CC chemokine 1 protein from Cromileptes altivelis (CaCC1) promotes antimicrobial immune defense.

Chemokines are a family of small signaling proteins that are secreted by various cells. In addition to their roles in immune surveillance, localization of antigen, and lymphocyte trafficking for the maintenance of homeostasis, chemokines also function in induce immune cell migration under pathological conditions. In the present study, a novel CC chemokine gene (CaCC1) from humpback grouper (Cromileptes altivelis) was cloned and characterized. CaCC1 comprised a 435 bp open reading frame encoding 144 amino acid residues. The putative molecular weight of CaCC1 protein was 15 kDa CaCC1 contains four characteristic cysteines that are conserved in other known CC chemokines. CaCC1 also shares 11.64%-90.28% identity with other teleost and mammal CC chemokines. Phylogenetic analysis revealed that CaCC1 is most closely related to Epinephelus coioides EcCC1, both of which are in a fish-specific CC chemokine clade. CaCC1 was constitutively expressed in all examined C. altivelis tissues, with high expression levels in skin, heart, liver, and intestine. Vibrio harveyi stimulation up-regulated CaCC1 expression levels in liver, spleen, and head-kidney. Functional analyses revealed that the recombinant protein (rCaCC1) could induce the migration of head-kidney lymphocytes from C. altivelis. Moreover, rCaCC1 significantly enhanced phagocytosis in head-kidney macrophages from C. altivelis. In addition, rCaCC1 exhibited antimicrobial activities against Staphylococcus aureus, Edwardsiella tarda, and V. harveyi. In vivo, CaCC1 overexpression improved bacterial clearance in V. harveyi infected fish. Conversely, CaCC1 knockdown resulted in a significant decrease of bacterial clearance. These results demonstrate the important roles that CaCC1 plays in homeostasis and in inflammatory response to bacterial infection.

Molecular characterization of the interferon regulatory factor (IRF) family and functional analysis of IRF11 in the large yellow croaker (Larimichthys crocea).

Interferon regulatory factors (IRFs) are a family of transcription factors involved in regulating interferon (IFN) responses and immune cell development. A total of 11 IRFs have been identified in teleost fish. Here, a complete repertoire of 11 IRFs (LcIRFs) in the large yellow croaker (Larimichthys crocea) was characterized with the addition of five newly identified members, LcIRF2, LcIRF5, LcIRF6, LcIRF10, and LcIRF11. These five LcIRFs possess a DNA-binding domain (DBD) at the N-terminal that contains five to six conserved tryptophan residues and an IRF-association domain (IAD) or IAD2 at the C-terminal that is responsible for interaction with other IRFs or co-modulators. Phylogenetic analysis showed that the 11 LcIRFs were divided into four clades including the IRF1 subfamily, IRF3 subfamily, IRF4 subfamily, and IRF5 subfamily. These are evolutionarily related to their respective counterparts in other fish species. The 11 LcIRFs were constitutively expressed in all examined tissues, although at different expression levels. Upon polyinosinic: polycytidylic acid (poly (I:C)) stimulation, the expression of all 11 LcIRFs was significantly induced in the head kidney and reached the highest levels at 6 h post-stimulation (except LcIRF4). LcIRF1, LcIRF3, LcIRF7, LcIRF8, and LcIRF10 were more strongly induced by poly (I:C) than the other LcIRFs. Significant induction of all LcIRFs was observed in the spleen, with LcIRF2, LcIRF5, LcIRF6, LcIRF7, LcIRF9, and LcIRF11 reaching their highest levels at 48 h LcIRF3 and LcIRF11 showed a stronger response to poly (I:C) in the spleen than the other LcIRFs. In addition, LcIRF1, LcIRF3, LcIRF7, LcIRF9, LcIRF10, and LcIRF11 were significantly induced by Vibro alginolyticus in both the spleen and the head kidney, with LcIRF1 strongly induced. Thus, LcIRFs exhibited differential inducible expression patterns in response to different stimuli in different tissues, suggesting that LcIRFs have different functions in the regulation of immune responses. Furthermore, overexpression of LcIRF11 activated the promoters of LcIFNc, LcIFNd, and LcIFNh, and differentially induced the expression levels of LcIFNs and IFN-stimulated genes (ISGs). Overexpression of LcIRF11 in epithelioma papulosum cyprinid (EPC) cells inhibited the replication of viral genes after infection of spring viremia of carp virus (SVCV). These data suggested that LcIRF11 may function as a positive regulator in regulating the cellular antiviral response through induction of type I IFN expression. Taken together, the present study reported molecular characterization and expression analysis of 11 IRFs in the large yellow croaker, and investigated the role of LcIRF11 in the antiviral response, which laid a good foundation for further study on the evolution and functional characterization of fish IRFs.

