Genomic organization, gene duplication, and expression analysis of interleukin-1beta in channel catfish (Ictalurus punctatus)

Interleukin-1beta (IL-1beta) is one of the pivotal early response pro-inflammatory cytokines that enables organisms to respond to infection and induces a cascade of reactions leading to inflammation. In spite of its importance and two decades of studies in the mammalian species, genes encoding IL-1beta were not identified from non-mammalian species until recently. Recent research, particularly with genomic approaches, has led to sequencing of IL-1beta from many species. Clinical studies also suggested IL-1beta as an immunoregulatory molecule potentially useful for enhancing vaccination. However, no IL-1beta genes have been identified from channel catfish, the primary aquaculture species from the United States. In this study, we identified two distinct cDNAs encoding catfish IL-1beta. Their encoding genes were identified, sequenced, and characterized. The catfish IL-1beta genes were assigned to bacterial artificial chromosome (BAC) clones. Genomic studies indicated that the IL-1beta genes were tandemly duplicated on the same chromosome. Phylogenetic analysis of various IL-1beta genes indicated the possibility of recent species-specific gene duplications in channel catfish, and perhaps also in swine and carp. Expression analysis indicated that both IL-1beta genes were expressed, but exhibited distinct expression profiles in various catfish tissues, and after bacterial infection with Edwardsiella ictaluri.     

Catfish hepcidin gene is expressed in a wide range of tissues and exhibits tissue-specific upregulation after bacterial infection

Antimicrobial peptides (AMPs) are important components of the host innate immune response against microbial invasion. The cysteine-rich AMPs such as defensin and hepcidin have been extensively studied from various organisms, but their role in disease defense in catfish is unknown. As a first step, we sequenced a hepcidin cDNA from both channel catfish and blue catfish, and characterized the channel catfish hepcidin gene. The channel catfish hepcidin gene consists of two introns and three exons that encode a peptide of 96 amino acids. The amino acid sequences and gene organization were conserved between catfish and other organisms. In contrast to its almost exclusive expression in the liver in humans, the channel catfish hepcidin gene was expressed in a wide range of tissues except brain. Its expression was detected early during embryonic and larval development, and induced after bacterial infection with Edwardsiella ictaluri, the causative agent of enteric septicemia of catfish (ESC) in a tissue-specific manner. The upregulation was observed in the spleen and head kidney, but not in the liver. The expression of hepcidin was upregulated 1--3 days after challenge, but returned to normal levels at 7 days after challenge. The expression profile of the catfish hepcidin gene during the course of bacterial infection mirrors those of inflammatory proteins such as chemokines, suggesting an important role for hepcidin during inflammatory responses.     

Molecular identification and expression analysis of tumor necrosis factor in channel catfish (Ictalurus punctatus)

A tumor necrosis factor (TNF) -like gene, encoding a propeptide of 230 amino acids and a mature (soluble) peptide of 162 amino acids, was identified in channel catfish (Ictalurus punctatus). While the catfish protein shared features in common with both mammalian TNF and TNFβ homologs, overall sequence identity/similarity was slightly higher vs. TNF genes when mature TNF sequences were compared. Phylogenetic analysis placed catfish and other fish TNF sequences within their own cluster apart from mammalian TNF and β genes, and supported the suggestion that TNF and β genes separated after the divergence of mammals and teleosts. In contrast to trout and carp, but similar to flounder, catfish TNF was present as a single copy gene. Expression studies demonstrated that catfish TNF mRNA was present in all tested tissues (i.e. liver, spleen, head kidney, mesonephros, gill, thymus, and PBLs) from an unstimulated fish. Moreover, catfish TNF was constitutively expressed in actively proliferating, but otherwise unstimulated, macrophage (42TA) and T cell (G14D; TS32.17) lines, but not in B cell (1G8 or 3B11) or fibroblast lines. TNF expression was upregulated in PBLs, and in G14D and 42TA cells, but not in 3B11 cells, by PMA/calcium ionophore treatment. These results demonstrate that a catfish homolog of TNF has been identified, and indicate that catfish TNF is expressed in catfish in a manner similar to that seen in mammals.     

