Molecular cloning, promoter analysis and induced expression of the complement component C9 gene in the grass carp Ctenopharyngodon idella

Complement-mediated killing of pathogens through lytic pathway is an important effector mechanism of innate immune response. C9 is the ninth member of complement components, creating the membrane attack complex (MAC). In the present study, a putative cDNA sequence encoding the 650 amino acids of C9 and its genomic organization were identified in grass carp Ctenopharyngodon idella. The deduced amino acid sequence of grass carp C9 (gcC9) showed 48% and 38.5% identity to Japanese flounder and human C9, respectively. Domain search revealed that gcC9 contains a LDL receptor domain, an EGF precursor domain, a MACPF domain and two TSP domain located in the N-terminal and C-terminal, respectively. Phylogenetic analysis demonstrated that gcC9 is clustered in a same clade with Japanese flounder, pufferfish and rainbow trout C9. The gcC9 gene consists of 11 exons with 10 introns, spacing over approximately 7 kb of genomic sequence. Analysis of gcC9 promoter region revealed the presence of a TATA box and some putative transcription factor such as C/EBP, HSF, NF-AT, CHOP-C, HNF-3B, GATA-2, IK-2, EVI- 1, AP-1, CP2 and OCT-1 binding sites. The first intron region contains C/EBPb, HFH-1 and Oct-1 binding sites. RT-PCR and Western blotting analysis demonstrated that the mRNA and protein of gcC9 gene have similar expression patterns, being constitutively expressed in all organs examined of healthy fish, with the highest level in hepatopancreas. By real-time quantitative RT-PCR analysis, gcC9 transcripts were significantly up-regulated in head kidney, spleen, hepatopancreas and down-regulated in intestine from inactivated fish bacterial pathogen Flavobacterium columnare-stimulated fish, demonstrating the role of C9 in immune response. (c) 2007 Elsevier B.V. All rights reserved.

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

Interleukin-1 beta (IL-1 beta) 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-1 beta were not identified from non-mammalian species until recently. Recent research, particularly with genomic approaches, has led to sequencing of IL-1 beta from many species. Clinical studies also Suggested IL-1 beta as an immunoreagulatory molecule potentially useful for enhancing vaccination. However, no IL-1 beta 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-1 beta. Their encoding genes were identified, sequenced, and characterized. The catfish IL-1 beta genes were assigned to bacterial artificial chromosome (BAC) clones. Genomic studies indicated that the IL-1 beta genes were tandemly duplicated on the same chromosome. Phylogenetic analysis of various IL-1 beta 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-1 beta genes were expressed, but exhibited distinct expression profiles in various catfish tissues, and after bacterial infection with Edwardsiella ictaluri. (c) 2005 Elsevier Ltd. All rights reserved.

Cloning of biologically active genomes from a Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus isolate by using a bacterial artificial chromosome

Purification of genotypes from baculovirus isolates provides understanding of the diversity of baculoviruses and may lead to the development of better pesticides. Here, we report the cloning of different genotypes from an isolate of Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HaSNPV) by using a bacterial artificial chromosome (BAC). A transfer vector (pHZB10) was constructed which contained an Escherichia coli mini-F replicon cassette within the upstream and downstream arms of HaSNPV polyhedrin gene. Hz2e5 cells were co-transfected with wild-type HaSNPV DNA and pHZB10 to generate recombinant viruses by homologous recombination. The DNA of budded viruses (BVs) was used to transform E. coli. One of the bacmid colonies, HaBacHZ8, has restriction enzyme digestion profiles similar to an in vivo cloned strain HaSNPV-G4, the genome of which has been completely sequenced. For testing the oral infectivity, the polyhedrin gene of HaSNPV was reintroduced into HaBacHZ8 to generate the recombinant bacmid HaBacDF6. The results of one-step growth curves, electron microscopic examination, protein expression analysis and bioassays indicated that HaBacDF6 replicated as well as HaSNPV-G4 in vitro and in vivo. The biologically functional HaSNPV bacmids obtained in this research will facilitate future studies on the function genomics and genetic modification of HaSNPV. (C) 2003 Elsevier B.V. All rights reserved.