Large yellow croaker peroxiredoxin IV protect cells against oxidative damage and apoptosis.

Oxidative stress and inflammation lead to cell damage and are implicated in many disease states. High concentrations of hydrogen peroxide (H2O2) may mediate cells apoptosis by increasing intracellular reactive oxygen species (ROS) levels. In this study, we established a LYCK-PrxIV cell line (large yellow croaker head kidney cell line stably expressing peroxiredoxin IV). The level of nitric oxide (NO), superoxide anion and hydrogen peroxide (H2O2) in this LYCK-PrxIV cells were significantly lower than those in control cells of LYCK-pcDNA3.1 (LYCK cell line stably transfected by pcDNA3.1 vector). Additionally, when exposed to H2O2, cell apoptosis was significantly alleviated in LYCK-PrxIV than in control cells. Meanwhile, the ROS level and ATP content were maintained more stable in LYCK-PrxIV than in LYCK-pcDNA3.1. The over-expression of LcPrxIV in LYCK-PrxIV cells induced a declined mRNA expression of LcCXC, LcCC, LcIL-8 and LcTNF-α2, as well as an increase of LcIL-10 mRNA expression, when compared to LYCK-pcDNA3.1. On the other hand, the expression of chemokine LcCXC, LcCC and LcTNF-a2 increased in LYCK-pcDNA3.1 after H2O2 stimulation, while that of LcIL-8 and LcIL-10 decreased. The regualtion of gene expression in LYCK-PrxIV cells was almost the same as that in LYCK-pcDNA3.1, but the change fold was much more moderate. These results suggest that LcPrxIV may be an indispensable ROS scavenger protecting LYCK cells against oxidative damage as well as the subsequent apoptosis and inflammatory response, which provides a clue that LcPrxIV may be an assist in fish immune response.

Molecular characterization of a new fish specific chemokine CXCL_F6 in large yellow croaker (Larimichthys crocea) and its role in inflammatory response.

Chemokines are a superfamily of structurally related chemotactic cytokines exerting significant roles in regulating cell migration and activation. Currently, five subgroups of fish specific CXC chemokines, named CXCL_F1-CXCL_F5, have been identified in teleost fish. However, understanding of the functions of these fish specific CXC chemokines is still limited. Here, a new member of fish specific CXC chemokines, LcCXCL_F6, was cloned from large yellow croaker Larimichthys crocea. Its open reading frame (ORF) is 369 nucleotides long, encoding a peptide of 122 amino acids (aa). The deduced LcCXCL_F6 protein contains a 19-aa signal peptide and a 103-aa mature polypeptide, which has four conserved cysteine residues (C28, C30, C56, and C72), as found in other known CXC chemokines. Phylogenetic analysis showed LcCXCL_F6 formed a separate clade with sequences from other fish species, tentatively named CXCL_F6, distinct from the clades formed by fish CXCL_F1-5 and mammalian CXC chemokines. The LcCXCL_F6 transcripts were constitutively expressed in all examined tissues and significantly up-regulated in the spleen and head kidney tissues by poly (I:C) and Vibrio alginolyticus. Its transcripts were also detected in primary head kidney leukocytes (HKLs), peripheral blood leucocytes (PBLs), and large yellow croaker head kidney (LYCK) cell line, and significantly up-regulated by poly(I:C), lipopolysaccharide (LPS), and peptidoglycan (PGN) in HKLs. Recombinant LcCXCL_F6 protein (rLcCXCL_F6) could not only chemotactically attract monocytes/macrophages and lymphocytes from PBLs, but also enhance NO release and expression of proinflammatory cytokines (TNF-α, IL-1ß, and CXCL8) in monocytes/macrophages. These results indicate that LcCXCL_F6 plays a role in mediating the inflammatory response.