Organization and expression of thirteen alternatively spliced exons in catfish CD45 homologs

CD45, also known as LCA, is a transmembrane protein tyrosine phosphatase encoded by the PTPRC gene. In mammals, it plays an important role in T and B cell receptor and cytokine signaling by maintaining receptor associated kinases in an active state. A prominent CD45 feature is alternative splicing of exons encoding the N-terminus, resulting in the generation of several isoforms. The expression of isoforms is tightly regulated and dependent on the developmental/activation state of the lymphocyte. Nevertheless, the significance of these multiple isoforms in mammals is poorly understood. In this study, the channel catfish CD45 homolog was sequenced and found to be similar to CD45 of other species. However, unlike mammalian CD45, it appears that up to 13 exons are used in producing multiple alternatively spliced CD45 variants in catfish cells. These 13 alternatively spliced exons variably encode for O-linked glycosylation sites. Several of the exons are identical or very similar, suggesting gene duplication of a block of four exons. As demonstrated by RT-PCR, many of the alternatively spliced forms of catfish CD45 are differentially expressed in lymphoid cell lines with B cells expressing larger isoforms than do T cells. Furthermore, immunoprecipitation experiments utilizing anti-catfish CD45 mAbs substantiated that different size CD45 isoforms are expressed at the protein level on catfish T and B cells.     

Constitutive expression of three novel catfish CXC chemokines: homeostatic chemokines in teleost fish

Chemokines are best known for their vital role in leukocyte chemotaxis, as part of the larger inflammatory response. Expression analysis and functional characterization of chemokines in mammalian species have often overlooked the role of these proteins under homeostatic conditions. Recent investigations of chemokine diversity in teleost fish have also centered on the immune-related functions of chemotactic cytokines, such as CXCL8 and CXCL10. While a disease-based approach to chemokines is essential to the development of remediative therapies for both human and animal infections, it may be a poor measure of the overall complexity of chemokine functions. As part of a larger effort to assess the conservation of chemokine diversity in teleost fish, we report here the identification of three novel, constitutively expressed CXC chemokines from channel catfish (Ictalurus punctatus). Phylogenetic analyses indicated that two of the three CXC chemokines were orthologues for mammalian CXCL12 and CXCL14, respectively. Whereas a clear orthology could not yet be established for the third CXC chemokine, it shared highest amino acid identity with mammalian CXCL2. All three CXC chemokines show expression in a wide range of tissues, and early expression during development was observed for CXCL12. The expression of this new set of catfish CXC chemokines was not induced during challenge by infection of Edwardsiella ictaluri, the causative agent of the fish pathogen enteric septicemia of catfish. In contrast to the gene duplication of CXCL12 in carp and zebrafish, Southern blot analysis indicated that all three catfish CXC chemokines exist as single copy genes in the catfish genome suggesting that gene duplication of CXC chemokines in specific teleost fish was a recent evolutionary event.     

The CC and CXC chemokine receptors in channel catfish (Ictalurus punctatus) and their involvement in disease and hypoxia responses

