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Title: Molecular cloning, characterization and expression analysis of TLR9, MyD88 and TRAF6 genes in common carp (Cyprinus carpio)

item KONGCHUM, PAWAPOL - Virginia Polytechnic Institution & State University
item DAVID, LIOR - Hebrew University Of Jerusalem
item HALLERMAN, ERIC - Virginia Polytechnic Institution & State University
item HULATA, GIDEON - Agricultural Research Organization Of Israel
item Palti, Yniv

Submitted to: Fish and Shellfish Immunology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/1/2010
Publication Date: 1/3/2011
Citation: Kongchum, P., David, L., Hallerman, E., Hulata, G., Palti, Y. 2011. Molecular cloning, characterization and expression analysis of TLR9, MyD88 and TRAF6 genes in common carp (Cyprinus carpio). Fish and Shellfish Immunology. 30: 361-371.

Interpretive Summary: Common carp is an economically important food fish that has the longest history of domestication and is cultured worldwide. Fancy carp or koi are members of a subgroup of this species and are an important component of the U.S. aquaculture industry. Over the past few years, common carp and koi production enterprises have suffered from enormous losses due to disease outbreaks caused by cyprinid herpes virus-3 (CyHV-3). Toll-like receptors (TLRs) are a family of transmembrane proteins that recognize conserved pathogen structures to induce innate immune effector molecules. In vertebrates, TLRs can also distinguish among classes of pathogens and serve an important role in orchestrating the appropriate adaptive immune responses. We have mapped and annotated TLR genes in common carp and koi and developed genetic markers for the different TLR genes to provide useful tools for genetic improvement of disease resistance in this economically important carp species.

Technical Abstract: Induction of innate immune pathways is critical for early host defense but there is limited understanding of how teleost fish recognize pathogen molecules and activate these pathways. In mammals, cells of the innate immune system detect pathogenic molecular structures using pattern recognition receptors (PRRs). TLR9 functions as PRR that recognizes CpG motifs in bacterial and viral DNA and requires adaptor molecules MyD88 and TRAF6 for signal transduction. A disease caused by the DNA virus cyprinid herpesvirus 3 (CyHV-3) is highly contagious and virulent in common carp (Cyprinus carpio). The ability of TLR9 to sense viral DNA and the important roles of MyD88 and TRAF6 in the TLR9 signaling pathway make them candidates for involvement in the host antiviral response to CyHV-3. Here we report the full-length cDNA isolation, structural characterization and tissue mRNA expression analysis of the common carp (cc) TLR9, MyD88 and TRAF6 gene orthologs. Sequence analyses revealed that MyD88 and TRAF6 genes are duplicated in common carp. To our knowledge this is the first report of MyD88 and TRAF6 gene duplication in vertebrates. The ccTLR9 open reading frame (ORF) is predicted to encode a 1,064 amino acid (aa) protein with 80% identity to the zebrafish TLR9. The ccMyD88a and b ORFs are predicted to encode 288 aa and 284 aa peptides, respectively. They share 91% of aa sequence identity between paralogues and 90% and 88% respective identity to the zebrafish MyD88. The ccTRAF6a and b ORFs are both predicted to encode a 543 aa peptide; sharing 95% aa sequence identity between paralogues and 88% identity to the zebrafish TRAF6. The ccTLR9 gene was contained in a single large exon. The ccMyD88a and ccMyD88b coding sequences spanned 5 short exons ranging from 91 to 304 bp. The TRAF6b gene spans 6 exons (302, 151, 159, 72, 78 and 870 bp). PCR amplification to obtain the entire coding sequence of ccTRAF6a from genomic DNA was not successful. The 2104 bp fragment amplified covered the 3' end of the gene and contains a partial sequence of one exon (37 bp) and 3 complete exons (72, 78 and 870 bp). The predicated protein domains of the common carp TLR9, MyD88 and TRAF6 are conserved and resemble orthologs from other vertebrates. Real-time quantitative PCR assays of the gene transcripts in healthy common carp indicated that mRNA expression varies between tissues. The expression of ccTLR9 was highest in the kidney while the ccMyD88a and b and ccTRAF6a and b levels of expression were highest in the liver and gills. Differential expressions occurred between the paralogous transcripts of ccMyD88 and ccTRAF6 in white and red muscles, suggesting that one isotype of each gene may have evolved and attained a new function. The genomic information we describe in this paper provide evidence of sequence and structural conservation of immune response genes in common carp and will be useful for identifying gene polymorphisms that may be associated with differential immune responses and susceptibility to CyHV-3 infection.