Characterization and function of a group II type I interferon in the perciform fish, large yellow croaker (Larimichthys crocea).

Teleost fish possess two groups of type I interferons (IFNs) with two (group I IFNs) or four (group II IFNs) conserved cysteines, which are further classified into seven subgroups. In our previous study, two group I type I IFNs, LcIFNd and LcIFNh (a new subgroup member), were identified in the perciform fish, large yellow croaker (Larimichthys crocea). Here, we identified a group II type I IFN, LcIFNc, in this species. The deduced LcIFNc contained six cysteines, four of which are highly conserved (C1: C28, C2:C53, C3: C130, and C4:C159) in the fish group II type I IFNs, and a typical type I IFN signature motif was also found in it. Phylogenetic analysis indicated that LcIFNc belongs to the IFNc subgroup of fish group II type I IFNs. LcIFNc was constitutively expressed in all examined tissues, and was rapidly up-regulated in spleen and head kidney by poly(I:C) and Aeromonas hydrophila. Recombinant LcIFNc protein (rLcIFNc) could increase the expression of antiviral genes, Mx1, PKR and ISG15, in large yellow croaker peripheral blood leukocytes (PBLs). The rLcIFNc also exhibited obvious antiviral activity based on less cytopathic effect (CPE) and decreased expression levels of several viral genes in the rLcIFNc-treated grouper spleen (GS) cells following Singapore grouper iridovirus (SGIV) infection. Additionally, rLcIFNc was able to induce the expression of LcIFNc, as well as LcIFNd and LcIFNh in the PBLs and primary head kidney cells (HKCs) from large yellow croaker. These results therefore indicated that LcIFNc not only had antiviral activity, but also mediated the regulation of type I IFN response.

Identification of two IL-4/13 homologues in large yellow croaker (Larimichthys crocea) revealed their similar roles in inducing alternative activation of monocytes/macrophages.

Mammalian interleukin-4 (IL-4) and -13 (IL-13), two anti-inflammatory T helper cell type 2 (Th2) cytokines, play the central roles in mediating the alternative activation of monocytes/macrophages (MO/Mφs). However, exact functions in MO/Mφs polarization of IL-4/13 homologues in teleost fish remain largely unknown. In this study, we identified two IL-4/13 homologues from large yellow croaker Larimichthys crocea, LcIL-4/13A and LcIL-4/13B, which share low amino acid sequence identities to the known fish IL-4/13 molecules. Phylogenetic analysis showed that LcIL-4/13A is evolutionarily closely related to Dicentrarchus labrax IL-4/13A, and LcIL-4/13B to Takifugu rubripes IL-4/13B. The two LcIL-4/13 genes were constitutively expressed in all examined tissues, but with different expression levels. Both LcIL-4/13A and LcIL-4/13B were up-regulated by inactivated trivalent bacterial vaccine in the head kidney, and LcIL-4/13B appeared more responsive to bacterial vaccine than LcIL-4/13A. Recombinant LcIL-4/13A and LcIL-4/13B proteins (rLcIL-4/13A and rLcIL-4/13B) produced in Escherichia coli could significantly decrease production of reactive oxygen species (ROS) and nitrogen oxide (NO) in the head kidney MO/Mφs from large yellow croaker. Furthermore, rLcIL-4/13A and rLcIL-4/13B obviously down-regulated expression of pro-inflammatory cytokine (IL-1ß and TNF-α) and inducible NO synthase (iNOS) genes in MO/Mφs, while they increased mRNA expression of anti-inflammatory cytokines (TGF-ß and VEGF) and arginase-2. Additionally, the phagocytic activity of MO/Mφs was also inhibited by rLcIL-4/13A or rLcIL-4/13B. All these results therefore indicated that both LcIL-4/13A and LcIL-4/13B, although exhibiting a lower degree of sequence identity of 15.6% and differential expression pattern, have the similar roles in promoting alternative activation of head kidney MO/Mφs.

Molecular characterization and functional activity of CXCL8_L3 in large yellow croaker Larimichthys crocea.