Chemokines are vital regulators of cell mobilization for immune surveillance, inflammation, and development. Chemokines signal through binding to their receptors that are a superfamily of seven-transmembrane domain G-coupled receptors. Recently, a complete repertoire of both CC and CXC chemokines have been identified in channel catfish, but nothing is known about their receptors. In this study, a set of 29 CC chemokine receptor (CCR) genes and 8 CXC chemokine receptor (CXCR) genes were identified and annotated from the channel catfish genome. Extensive phylogenetic and comparative genomic analyses were conducted to annotate these genes, revealing fish-specific CC chemokine receptors, and lineage-specific tandem duplications of chemokine receptors in the teleost genomes. With 29 genes, the channel catfish genome harbors the largest numbers of CC chemokine receptors among all the genomes characterized. Analysis of gene expression after bacterial infections indicated that the chemokine receptors were regulated in a gene-specific manner. Most differentially expressed chemokine receptors were up-regulated after Edwardsiella ictaluri and Flavobacterium columnare infection. Among which, CXCR3 and CXCR4 were observed to participate in immune responses to both bacterial infections, indicating their potential roles in catfish immune activities. In addition, CXCR3.2 was significantly up-regulated in ESC-susceptible fish, and CXCR4b was mildly induced in ESC-resistant fish, further supporting the significant roles of CXCR3 and CXCR4 in catfish immune responses. CXCR4b and CCR9a were both up-regulated not only after bacterial infection, but also after hypoxia stress, providing the linkage between bacterial infection and low oxygen stresses. These results should be valuable for comparative immunological studies and provide insights into their roles in disease and stress responses.     

Identification of a beta 2 (CD18) molecule in a teleost species, Ictalurus punctatus Rafinesque

Beta 2, in combination with the alpha subunit, is responsible for tight adhesion of leukocytes, especially neutrophils and macrophages, in areas of inflammation. Although identified in mammalian and avian species; the beta 2 or CD18 molecule has yet to be identified in fish. The present investigation has identified a full-length channel catfish, Ictalurus punctatus, cDNA beta 2 molecule composed of 2.8 kb and a deduced amino acid sequence of 772 amino acids. The catfish molecule has an amino acid homology ranging from 54 to 63% with mouse, bovine, rabbit, human and chicken. The channel catfish molecule retains several characteristics of mammalian beta 2 molecules, such as cysteine-rich repeat regions, N-linked glycosylation sites, and several proposed signal sequences. Expression of the beta 2 molecule on the catfish neutrophil cytoplasmic membranes is increased upon phorbol dibutyrate stimulation of the cells. Based on Western blotting and the immunoprecipitation test, the channel catfish beta 2 molecule has a molecular mass of approximately 95 kD, essentially the same as that for mammalian species. However, two additional molecules, perhaps chains, of unexpected molecular mass appear to co-precipitate in the SPIT with the 95 kD CD18 molecule. These results confirm the existence and expression of a beta 2 gene in channel catfish, a species phylogenetically distant from mammalian species.     

Characterization of the molecular chaperone calnexin in the channel catfish, Ictalurus punctatus, and its association with MHC class II molecules

Folding and assembly of MHC molecules in mammals occurs in the endoplasmic reticulum (ER), but has not been studied in teleosts. Calnexin (CNX) is an ER chaperone that associates with glycoproteins bearing a monoglucosylated N-linked oligosaccharide side chain. Here we report the first identification and characterization of a full-length CNX cDNA clone in a teleost, and the association of the CNX chaperone with MHC class II in a channel catfish T cell line. The 1.8 kb CNX clone encodes a protein of 607 amino acids that is 72% identical to the consensus sequence of mammalian CNXs. The association of CNX with class II is of particular interest because the native MHC class II alpha chain of Ictalurus punctatus does not bear any N-linked oligosaccharide consensus glycosylation sequences. Thus the assembly of class II molecules in the catfish probably proceeds via different steps than occurs in mammals.     

The two channel catfish intelectin genes exhibit highly differential patterns of tissue expression and regulation after infection with Edwardsiella ictaluri