Regulation of autoinducer 2 production and luxS expression in a pathogenic Edwardsiella tarda strain

Edwardsiella tarda is a bacterial pathogen that can infect both humans and animals. TX1, an Ed. tarda strain isolated from diseased fish, was found to produce autoinducer 2 (Al-2)-like activity that was growth phase dependent and modulated by growth conditions. The gene coding for the Al-2 synthase was cloned from TX1 and designated luxS(Et). LuxS(Et) was able to complement the Al-2 mutant phenotype of Escherichia coli strain DH5 alpha. Expression Of luxS(Et) correlated with Al-2 activity and was increased by glucose and decreased by elevated temperature. The effect of glucose was shown to be mediated through the cAMP-CRP complex, which repressed luxS(Et) expression. Overexpression of luxS(Et) enhanced Al-2 activity in TX1, whereas disruption of luxS(Et) expression by antisense RNA interference (i) reduced the level of Al-2 activity, (ii) impaired bacterial growth under various conditions, (iii) weakened the expression of genes associated with the type III secretion system and biofilm formation, and (iv) attenuated bacterial virulence. Addition of exogenous Al-2 was able to complement the deficiencies in the expression of TTSS genes and biofilm production but failed to rescue the growth defects. Our results (i) demonstrated that the Al-2 activity in TX1 is controlled at least in part at the level of luxS(Et) expression, which in turn is regulated by growth conditions, and that the temporal expression of luxS(Et) is essential for optimal bacterial infection and survival; and (ii) suggested the existence in Ed. tarda of a LuxS/Al-2-mediated signal transduction pathway that regulates the production of virulence-associated elements.

Regulation of autoinducer 2 production and luxS expression in a pathogenic Edwardsiella tarda strain

Edwardsiella tarda is a bacterial pathogen that can infect both humans and animals. TX1, an Ed. tarda strain isolated from diseased fish, was found to produce autoinducer 2 (Al-2)-like activity that was growth phase dependent and modulated by growth conditions. The gene coding for the Al-2 synthase was cloned from TX1 and designated luxS(Et). LuxS(Et) was able to complement the Al-2 mutant phenotype of Escherichia coli strain DH5 alpha. Expression Of luxS(Et) correlated with Al-2 activity and was increased by glucose and decreased by elevated temperature. The effect of glucose was shown to be mediated through the cAMP-CRP complex, which repressed luxS(Et) expression. Overexpression of luxS(Et) enhanced Al-2 activity in TX1, whereas disruption of luxS(Et) expression by antisense RNA interference (i) reduced the level of Al-2 activity, (ii) impaired bacterial growth under various conditions, (iii) weakened the expression of genes associated with the type III secretion system and biofilm formation, and (iv) attenuated bacterial virulence. Addition of exogenous Al-2 was able to complement the deficiencies in the expression of TTSS genes and biofilm production but failed to rescue the growth defects. Our results (i) demonstrated that the Al-2 activity in TX1 is controlled at least in part at the level of luxS(Et) expression, which in turn is regulated by growth conditions, and that the temporal expression of luxS(Et) is essential for optimal bacterial infection and survival; and (ii) suggested the existence in Ed. tarda of a LuxS/Al-2-mediated signal transduction pathway that regulates the production of virulence-associated elements.

