Molecular characterization of nucleotide binding and oligomerization domain (NOD)-2, analysis of its inductive expression and down-stream signaling following ligands exposure and bacterial infection in rohu (Labeo rohita) B. Swain a , M. Basu a , B.R. Sahoo a , N.K. Maiti a , P. Routray b , A.E. Eknath a , M. Samanta a,⇑ a Fish Health Management Division, Central Institute of Freshwater Aquaculture (CIFA), Kausalyaganga, Bhubaneswar, Orissa 751002, India b Aquaculture Production and Environment Division, Central Institute of Freshwater Aquaculture (CIFA), Kausalyaganga, Bhubaneswar, Orissa 751002, India article info Article history: Received 13 May 2011 Revised 16 June 2011 Accepted 17 June 2011 Available online 13 July 2011 Keywords: Indian major carp Labeo rohita NOD-2 RICK IFN-c Aeromonas hydrophila Edwardsiella tarda abstract Nucleotide-binding and oligomerization domain (NOD)-2 is a cytoplasmic pattern recognition receptor (PRR) and is a member of NOD like receptor (NLR) family. It senses a wide range of bacteria and viruses or their products and is involved in innate immune responses. In this report, NOD-2 gene was cloned and characterized from rohu (Labeo rohita) which is highly commercially important fish species in the Indian subcontinent. The full length rohu NOD-2 (rNOD-2) cDNA comprised of 3176 bp with a single open read- ing frame (ORF) of 2949 bp encoding a polypeptide of 982 amino acids (aa) with an estimated molecular mass of 109.65 kDa. The rNOD-2 comprised two N-terminal CARD domains (at 4–91 aa and 111–200 aa), one NACHT domain (at 271–441 aa) and seven C-terminal leucine rich repeat (LRR) regions. Phylogenet- ically, rNOD-2 was closely related to grass carp NOD-2 (gcNOD2) and exhibited significant similarity (94.2%) and identity (88.6%) in their amino acids. Ontogeny analysis of rNOD-2 showed its constitutive expression across the developmental stages, and highlighted the embryonic innate defense system in fish. Tissue specific analysis of rNOD-2 by quantitative real-time PCR (qRT-PCR) revealed its wide distri- bution; highest expression was in liver followed by blood. In response to PGN and LTA stimulation, Aeromonas hydrophila and Edwardsiella tarda infection, and poly I:C treatment, expression of rNOD-2 and its associated downstream molecules RICK and IFN-c were significantly enhanced in the treated fish compared to control. These findings suggested the key role of NOD-2 in augmenting innate immunity in fish in response to bacterial and viral infection. This study may be helpful for the development of preven- tive measures against infectious diseases in fish. Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction Innate immune system is the first line of defense system against pathogens and pathogenic products. While higher vertebrates like birds and mammals show presence of higher level of immune de- fense mechanism like adaptive immunity, which is a slower pro- cess, insects and other lower vertebrates like fish and amphibian depend upon innate immune system, a more faster one but less specific than adaptive immunity (Akira et al., 2006; Magnadottir, 2006). Innate immune system recognizes pathogens or conserved pathogen derived structures like flagellin, lipopolysaccharides (LPS), lipoproteins, peptidoglycan (PGN) and nucleic acid by germ-line-encoded pattern recognition receptors (PRRs) that are distributed in extracellular, membrane and cytoplasmic compart- ments. Three major classes of PRRs have been identified: (a) the Toll-like receptors (TLRs) that recognize ligand on either the extra- cellular surface or within the endosome, (b) the NOD-like receptors (NLRs) that function as cytoplasmic sensors, and (c) RIG I-like receptors (RLRs) class of cytoplasmic PRR that recognize viruses (Akira et al., 2006; Franchi et al., 2009; Chen et al., 2009). NLR is more recently identified member in the intracellular PRR family. This family of receptors is characterized with presence of three domains. At C-terminal, a leucin rich repeat (LRR) domain, at the center a nucleotide oligomerization (NACHT) domain, and a protein–protein interaction domain at the N-terminal end (Chen et al., 2009; Benko et al., 2008). The C-terminal LRR domain recog- nizes specific ligand to which the NLR is to be associated, the NACHT domain mediates self-regulation and oligomerization and the N-terminal domain generates downstream signals (Jin and Flavell, 2010). Based on various N-terminal domains, like pyrin do- main (PYD), caspase recruitment domain (CARD) or baculovirus inhibitor of apoptosis repeat domain (BIR), NLR is subdivided into three classes viz., NALP (NACHT, LRR and PYD containing proteins), NOD (NACHT, LRR and CARD containing proteins) and NAIP (for neuronal apoptosis inhibitor protein) (Akira et al., 2006; Wilman- ski et al., 2008; Opitz et al., 2009; Proell et al., 2008; Fritz et al., 2006). NOD subfamily consists of five members NOD1, NOD2, 0145-305X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.dci.2011.06.018 ⇑ Corresponding author. Tel.: +91 674 2465421; fax: +91 674 2465407. E-mail address: msamanta1969@yahoo.com (M. Samanta). Developmental and Comparative Immunology 36 (2012) 93–103 Contents lists available at SciVerse ScienceDirect Developmental and Comparative Immunology journal homepage: www.elsevier.com/locate/dci