Intelectins (IntL) are Ca(2+)-dependent secretory glycoproteins that play a role in the innate immune response. The mammalian IntL is also known as lactoferrin receptor (LfR) that is involved in iron metabolism. The objective of this study was to characterize the intelectin genes in both channel catfish and blue catfish, to determine their genomic organization and copy numbers, to determine their patterns of tissue expression, and to establish if they are involved in defense responses of catfish after bacterial infection. Two types of IntL genes have been identified from catfish, and IntL2 was completely sequenced. The genomic structure and organization of IntL2 were similar to those of the mammalian species and of zebrafish and grass carp, but orthologies cannot be established with mammalian IntL genes. The IntL genes are highly conserved through evolution. Sequence analysis also indicated the presence of the fibrinogen-related domain in the catfish IntL genes, suggesting their structural conservations. Phylogenetic analysis suggested the presence of at least two prototypes of IntL genes in teleosts, but only one in mammals. The catfish IntL genes exhibited drastically different patterns of expression as compared to those of the mammalian species, or even with the grass carp gene. The catfish IntL1 gene is widely expressed in various tissues, whereas the channel catfish IntL2 gene was mainly expressed in the liver. While the catfish IntL1 is constitutively expressed, the catfish IntL2 was drastically induced by intraperitoneal injection of Edwardsiella ictaluri and/or iron dextran. Such induction was most dramatic when the fish were treated with both the bacteria and iron dextran. While IntL1 was expressed in all leukocyte cell lines, no expression of IntL2 was detected in any of the leukocyte cell lines, suggesting that the up-regulated channel catfish IntL2 expression after bacterial infection may be a consequence of the initial immune response, and/or a downstream immune response rather than a part of the primary immune responses.     

Genomic sequences comparison and differential expression of miiuy croaker MHC class I gene, after infection by Vibrio anguillarum

Major histocompatibility complex (MHC) has a central role in the adaptive immune system by presenting foreign peptide to the T-cell receptor. MHC gene family contains two main subgroups of immunologically active molecules. In order to study the molecular function and genomic characteristic of class I gene in teleost, the full lengths of MHC class Iα cDNA and genomic sequence were cloned from miiuy croaker (Miichthys miiuy). Seven exons and six introns were identified in miiuy croaker class Iα gene. This genomic structural feature of miiuy croaker is similar to that present in some fishes such as Japanese flounder and Atlantic salmon, but different from that present in some other fishes such as half-smooth tongue sole and channel catfish. The deduced amino acid sequence of class Iα gene had 25.9-54.1% identity with those of mammal and teleost. Real-time quantitative RT-PCR demonstrated that the MHC class Iα gene was ubiquitously expressed in 10 normal tissues; expression levels of MHC Iα gene were found first upregulated and then downregulated throughout the pathogenic bacteria infection process in spleen and kidney.     

The chemokinome superfamily: II. The 64 CC chemokines in channel catfish and their involvement in disease and hypoxia responses

Chemokines are a superfamily of structurally related chemotactic cytokines exerting significant roles in regulating cell migration and activation. Based on the arrangement of the first four cysteine residues, they are classified into CC, CXC, C and CX3C subfamilies. In this study, a complete set of 64 CC chemokine ligand (CCL) genes was systematically identified, annotated, and characterized from the channel catfish genome. Extensive phylogenetic and comparative genomic analyses supported their annotations, allowing establishment of their orthologies, revealing fish-specific CC chemokines and the expansion of CC chemokines in the teleost genomes through lineage-specific tandem duplications. With 64 genes, the channel catfish genome harbors the largest numbers of CC chemokines among all the genomes characterized to date, however, they fall into 11 distinct CC chemokine groups. Analysis of gene expression after bacterial infections indicated that the CC chemokines were regulated in a gene-specific and time-dependent manner. While only one member of CCL19 (CCL19a.1) was significantly up-regulated after Edwardsiella ictaluri infection, all CCL19 members (CCL19a.1, CCL19a.2 and CCL19b) were significantly induced after Flavobacterium columnare infection. In addition, CCL19a.1, CCL19a.2 and CCL19b were also drastically up-regulated in ESC-susceptible fish, but not in resistant fish, suggesting potential significant roles of CCL19 in catfish immune responses. High expression levels of certain CC appeared to be correlated with susceptibility to diseases and intolerance to hypoxia.