Molecular cloning, characterization and expression of heat shock protein 90 gene in the haemocytes of bay scallop Argopecten irradians

Heat shock protein 90 (HSP90) is a highly conserved molecular chaperone that plays key roles in the folding, maintenance of structural integrity and regulation of a subset of cytosolic proteins. In the present study, the cDNA of Argopecten irradians HSP90 (designated AiHSP90) was cloned by the combination of homology cloning and rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of AiHSP90 was of 2669 bp, including an open reading frame (ORF) of 2175 bp encoding a polypeptide of 724 amino acids with predicted molecular weight of 83.08 kDa and theoretical isoelectric point of 4.81. BLAST analysis revealed that AiHSP90 shared high similarity with other known HSP90s, and the five conserved amino acid blocks defined as HSP90 protein family signatures were also identified in AiHSP90, which indicated that AiHSP90 should be a cytosolic member of the HSP90 family. Fluorescent real-time quantitative PCR was employed to examine the expression pattern of AiHSP90 mRNA in haemocytes of scallops challenged by Gram-negative bacteria Vibrio anguillarum and Gram-positive bacteria Micrococcus luteus. In both bacterial challenged groups, the relative expression level of AiHSP90 transcript was up-regulated and reached maximal. level at 9 h after injection, and then dropped progressively to the original level at about 48 h post challenge. The results indicated that AiHSP90 was potentially involved in the immune responses against bacteria challenge in scallop A. irradian. (c) 2007 Elsevier Ltd. All rights reserved.

A transglutaminase from Chinese shrimp (Fenneropenaeus chinensis), full-length cDNA cloning, tissue localization and expression profile after challenge

Transglutaminase can catalyze the cross-linking reaction between soluble clotting protein molecules from the plasma for prevention of excess blood loss from a wound and obstructing micro-organisms from invading the wound in crustaceans. A novel transglutaminase (FcTG) gene was cloned from hemocytes of Chinese shrimp Fenneropenaeus chinensis by 3' and 5' rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA consists of 2972 bp, encoding 757 amino acids with a calculated molecular mass of 84.96 kDa and a theoretical isoelectric point of 5.61. FcTG contains a typical transglutaminase-like homologue (TGc domain: E-value = 1.94e-38). Three catalytic sites (Cys-324, His-391 and Asp-414) are present in this domain. The deduced amino acid sequence of FcTG showed high identity with black tiger shrimp TG, kuruma shrimp TG and crayfish TG. Transcripts of FcTG mRNA were mainly detected in gill, lymphoid organ and hemocytes by RT-PCR. RNA in situ hybridization further confirmed that FcTG was constitutively expressed in hemocytes both in the circulatory system and lymphoid organ. The variation of mRNA transcription level in hemocytes and lymphoid organ following injection of killed bacteria or infection with white spot syndrome virus (WSSV) was quantified by RT-PCR. The up-regulated expression of FcTG in shrimp lymphoid organ following injection of bacteria indicates that it is inducible and might be associated with bacterial challenge. (c) 2006 Elsevier Ltd. All rights reserved.

cDNA cloning, characterization and expression analysis of the antioxidant enzyme gene, catalase, of Chinese shrimp Fenneropenaeus chinensis

Catalase is an important antioxidant protein that protects organisms against various oxidative stresses by eliminating hydrogen peroxide. The full-length catalase cDNA of Chinese shrimp Fenneropenaeus chinensis was cloned from the hepatopancreas using degenerate primers by the method of 3' and 5' rapid amplification of cDNA ends PCR. The cDNA sequence consists of 1892 bp with a 1560 bp open reading frame, encoding 520 amino acids with high identity to invertebrate, vertebrate and even bacterial catalases. The sequence includes the catalytic residues His71, Asn144, and Tyr354. The molecular mass of the predicted protein is 58824.04 Da with an estimated pl of 6.63. Sequence comparison showed that the deduced amino acid sequence of F. chinensis catalase shares 96%, 73%, 71% and 70% identity with that of Pacific white shrimp Litopenaeus vannamei, Abalone Haliotis discus hannai, Zhikong scallop Chlamys farreri and Human Homo sapiens, respectively. Catalase transcripts were detected in hepatopancreas, hemocytes, lymphoid organ, intestine, ovary, muscle and gill. by real-time PCR. The variation of catalase mRNA transcripts in hemocytes and hepatopancreas was also quantified by real-time PCR and the result indicated that the catalase showed up-regulated expression trends in hemocytes at 14 h and in hepatopancreas at 37 h after injection with white spot syndrome virus (WSSV). (c) 2008 Elsevier Ltd. All rights reserved.

