Characterization of three Nod-like receptors and their role in antimicrobial responses of goldfish (Carassius auratus L.) macrophages to Aeromonas salmonicida and Mycobacterium marinum Jiasong Xie a , Jordan W. Hodgkinson a , Barbara A. Katzenback a , Nikolina Kovacevic a , Miodrag Belosevic a,b,⇑ a Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada b School of Public Health, University of Alberta, Edmonton, Alberta, Canada article info Article history: Received 18 October 2012 Revised 14 November 2012 Accepted 15 November 2012 Available online 27 November 2012 Keywords: Aeromonas Mycobacterium Macrophage Fish Nod receptors Nod1 Nod2 Nlrx1 NLRs Gene expression Antimicrobial-response abstract The nucleotide-binding oligomerization domain proteins Nod1, Nod2 and Nlrx1 are cytoplasmic patho- gen recognition receptors (PRRs) of the Nod-like receptor (NLR) family. In this report, goldfish Nod1 (gfNod1), Nod2 (gfNod2) and Nlrx1 (gfNlrx1) genes were cloned and characterized. The full length of gfNod1, gfNod2 and gfNlrx1 were 3234 bp, 3129 bp and 4900 bp, encoding 937, 982 and 1008 amino acids, respectively. The three Nod-like receptors have a NACHT domain and C-terminal leucine rich repeat (LRR) domains. In addition to these, gfNod1 and gfNod2 also had an N-terminal CARD domain (two in gfNod2). Phylogenetic analysis showed that the three NLRs are highly conserved. Quantitative gene expression analysis of the three receptors revealed the greatest mRNA levels in the spleen, and in isolated neutrophils and splenocytes. Furthermore, treatment of goldfish macrophages with LPS, Poly I:C, MDP, PGN, heat-killed Aeromonas salmonicida or Mycobacterium marinum differentially altered the expression of the Nod-like receptors. Our results indicate that Nod-like receptors are functionally highly conserved and that they play a pivotal role in recognition of fish pathogens such as A. salmonicida and M. marinum. Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction The conserved microbial structures known as pathogen-associ- ated molecular patterns (PAMPs) are recognized by germ line- encoded innate immune receptors (PRRs), which exist as four ma- jor classes, including the Toll-like receptors (TLRs), the Nod-like receptors (NLRs), the retinoid acid-inducible gene-1 (RIG-1)-like receptors (RLRs) and the C-type lectin receptors (CLRs). PAMPs include lipopolysaccharides (LPS), polyinosinic: polycytidylin acid (Poly: IC), lipopeptide, peptidoglycan (PGN), flagellin, dsRNA, ssRNA and CpG DNA, among others. Recognition of these PAMPs by PRRs triggers the activation of signaling pathways including nu- clear factor-jB (NF-jB), mitogen-activated protein kinases (MAP- Ks) and type I interferons (IFN), which promote inflammatory and antimicrobial responses. The TLRs are the best known group of the innate immune recep- tors whose function has been reasonably well characterized in dif- ferent infectious diseases. TLRs play a fundamental role in innate immune responses by sensing the molecular signatures of micro- bial pathogens that recognize structural components shared by many bacteria, viruses and fungi (Takeda et al., 2003). Structurally, TLRs are type I membrane proteins characterized by an ectodomain composed of leucine rich repeats (LRR) that are responsible for rec- ognition of PAMPs, and a cytoplasmic domain known as the TIR do- main homologous to the cytoplasmic region of the IL-1 receptor, which is required for the activation of defense responses against an invading organism (Uematsu and Akira, 2007). To date, 10 members of TLRs have been identified in humans, and 13 in mice. A number of studies have identified their respective ligands that include LPS (TLR4), lipoproteins (TLR2), flagellin (TLR5), non-meth- ylated CpG motifs of DNA (TLR9), double-stranded RNA (TLR3), and single-stranded RNA (TLR7 and TLR8) (Akira et al., 2001; Takeda and Akira, 2005). After recognition of microbial pathogens, TLRs trigger intracellular signaling pathways that result in the induction of type I interferons (IFN) and chemokines, as well as other signal- ing pathways that play a role in the generation of antimicrobial 0145-305X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.dci.2012.11.005 ⇑ Corresponding author. Address: CW-405 Biological Sciences, University of Alberta, Edmonton, Canada AB T6G 2E9. Tel.: +1 780 492 1266; fax: +1 780 492 9234. E-mail address: mike.belosevic@ualberta.ca (M. Belosevic). Developmental and Comparative Immunology 39 (2013) 180–187 Contents lists available at SciVerse ScienceDirect Developmental and Comparative Immunology journal homepage: www.elsevier.com/locate/dci