CXCL8, also called interleukin-8, is a typical CXC chemokine that plays a key role in promoting inflammation. Phylogenetically, fish CXCL8 chemokines can be divided into three subgroups, CXCL8_L1, CXCL8_L2, and CXCL8_L3, of which CXCL8_L3 is a new subgroup. The CXCL8_L3 gene sequences have been reported in many fish species, but their function remains unknown. Here, a CXCL8_L3 (LycCXCL8_L3) gene was cloned from large yellow croaker Larimichthys crocea. Its open reading frame (ORF) was 309 nucleotides long, encoding a peptide of 102 amino acids. The deduced LycCXCL8_L3 protein contains an 18-aa signal peptide and an 84-aa mature polypeptide, which has four conserved cysteine residues (C30, C32, C57, and C73) as found in other known CXCL8 chemokines. Phylogenetic analysis showed LycCXCL8_L3 formed a major clade with CXCL8_L3 sequences from other fish species. The LycCXCL8_L3 transcript was constitutively expressed in all examined tissues and significantly up-regulated in the spleen and head kidney tissues by inactivated trivalent bacterial vaccine. The LycCXCL8_L3 transcript was also detected in peripheral blood leukocytes (PBLs), primary head kidney macrophages (PKM), and large yellow croaker head kidney cell line (LYCK), with the highest levels in PKM. Recombinant LycCXCL8_L3 (rLycCXCL8_L3) protein could not only chemotactically attract lymphocytes and eosinophils in PBLs, but also enhance the respiratory burst activity of PKM. These results indicate that LycCXCL8_L3 may play an important role in the inflammatory response of large yellow croaker. To our knowledge, this is the first report on functional study of the CXCL8_L3 in fish.

Comparative study of interleukin-17C (IL-17C) and IL-17D in large yellow croaker Larimichthys crocea reveals their similar but differential functional activity.

Interleukin 17 (IL-17) family members are key players in regulating the immune response in mammals. Here, we identified the IL-17C and IL-17D homologs from large yellow croaker (Larimichthys crocea), named LcIL-17C and LcIL-17D, respectively. The deduced LcIL-17C and LcIL-17D proteins possessed the typical IL-17 domain and shared a conserved arrangement of eight cysteine residues. Both LcIL-17C and LcIL-17Dc genes were constitutively expressed in all tissues examined, although at different levels. After challenge with Aeromonas hydrophila, the expression of LcIL-17C and LcIL-17D was significantly increased in gills, head kidney, and spleen. In the peripheral blood leukocytes (PBLs), the recombinant LcIL-17C (rLcIL-17C) could strongly promote the expression of chemokines (CXCL8, CXCL12, and CXCL13), proinflammatory factors (TNF-α, IL-1ß, IL-6, and IFNg), and antibacterial peptide hepcidin, whereas rLcIL-17D induced a weaker expression of these chemokines. Consistently, the culture supernatants from the PBLs treated by rLcIL-17C showed a stronger ability to induce the migration of PBLs than those treated by rLcIL-17D. Furthermore, both rLcIL-17C and rLcIL-17D could activate the NF-κB signalling in the epithelioma papulosum cyprini (EPC) cells. Taken together, these results indicated that LcIL-17C and LcIL-17D, although differing in their ability to mediate chemotaxis for PBLs, may promote the inflammatory response and host defence via activating NF-κB signalling. To our knowledge, this is the first report on functional identification of a IL-17C in teleost.

Molecular characterization and expression analysis during embryo development of CD4-1 homologue in large yellow croaker Larimichthys crocea.