Molecular cloning, expression of a big defensin gene from bay scallop Argopecten irradians and the antimicrobial activity of its recombinant protein

Antimicrobial peptides are important components of the host innate immune responses by exerting broad-spectrum microbicidal activity against pathogenic microbes. The first mollusk big defensin (designated AiBD) cDNA was cloned from bay scallop Argopecten irradians by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) techniques. The scallop AiBD consisted of 531 nucleotides with a canonical polyadenylation signal sequence AATAAA and a poly(A) tail, encoding a polypeptide of 122 amino acids. The high similarity of AiBD deduced amino acid sequence with big defensin from Tachypleus tridentatus and Branchiostoma belcheri tsingtaunese indicated that AiBD should be a member of big defensin family. The expression of AiBD in various tissues was measured by using Northern blotting analysis. mRNA transcripts of AiBD could be detected in haemocytes of unchallenged scallops. The temporal expression of AiBD in haemolymph after Vibrio anguilarum challenge was recorded by quantitative real time PCR. The relative expression level of AiBD in haemolymph was up-regulated evenly in the first 8 h, followed by a drastic increase, and increased 131.1-fold at 32 h post-injection. These results indicated that AiBD could be induced by bacterial challenge, and it should participate in the immune responses of A. irradians. Biological activity assay revealed that recombinant AiBD could inhibit the growth of both Gram-positive and Gram-negative bacteria, and also showed strong fungicidal activity towards the expression host. Recombinant expression of AiBD made it possible to further characterize its functions involved in immune responses, and also provided a potential therapeutic agent for disease control in aquaculture. (c) 2006 Elsevier Ltd. All rights reserved.

Analysis of the expression and antioxidative property of a peroxiredoxin 6 from Scophthalmus maximus

Peroxiredoxins (Prxs) are a group of antioxidant proteins that protect cells from oxidative damage caused by various peroxides. To date, six different isoforms of peroxiredoxin (Prx1 to Prx6) have been identified, of which, Prx6 belongs to the 1-Cys Prx subfamily. Although Prx6 of several fish species have been reported at sequence level, there are very few documented studies on the potential function of fish Prx6. In this report, we describe the identification and analysis of a Prx6 homologue, SmPrx6, from turbot Scophthalmus maximus. The full length cDNA of SmPrx6 contains a 5'- untranslated region (UTR) of 60 bp, an open reading frame of 666 bp, and a 3'-UTR of 244 bp. The deduced amino acid sequence of SmPrx6 shares 81-87% overall identities with known fish Prx6. In silico analysis identified in SmPrx6 a conserved Prx6 catalytic motif, PVCTTE, and the catalytic triads putatively involved in peroxidase and phospholipase A2 activities. Expression of SmPrx6 was detected in most fish organs, with the highest expression levels found in blood and heart and the lowest level in spleen. Experimental challenges with bacterial pathogens and poly(I:C) upregulated SmPrx6 expression in liver and spleen in a manner that is dependent on the challenging agent and the tissue type. Treatment of cultured primary hepatocytes with H2O2 enhanced SmPrx6 expression in a dose-dependent manner. Recombinant SmPrx6 expressed in and purified from Escherichia coli exhibited thiol-dependent antioxidant activity and could protect cultured hepatocytes from H2O2-induced oxidative damage. Taken together, these results indicate that SmPrx6 is a Prx6 homologue with antioxidative property and is likely to be involved in both cellular maintenance and protective response during host immune defense against bacterial infection. (C) 2010 Elsevier Ltd. All rights reserved.

