish Shellfish Immunol. 47 (2015) 397–406. [45] J. Xie, J.W. Hodgkinson, B.A. Katzenback, N. Kovacevic, M. Belosevic, Characterization of three Nod-like receptors and their role in antimicrobial responses of goldfish (Carassius auratus L.) macrophages to Aeromonas salmonicida and Mycobacterium marinum, Dev. Comp. Immunol. 39 (2013) 180–187.
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Table1. Similarities (%) and identity (%) of turbot NLRC3a and NLRC3b sequences with other
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species (MatGAT analysis).
Half -smooth tongue sole NLRC3a Fugu NLRC3a
Medaka NLRC3a Nile tilapia NLRC3a
Atlantic salmon NLRC3b Half-smooth tongue sole NLRC3b
Medaka NLRC3b
537 538 539 540 541 542 543 544
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Nile tilapia NLRC3b 535
25.6 24.8 25.2 24.8 24.3 23.1
81.1 80.3
43.7 43.3
25.6 24.7
92.2 93.7 45.4
82.7 86.5 24.8
42.3 44.2 66.2
23.8 24.5 60.1
40.0 42.5 43.8 44.3 40.7
22.3 23.7 24.5 23.1 23.7
72.8 78.7 85.1 79.2 69.3
61.7 63.9 72.7 64.6 58.1
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Fugu NLRC3b Large yellow croaker NLRC3b
90.7 89.5
45.8 44.4 44.5 45.9 43.7 41.5
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Western clawed frog NLRC3 Zebrafish NLRC3
24.1 48.1 47.7 50.5 51.0 45.7 51.3
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Green sea turtle NLRC3
43.1 67.6 67.8 72.0 70.5 64.8 73.1
Turbot NLRC3b Similarity Identity
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Turbot NLRC3a Similarity Identity
Species Turbot NLRC3a Turbot NLRC3b Human NLRC3 Mouse NLRC3 Chicken NLRC3
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Fig. 1. Alignment of the deduced amino acid sequences of turbot NLRC3a with other species.
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Dashes represent amino acid deletions. Asterisks indicated identical amino acids; colons
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indicated similar amino acids and empty spaces represented absence or low level of similarities.
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Fig. 2. Alignment of the deduced amino acid sequences of turbot NLRC3b with other species.
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Dashes represent amino acid deletions. Asterisks indicated identical amino acids; colons
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indicated similar amino acids and empty spaces represented absence or low level of similarities.
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Fig. 3. Phylogenetic tree for the turbot NLRC3 genes. The phylogenetic tree was constructed
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based on the amino acid sequences of NLRC3 from fish and other vertebrate species using the
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neighbor-joining method in MEGA 6. Gaps were removed by complete deletion, the
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phylogenetic tree was evaluated with 10,000 bootstrap replications, and the bootstrapping values
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were indicated by numbers at the nodes. Dark solid circles indicated the newly characterized
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turbot NLRC3 genes (NLRC3a and NLRC3b).
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Fig. 4. Basal gene expression of the NLRC3 genes (NLRC3a and NLRC3b) in different turbot
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tissues. Expression levels were calibrated against tissue of liver which had the lowest expression
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level, and 18S rRNA was used as the reference gene. The Ct values of liver was set as the control
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to determine the gene expression patterns in healthy turbot tissues. HK was the abbreviation for
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head kidney.
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Fig. 5. Real-time qPCR analysis for NLRC3 expression levels following Vibrio anguillarum
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infection. The NLRC3 expression was measured in the mucosal tissues including skin, gill, and
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intestine at the time points of 2 h, 6 h, 12 h, and 24 h post-infection. Fold change was calculated
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by the change in expression at a given time point relative to the untreated control and normalized
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by change in the 18S rRNA reference gene. The results were presented as mean ± SE of fold
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changes and * indicated statistical significance at P < 0.05. (A) Relative gene expression level of
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NLRC3a following V. anguillarum infection in the mucosal tissues at different time points; (B)
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Relative gene expression level of NLRC3b following V. anguillarum infection in the mucosal
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tissues at different time points
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Fig. 6. Real-time qPCR analysis for NLRC3 expression in the mucosal tissues of turbot
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following Streptococcus iniae infection at the time points of 2 h, 4 h, 8 h and 12 h. Fold change
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was calculated by the change in expression at a given time point relative to the untreated control
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and normalized by changes in the 18S rRNA housekeeping gene. The results were presented as
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mean ± SE of fold changes and the * indicated statistical significance at P < 0.05. (A) Relative
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gene expression level of NLRC3a following S. iniae infection in the mucosal tissues at different
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time points; (B) Relative gene expression level of NLRC3b following S. iniae infection in the
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mucosal tissues at different time points.
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NLRC3a and NLRC3b were identified in turbot. Turbot NLRC3a is homologous to their counterparts in other vertebrates. NLRC3b appears to be unique to teleost.
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NLRC3a and NLRC3b were ubiquitously expressed in turbot tissues.
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Expression of NLRC3a and NLRC3b were significantly induced after bacterial challenge.