CD4+ helper T (Th) cells are a master component of the adaptive immune response. CD4 is one of the most effective surface markers for identifying Th cells. In the present study, we cloned and characterized a CD4-1 homologue, LycCD4-1, from large yellow croaker Larimichthys crocea. The full-length cDNA of LycCD4-1 is 1695 bp long, encoding a protein of 462 amino acids. The deduced LycCD4-1 protein has a typical domain architecture as found in mammalian CD4 molecules, including a signal peptide, four extracellular immunoglobulin-like (Ig-like) domains, a transmembrane region, and a CXC signaling motif in the cytoplasmic tail. Four N-glycosylation sites and 10 cysteine residues were also found in LycCD4-1, which may be essential for its tertiary structure and succeeding function. Homology comparison showed that LycCD4-1 has 27.9-58.4% identity to other teleost fish CD4-1 molecules, and 16.4-20% identity to those of higher vertebrates. Genomic analysis revealed that the LycCD4-1 gene consisted of nine exons and eight introns and exhibited a similar exon-intron organization to other species CD4 genes except for a different intron length. Phylogenetic analysis showed that LycCD4-1 form a cluster with CD4-1 molecules in other fish species. The LycCD4-1 was constitutively expressed in all tissues tested, with a higher expression in gills and spleen. LycCD4-1 mRNA expression in the spleen and head kidney tissue was increased by poly (I:C) at 48 h, whereas its expression levels were somewhat down-regulated at 6 h and 72 h after bacterial vaccine induction in spleen. Unexpectedly, LycCD4-1 mRNA could be detected in each stage of early embryo development since fertilized eggs, with a higher level before mid-gastrula and the highest level in high blastocysts. These results will be helpful for better understanding molecular characteristics of CD4-1 and tracing origin of CD4-1+ cell precursors in fish.

Identification of a fish specific chemokine CXCL_F2 in large yellow croaker (Larimichthys crocea) reveals its primitive chemotactic function.

Chemokines are a superfamily of cytokines regulating immune cell migration under both inflammatory and normal physiological conditions. Currently, a number of fish specific CXC chemokines, named as CXCL_F1-5, have been identified in several species. However, understanding of their functional characteristics is still limited. In this study, we identified a fish specific chemokine CXCL_F2 (LycCXCL_F2) from large yellow croaker (Larimichthys crocea). The open reading frame (ORF) of LycCXCL_F2 is 348 nucleotides long, encoding a protein of 115 amino acids (aa). The deduced LycCXCL_F2 protein contains a 20-aa signal peptide and a 95-aa mature polypeptide. Phylogenetic analysis showed that LycCXCL_F2 fell into a major clade formed by CXCL_F2 sequences and was separated from CXCL_F1 and CXCL_F3-5 subgroups. LycCXCL_F2 mRNA transcript was constitutively expressed in various tissues, with the highest levels in spleen and head kidney. After stimulation with inactivated trivalent bacterial vaccines, LycCXCL_F2 mRNA transcription was significantly increased in both spleen and head kidney. Moreover, recombinant LycCXCL_F2 protein exhibited obvious chemotaxis to monocytes, lymphocytes and eosnophils of PBLs isolated from large yellow croaker, but could not induce the respiratory burst of macrophages. These results indicate that this fish specific CXC chemokine LycCXCL_F2 possesses primitive chemotactic activity and may play a role in immune response in large yellow croaker.

A cystatin F homologue from large yellow croaker (Larimichthys crocea) inhibits activity of multiple cysteine proteinases and Ii chain processing in vitro.

Cystatin F, a member of the family II cystatins, plays important roles in immune response-related processes through inhibiting specific enzyme targets. In this study, a cystatin F homologue, LycCysF, was identified and characterized from large yellow croaker (Larimichthys crocea). The deduced LycCysF protein exhibits a typical structural feature of type II cystatins, including three evolutionally conserved motifs, Gly(35), QVVRG(79-83) and PW(130-131). Tissue expression analysis showed that LycCysF mRNA was expressed in all tissues examined, albeit at different levels. Recombinant LycCysF (rLycCysF) produced in Pichia pastoris could inhibit the activity of multiple cysteine proteases, including papain, legumain and recombinant large yellow croaker cathepsin B, L and S. Moreover, rLycCysF could inhibit the Ii chain processing by recombinant cathepsin S in vitro. These data suggest that LycCysF may participate in regulation of cathepsins and MHC-II associated Ii chain processing. In addition, mammalian cystatin F is produced as an inactive dimer, becoming activated by proteolysis in the endo/lysosome of immune cells and then exerts its function of regulating downstream proteases activity. However, the N-terminal extension and two additional cysteine residues responsible for dimer formation are absent in LycCysF and cystatin F from other fish species, reptiles and Aves, indicating that these proteins can not form dimer and may regulate the proteases activity via an alternate pathway distinct from mammalian cystatin F. To our knowledge, this is the first report on molecular characteristics of a teleost cystatin F and its role in Ii chain processing.

Molecular and functional identification of three interleukin-17A/F (IL-17A/F) homologues in large yellow croaker (Larimichthys crocea).