Identification and analysis of the immune effects of CpG motifs that protect Japanese flounder (Paralichthys olivaceus) against bacterial infection

CpG-containing oligodeoxynucleotides (ODNs) are known to be immunostimulatory in vertebrate systems and can activate both innate and adaptive immune responses. In this report, we described the selection, identification, and analysis of CpG motifs with immunoprotective effects in Japanese flounder. Sixteen CpG ODNs were synthesized and examined for the ability to inhibit bacterial dissemination in Japanese flounder blood. Four ODNs with the strongest inhibitory effects were selected and mixed to form ODNs 4M. In addition, a plasmid, pCN6, was constructed that contains the sequences of the four selected ODNs. When administered into Japanese flounder via intraperitoneal injection, both ODNs 4M and pCN6 could, in dose and time dependent manners, afford short-term protection against the infections of two different bacterial pathogens. Immunological analyses showed that ODNs 4M and, especially, pCN6 activated head kidney macrophages and enhanced serum bactericidal activity via probably the alternative pathway of complement activation. When used as a DNA vaccine to immunize Japanese flounder, pCN6 conferred apparent protections (42.9% and 52.6%, respectively, in terms of relative percent survival) against the challenges of two different fish pathogens at 4-week post-vaccination. Transcriptional analysis showed that vaccination with pCN6 upregulated the expression of the genes encoding NKEF, MHC II alpha, IL-1 beta, Mx, and MHC I alpha. These results demonstrate that ODNs 4M and pCN6 are immunostimulatory in Japanese flounder and can induce short- and long-term nonspecific protections against bacterial infections. (C) 2010 Elsevier Ltd. All rights reserved.

Identification and analysis of a CpG motif that protects turbot (Scophthalmus maximus) against bacterial challenge and enhances vaccine-induced specific immunity

Oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs in certain contexts are known to be immunostimulatory in vertebrate systems. CpG ODNs with immune effects have been identified for many fish species but, to our knowledge, not for turbot. In this study, a turbot-effective CpG ODN, ODN 205, was identified and a plasmid, pCN5, was constructed which contains the CpG motif of ODN 205. When administered into turbot via intraperitoneal (i.p.) injection, both ODN 205 and pCN5 could (i) inhibit bacterial dissemination in blood in dose and time dependent manners, and (ii) protect against lethal bacterial challenge. Immunological analyses showed that in vitro treatment with ODN 205 stimulated peripheral blood leukocyte proliferation, while i.p. injection with ODN 205 enhanced the respiratory burst activity, chemiluminescence response, and acid phosphatase activity of turbot head kidney macrophages. pCN5 treatment-induced immune responses similar to those induced by ODN 205 treatment except that pCN5 could also enhance serum bactericidal activity in a calcium-independent manner. To examine whether ODN 205 and pCN5 had any effect on specific immunity, ODN 205 and pCN5 were co-administered into turbot with a Vibrio harveyi subunit vaccine, DegQ. The results showed that pCN5, but not ODN 205, significantly increased the immunoprotective efficacy of DegQ and enhanced the production of specific serum antibodies in the vaccinated fish. Further analysis indicated that vaccination with DegQ in the presence of pCN5 upregulated the expression of the genes encoding MHC class II alpha, IgM, Mx, and IL-8 receptor. Taken together, these results demonstrate that ODN 205 and pCN5 can stimulate the immune system of turbot and induce protection against bacterial challenge. In addition, pCN5 also possesses adjuvant property and can potentiate vaccine-induced specific immunity. (C) 2010 Elsevier Ltd. All rights reserved.

Identification and analysis of a Sciaenops ocellatus ISG15 homologue that is involved in host immune defense against bacterial infection

