M.X. Chang

Molecular characterization and expression analysis of nuclear oligomerization domain proteins NOD1 and NOD2 in grass carp Ctenopharyngodon idella.

Nuclear oligomerization domains (NODs) are cytosolic pattern recognition receptors (PRRs) to detect bacterial component. In this study, the molecular cloning and genomic characterization of grass carp NOD1 (gcNOD1) and grass carp NOD2 (gcNOD2) were reported. The complete open reading frame of gcNOD1 contains 2814 bp, encoding a 937-amino acid polypeptide. The gcNOD2 cDNA sequence encodes 982-amino acid polypeptide. Both gcNOD1 and gcNOD2 possess three conserved domains: carboxy terminal leucine rich repeat (LRR) domains, a central NOD, NBS or NACHT domain, and an amino terminal CARD domain (two in the case of NOD2). At the genomic level, gcNOD1 consists of 11 exons, with 10 intervening introns, spanning approximately 9 kb of genomic sequence. Whereas gcNOD2 has a length of approximately 5 kb with 9 intervening introns. Real time PCR analysis showed gcNOD1 and gcNOD2 were ubiquitously expressed in adult tissues. The highest transcript level of gcNOD1 was detected in brain, but in head kidney for gcNOD2. Grass carp reovirus significantly induced the expression of gcNOD1 and gcNOD2 in spleen (from days 1 to 6). However, expression profiles differed in time course response. Induction experiments with lipopolysaccharide (LPS), peptidoglycan (PGN) and polyI:C revealed the differential expression and regulation of gcNOD1 and gcNOD2 in blood, head kidney, trunk kidney and spleen. All these data suggest a potential role of NOD1 and NOD2 in fish innate immune protection to bacterial and virus infections.

Molecular characterization and expression analysis of the IFN-gamma related gene (IFN-γrel) in grass carp Ctenopharyngodon idella

Interferon gamma (IFN-gamma), the only member of the type II class of interferons, has been identified in teleost fish. In addition to the IFN-gamma gene, fish possess an IFN-gamma related gene (IFN-gammarel) neighbouring the authentic IFN-gamma gene in the genome. In the present study, the cDNA sequence encoding 167 amino acids of IFN-gammarel and its genomic organization were identified in grass carp Ctenopharyngodon idella. The predicted protein sequence of grass carp IFN-gammarel (gcIFN-gammarel) showed 63% and 50% identities to zebrafish and common carp IFN-gammarel (previously termed as IFN-gamma1), respectively. The IFN-gammarel gene consists of 4 exons, with 3 intervening introns, spanning approximately 2kb of genomic sequence. The gcIFN-gammarel gene did not contain any polymorphic DNA repeats in the introns. Realtime PCR analysis showed that grass carp reovirus induced a high and long lasting (from days 1 to 7) expression of gcIFN-gammarel in spleen. The expression of gcIFN-gammarel in blood, head kidney, trunk kidney and spleen was also increased by bacterial peptidoglycan (PGN), lipopolysaccharide (LPS) and the interferon inducer polyI:C. The highest induction of gcIFN-gammarel expression by PGN was observed in spleen, then in blood and head kidney. Further analysis of the expression patterns of gcIFN-gammarel and PGN receptors, nucleotide oligomerization domains (NOD) 1 and 2, may suggest that IFN-gammarel was possibly activated in a NOD2-dependent mechanism.

Identification of immune genes in grass carp Ctenopharyngodon idella in response to infection of the parasitic copepod Sinergasilus major

The parasitic copepod Sinergasilus major is an important pathogen of grass carp Ctenopharyngodon idella. To understand the immune response of grass carp to the copepod infection, suppression subtractive hybridization method was employed to characterize genes up-regulation during the copepod infection in liver and gills of the fish. One hundred and twenty-two dot blot positive clones from infected subtracted library were sequenced. Searching available databases by using these nucleotide sequences revealed that 23 genes are immune-related, including known acute-phase reactants, and four novel genes encoding proteins such as source of immunodominant MHC-associated peptides (SIMP), TNF receptor-associated factor 2 binding protein (T2BP), poliovirus receptor-related protein 1 precursor, glycoprotein A repetitions predominant (GARP). The differential expression of seven immune genes, i.e. GARP, alpha-2-macroglobulin, MHC class I, C3, SIMP, T2BP, transferrin, as a result of infection was further confirmed by RT-PCR, with the up-regulation of alpha-2-macroglobulin, MHC class I, C3, SIMP and T2BP in the liver of infected fish, and down-regulation of SIMP in the gills of infected fish. The present study provides foundation for understanding grass carp immune response and candidate genes for further analysis.