The interleukin-17 (IL-17) cytokine family plays a central role in the coordination of inflammatory responses. In fish species, three genes that have a similar homology to both IL-17A and IL-17F were designated IL-17A/F1, 2, and 3. In this study, we identified three IL-17A/F homologues (LycIL-17A/F1, 2, and 3) from large yellow croaker (Larimichthys crocea). The deduced LycIL-17A/F1 and 3 had four cysteine residues conserved in teleost IL-17A/F1 and 3 homologues and shared a domain similar to the B chain of human IL-17F. The deduced LycIL-17A/F2 possessed the unique arrangement of six cysteine residues as teleost IL-17A/F2 (except Fugu IL-17A/F2) and higher vertebrate IL-17A and F, and shared a domain similar to the D/E chain of human IL-17A. Phylogenetic analysis showed that teleost IL-17A/F1 and 3 fall into a major clade, whereas IL-17A/F2 forms a separated clade and is clustered with IL-17N. Based on structural and phylogenetic analyses, we suggest that teleost IL-17A/Fs may be classified into two subgroups: one consisting of IL-17A/F1 and 3, and the other composed of IL-17A/F2. The three LycIL-17A/Fs were constitutively expressed in all tissues examined although at a different level. Following challenge with Aeromonas hydrophila, expression of these three LycIL-17A/Fs was rapidly increased in head kidney and gills. The in vivo assays showed that recombinant LycIL-17A/F1, 2, and 3 all were able to enhance the expression of pro-inflammatory cytokines (IL-1ß, IL-6, and TNF-α2), chemokines (CXCL8 and CXCL13), and antimicrobial peptide hepcidin in head kidney. Furthermore, LycIL-17A/Fs appeared to mediate pro-inflammatory responses via NF-κB signalling. These results therefore reveal similar functions between the two subgroup members，LycIL-17A/F1 and 3 and LycIL-17A/F2, in promoting inflammation and host defences.

Molecular Characterization and Biological Effects of a C-Type Lectin-Like Receptor in Large Yellow Croaker (Larimichthys crocea).

The C-type lectin-like receptors (CTLRs) play important roles in innate immunity as one type of pattern recognition receptors. Here, we cloned and characterized a C-type lectin-like receptor (LycCTLR) from large yellow croaker Larimichthys crocea. The full-length cDNA of LycCTLR is 880 nucleotides long, encoding a protein of 215 amino acids. The deduced LycCTLR contains a C-terminal C-type lectin-like domain (CTLD), an N-terminal cytoplasmic tail, and a transmembrane region. The CTLD of LycCTLR possesses six highly conserved cysteine residues (C1-C6), a conserved WI/MGL motif, and two sugar binding motifs, EPD (Glu-Pro-Asp) and WYD (Trp-Tyr-Asp). Ca(2+) binding site 1 and 2 were also found in the CTLD. The LycCTLR gene consists of five exons and four introns, showing the same genomic organization as tilapia (Oreochromis niloticus) and guppy (Poecilia retitculata) CTLRs. LycCTLR was constitutively expressed in various tissues tested, and its transcripts significantly increased in the head kidney and spleen after stimulation with inactivated trivalent bacterial vaccine. Recombinant LycCTLR (rLycCTLR) protein produced in Escherichia coli BL21 exhibited not only the hemagglutinating activity and a preference for galactose, but also the agglutinating activity against two food-borne pathogenic bacteria E. coli and Bacillus cereus in a Ca(2+)-dependent manner. These results indicate that LycCTLR is a potential galactose-binding C-type lectin that may play a role in the antibacterial immunity in fish.

Cathepsin S, but not cathepsin L, participates in the MHC class II-associated invariant chain processing in large yellow croaker (Larimichthys crocea).