ISG15 is an interferon-stimulated gene that encodes a ubiquitin-like protein. ISG15 homologues have been identified in a number of fish species, some of which are known to be regulated at expression level by virus infection and lipopolysacchande (LPS) treatment However, the relationship between ISG15 and live bacterial infection has not been investigated in piscine models. In this study, an ISG15 homologue, SoISG15, was identified from red drum Scraeriops ocellaws and analyzed at expression and functional levels The open reading frame ofSolSG15 is 477 base pairs (bp) and mtronless, with a 5'-untranslated region (UTR) of 91 bp and a 3'-UTR of 415 bp The deduced amino acid sequence of S0ISG15 shares 60-67% overall identities with the ISG15 of several fish species. S0ISG15 possesses two conserved ubiquinn-like domains and the canonical ubiquitin conjugation motif, LRGG, at the C-terminus. Expressional analysis showed that constitutive expression of SolSG15 was highest in blood and lowest in kidney Experimental challenges with LPS and bacterial pathogens induced significant S0ISG15 expression in the kidney but not in the liver Similar differential induction was also observed at cellular level with primary hepatocytes and head kidney (HK) lymphocytes. Poly(' C), however, effected drastic induction of S0ISG15 expression in kidney and liver at both tissue and cellular levels. Immunoblot analysis showed that S0ISG15 was secreted by cultured HK lymphocytes into the extracellular milieu. Recombinant S0ISG15 expressed in and purified from Eschenclua colt was able to enhance the respiratory burst activity, acid phosphatase activity, and bactericidal activity of HK macrophages. Taken together, the results of this study indicated that SoISG 15 possesses apparent immunological property and is likely to be involved in host immune defense against bacterial infection. (C)2010 Elsevier Ltd All rights reserved.

Peptidoglycan recognition protein of Chlamys farreri (CfPGRP-S1) mediates immune defenses against bacterial infection

Peptidoglycan recognition protein (PGRP) is an essential molecule in innate immunity for both invertebrates and vertebrates, owing to its prominent ability in detecting and eliminating the invading bacteria. Several PGRPs have been identified from mollusk, but their functions and the underlined mechanism are still unclear. In the present study, the mRNA expression profiles, location, and possible functions of PGRP-S1 from Zhikong scallop Chlamys farreri (CfPG RP-St) were analyzed. The CfPGRP-S1 protein located in the mantle, gill, kidney and gonad of the scallops. Its mRNA expression in hemocytes was up-regulated extremely after PGN stimulation (P < 0.01), while moderately after the stimulations of LPS (P < 0.01) and beta-glucan (P < 0.05). The recombinant protein of CfPGRP-S1 (designated as rCfPGRP-S1) exhibited high affinity to PGN and moderate affinity to LPS, but it did not bind beta-glucan. Meanwhile, rCfPGRP-S1 also exhibited strong agglutination activity to Gram-positive bacteria Micrococcus luteus and Bacillus subtilis and weak activity to Gram-negative bacteria Escherichia coli. More importantly, rCfPGRP-S1 functioned as a bactericidal amidase to degrade PGN and strongly inhibit the growth of E. coli and Staphyloccocus aureus in the presence of Zn2+. These results indicated that CfPGRP-S1 could not only serve as a pattern recognition receptor recognizing bacterial PGN and LPS, but also function as a scavenger involved in eliminating response against the invaders. (C) 2010 Elsevier Ltd. All rights reserved.

Scophthalmus maximus cystatin B enhances head kidney macrophage-mediated bacterial killing

Cystatins form a large family of cysteine protease inhibitors found in a wide arrange of organisms. Studies have indicated that mammalian cystatins play important roles under both physiological and pathological conditions. However, much less is known about fish cystatins. In this report, we described the identification and analysis of a cystatin B homologue, SmCytB, from turbot Scophthalmus maximus. The open reading frame of SmCytB is 300 bp, which encodes a 99-residue protein that shares high levels of sequence identities with the cystatin B of a number of fish species and contains the conserved cysteine protease inhibitor motif of cystatin B. Constitutive expression of SmCytB is high in muscle, brain, heart and liver, and low in spleen. blood, gill and kidney. Bacterial infection upregulates SmCytB expression in kidney, spleen, liver and brain but not in muscle or heart. Functional analysis showed that recombinant SmCytB purified from Escherichia colt exhibits apparent cysteine protease inhibitor activity. Transient overexpression of SmCytB in head kidney macrophages enhances macrophage bactericidal activity probably through a nitric oxide-independent mechanism. These results indicate that SmCytB is involved in the immune defense of turbot against bacterial infection. (C) 2010 Elsevier Ltd. All rights reserved.