RNAi suppression of zebrafish peptidoglycan recognition protein 6 (zfPGRP6) mediated differentially expressed genes involved in Toll-like receptor signaling pathway and caused increased susceptibility to Flavobacterium columnare

In Drosophila, Toll signaling cascade, which resembles the mammalian Toll-like receptor (TLR)/IL-1R signaling pathways and regulates the expression of anti-microbial peptide genes, mainly relies on peptidoglycan recognition proteins (PGRPs) for the detection of bacterial pathogens. To explore the effect of zebrafish peptidoglycan recognition protein 6 (zfPGRP6) on Toll-like receptor signaling pathway, RNA interference (siRNA) and real time quantitative PCR (RQ-PCR) methods were used to identify differentially expressed genes regulated by zfPGRP6. The target genes included TLR2, TLR3, TLR5, TLR7, TLR8, IL1R, Sterile-alpha and Armadillo motif containing protein (SARM), myeloid differentiation factor 88 (MyD88) and nuclear factor (NF)-kappa B2 (p100/p52). The results of RQ-PCR showed that RNAi-mediated suppression of zfPGRP6 significantly down-regulated the expression of TLR2, TLR5, IL1R, SARM, MyD88 and p100/p52. The expression of beta-defensin-1 was also down-regulated in those embryos silenced by zfPGRP6. In challenge experiments to determine the anti-bacterial response to Gram-negative bacteria, RNAi knock-down of zfPGRP6 markedly increased susceptibility to Flavobacterium columnare.

Ig heavy chain genes and their locus in grass carp Ctenopharyngodon idella.

The cDNA and genomic sequences of IgD and IgZ were characterized in grass carp Ctenopharyngodon idella in the present study, and with the identification of a BAC clone covering zeta, mu, and delta genes, the IgH locus containing these Ig genes and other V, D, J genes was also illustrated in this fish. Secretory and membrane-bound IgZ were identified, with two transmembrane exons spliced within the CH4 exon, as reported in IgM of mammals and IgZ in other teleost fish. The first and second constant domains of IgZ shows more than 90% nucleotide identity with respective domains of grass carp IgM. The IgD has a structure of delta1-(delta2-delta3-delta4)(2)-delta5-delta6-delta7-TM-UTR, with the repeat of delta2-delta3-delta4; but intron was not found between the two repeat, i.e. between the first delta2-delta3-delta4 (delta2.1-delta3.1-delta4.1) and the second delta2-delta3-delta4 (delta2.2-delta3.2-delta4.2), and the intron between delta3.1 and delta4.1 was much shorter than the intron between delta3.2 and delta4.2. The genomic organization of the IgH locus has a pattern of Vn-Dn-Jn-Czeta-Dn-Jn-Cmu-Cdelta, as reported in other teleost fish. Thirteen V(H), fourteen D, and twelve J(H) genes were observed in this locus, with the similarity of three D segments and four J(H) segments being the same in the upstream of Czeta and Cmu. The transcriptional enhancer located at the mu-delta intergenic region was also analyzed and it seems possible that this enhancer is functional as verified in zebrafish and channel catfish.

Characterization of C-C chemokine receptor subfamily in teleost fish.

Chemokines and their receptors play important roles in nervous and immune systems. Little information, however, exists concerning this gene family in teleost fish. In the present study, 17 C-C chemokine receptors genes were identified from Danio rerio, 9 from Gasterosteus aculeatus, 10 from Oryzias latipes, 8 from Takifugu rubripes and 5 from Tetraodon nigroviridis. Phylogenetic analysis showed that the orthologs to mammalian CCR6, 7, 8, 9 and CCRL1 receptors were evident in zebrafish, but the clear orthologs to mammalian CCR1, 2, 3, 4, 5 and 10 were not found in zebrafish. The gene structure of zebrafish CCR (zfCCR) was further analyzed. The open reading frame of zfCCR3-1, zfCCR3-3, zfCCR6-1, zfCCR6-2, zfCCR8-2 contain one exon, and two exons were identified for zfCCR2-1, zfCCR2-2, zfCCR4 and zfCCRL1-1, three exons for zfCCR3-2, zfCCR5 and zfCCR7, four exons for zfCCR8-1 and zfCCR9-1. The expression analyses showed that in zebrafish, most C-C chemokine receptor genes were expressed in fertilized eggs and oocytes, and all the receptor genes were expressed in larval stages. The zfCCR2-2, zfCCR3-1, zfCCR4 and zfCCR6-2 genes were expressed in all normal organs examined, whereas not for zfCCR2-1, zfCCR3-3, zfCCR6-1, zfCCR8-1, zfCCR9-2 and zfCCRL1-2. The expression of zfCCR3-2, zfCCR5, zfCCR7, zfCCR9-1 and zfCCRL1-1 were detected in the majority organs, and zfCCR8-2 and zfCCR8-3 detected only in brain. The differential expression pattern of different paralogues in organs may indicate their difference in function, which requires further investigation.

The gene and virus-induced expression of IRF-5 in grass carp Ctenopharyngodon idella.

The interferon regulatory factor 5 (IRF-5) is known to be involved in the innate immune response and in the regulation of DNA damage-induced apoptosis. In the present study, the cDNA and genomic sequences of IRF-5 were identified in grass carp (Ctenopharyngodon idella). The cDNA of grass carp IRF-5 (gcIRF-5) contains an open reading frame (ORF) of 1560 nucleotides, encoding a putative 519 amino acid protein, which showed 34.5-83.9% identity to IRF-5 homologues from mammals, amphibian, avian and fish, and 96.2% and 95.0% identity to zebrafish IRF-5 in the DNA-binding domain (DBD) and IRF association domain (IAD), respectively. The genomic DNA sequence of gcIRF-5 contains 6075bp consisting of 9 exons and 8 introns. The expression of gcIRF-5 was observed in all organs examined. The analysis of real-time quantitative RT-PCR revealed that grass carp reovirus (GCRV) induced the expression of gcIRF-5 in spleen and head kidney.

Structure and expression pattern of teleost caspase recruitment domain (CARD) containing proteins that are potentially involved in NF-κB signalling

Caspase-associated recruitment domain (CARD) proteins play critical roles in regulation of caspase activation, or regulation of NF-kappaB activation. In the present study, 9 CARD-genes, which may be involved in NF-kappaB activation, were identified in teleost fish. Phylogenetic analysis showed that the orthologs to mammalian RIPK2, NOD1, PYCARD, CARD9, CARD10, CARD11, CARD14, NOD2 and BCL10 were evident in teleost fish, but clear orthologs to mammalian CARD6 and CARD8 were not found in teleost fish. In zebrafish, most CARD-genes were expressed in embryos and larvae. In adult zebrafish, zfRIPK2, zfNOD1, zfPYCARD, zfCARD9 and zfNOD2 transcripts were constitutively expressed in kidney, gill, intestine, liver, brain and spleen whilst zfCARD11, zfCARD14 and BCL10 exhibited limited tissue distribution. A CARD-related gene (zfCARD-rel), containing a single CARD domain, was identified in the zebrafish genome and the EST databases and its transcripts were detected only in spleen and kidney. Phylogenetic analysis suggested that zfCARD-rel might be the ortholog of mammalian CARD8 or the short isoform of NLRP1. Overexpression of zfCARD-rel had a significantly inhibitory effect on NF-kappaB activity, demonstrating the zfCARD-rel protein might serve as a negative regulator of cell death and inflammatory response.

Zebrafish peptidoglycan recognition protein SC (zfPGRP-SC) mediates multiple intracellular signaling pathways

Insect PGRPs can function as bacterial recognition molecules triggering proteolytic and/or signal transduction pathways, with the resultant production of antimicrobial peptides. To explore if zebrafish peptidoglycan recognition protein SC (zfPGRP-SC) has such effects, RNA interference (siRNA) and high-density oligonucleotide microarray analysis were used to identify differentially expressed genes regulated by zfPGRP-SC. The mRNA levels for a set of genes involved in Toll-like receptor signaling pathway, such as TLRs, SARM, MyD88, TRAF6 and nuclear factor (NF)-kappa B2 (p100/p52), were examined by quantitative RT-PCR (QT-PCR). The results from the arrays and QT-PCR showed that the expression of 133 genes was involved in signal transduction pathways, which included Toll-like receptor signaling, Wnt signaling, BMP signaling, insulin receptor signaling, TGF-beta signaling, GPCR signaling, small GTPase signaling, second-messenger-mediated signaling, MAPK signaling, JAK/STAT signaling, apoptosis and anti-apoptosis signaling and other signaling cascades. These signaling pathways may connect with each other to form a complex network to regulate not just immune responses but also other processes such as development and apoptosis. When transiently over-expressed in HEK293T cells, zfPGRP-SC inhibited NF-kappaB activity with and without lipopolysacharide (LPS) stimulation.

Three goose-type lysozymes in the gastropod Oncomelania hupensis: cDNA sequences and lytic activity of recombinant proteins

Three goose-type (g-type) lysozymes, designated as OHLysG1, OHLysG2 and OHLysG3 were identified from expressed sequence tags (ESTs) of a gastropod Oncomelania hupensis, the intermediate host of Schistosoma japonicum. The full cDNA sequences of OHLysG1, OHLysG2 and OHLysG3 consisted of 735, 909 and 808 nucleotides, with an open reading frame of 198, 214 and 249 codons containing a 21, 7 and 8 amino acid (aa) signal peptide at the N-terminus, respectively. The three g-type lysozymes shared conserved features with other g-type lysozymes, such as the substrate binding sites, the catalytic residues critical for the fundamental structure and function of g-type lysozymes. It seems possible that g-type lysozymes in molluscs shared one conserved cysteine with those in birds and mammals, and six conserved cysteines were observed for mollusc g-type lysozymes, with two unique cysteines present in the g-type lysozymes of O. hupensis. The three lysozyme genes were expressed mainly in hepatopancreas, with relatively low expression level observed in head-foot muscle and intestine. When comparing S. japonicum-infected and uninfected snails, significant increase (P<0.05) was observed for all the three lysozymes in infected snails, with the highest increase detected in hepatopancreas, and lowest in intestine, implying their defensive role in the host-parasite, i.e. snail-trematode system. The three recombinant lysozymes expressed in Escherichia coli strain M15 showed lytic activity against Aeromonas hydrophila, Vibrio fluvialis, Aeromonas sobria and Micrococcus lysodeikticus. In conclusion, the finding of three g-type lysozymes in O. hupensis provides structural and functional evidence of multiple g-type lysozymes in gastropod, which may have evolutional implication in the snail-trematode system.