Two cysteine proteases, cathepsin S (CatS) and cathepsin L (CatL), have been identified as the key enzymes involved in the processing of invariant chain (Ii chain) in mammals. However, little is known about the roles of fish cathepsins in the Ii chain processing. In this study, large yellow croaker cathepsin S (LycCatS) and L (LycCatL) were identified and characterized. Based on the sequence comparison and phylogenetic analysis, both LycCatS and LycCatL are highly conserved to their counterparts in teleost. These two cathepsins were constitutively expressed in all tissues and immune-related cells tested, although at different levels. Both recombinant LycCatS (rLycCatS) and LycCatL (rLycCatL) possess the typical cysteine protease activity. Like other mammalian endopeptidase cathepsins, rLycCatS and rLycCatL could be autocatalytically activated to remove propeptides and release active mature peptides. On the other hand, the autocatalytic activation of rLycCatL could be inhibited by recombinant large yellow croaker Ii chain (rLyc-TR-Ii), but the autocatalytic activation of rLycCatS was not affected by rLyc-TR-Ii. Furthermore, the activated rLycCatS can efficiently process rLyc-TR-Ii in a stepwise manner in vitro, while the activated rLycCatL can not. These data indicate that cathepsin S may be the main cathepsin involved in the Ii chain processing in bony fish.

Molecular characterization and biological effects of a CXCL8 homologue in large yellow croaker (Larimichthys crocea).

CXCL8 also called interleukin-8, is a CXC-type chemokine that plays a key role in promoting inflammation. Three subgroups of CXCL8 homologues have been reported in teleost fish, including CXCL8_L1, CXCL8_L2 and CXCL8_L3. In the present study, we identified a CXCL8 homologue belonging to CXCL8_L1 subgroup (LycCXCL8_L1) in large yellow croaker (Larimichthys crocea) that shares low identity to the previously reported large yellow croaker CXCL8 (LycCXCL8). The full-length cDNA of LycCXCL8_L1 is 716 nucleotides (nt) long and encodes a protein consisting of 99 amino acids (aa) with a putative molecular weight of 11.2 kDa. The deduced LycCXCL8_L1 protein contains a 22-aa signal peptide and a 77-aa mature polypeptide, which possesses an arrangement of four cysteines typical of other known CXC chemokines (C(34), C(36), C(60), and C(77)). Genomic analysis revealed that the LycCXCL8_L1 gene consisted of four exons and three introns and exhibited a similar exon-intron organization to LycCXCL8 and other species CXCL8 genes except for a different intron length. Phylogenetic analysis showed that both LycCXCL8_L1 and LycCXCL8 belong to CXCL8_L1 subgroup. LycCXCL8_L1 mRNA was constitutively expressed in all tissues examined although at different levels. Upon bacterial vaccine induction, LycCXCL8_L1 mRNA expression was rapidly increased in the spleen and head kidney tissues. Recombinant LycCXCL8_L1 and LycCXCL8 proteins produced in Escherichia coli both induced chemotaxis and superoxide production in peripheral blood leucocytes from large yellow croaker. These results indicate that two CXCL8_L1 molecules exist in large yellow croaker and play roles in inflammatory response.

Molecular characterization and expression analysis of TLR 7 and TLR 8 homologs in large yellow croaker (Pseudosciaena crocea).

The two toll-like receptor (TLR) genes, LycTLR7 and LycTLR8, were cloned from large yellow croaker (Pseudosciaena crocea), an economically important marine fish in China. The full-length cDNAs of LycTLR7 and LycTLR8 are 3544 and 3593 bp, with an open reading frame (ORF) of 3165 and 3093 bp, encoding 1053 and 1030 amino acids, respectively. The TLR family motifs, such as leucine rich repeat (LRR) and Toll/interleukin (IL)-1 receptor (TIR) domain, are conserved in the LycTLR7 and LycTLR8, with 17 and 14 LRRs, and with a TIR domain, respectively. It is also noted that an LRR N-terminal domain (LRR-NT, residues 24-60) is present in the LycTLR7 but not in the LycTLR8. Both LycTLR7 and LycTLR8 contain a conserved extracellular CxRCxxxxxPCxxC motif, which was found in TLR7/TLR8 of other species and required for stimulus-induced signal transduction. Homology comparison shows that LycTLR7 has 79%, 71.9%, 65.9% and 65.8% identity to fugu, rainbow trout, carp and catfish TLR7, while LycTLR8 has 67.1%, 60.7%, 60.6%, 52.4%, and 51.5% identity to fugu TLR8, rainbow trout TLR8a1, rainbow trout TLR8a2, catfish TLR8-2, and catfish TLR8-1, respectively. Subcellular localization analysis revealed that both LycTLR7 and LycTLR8 are located in the endoplasmic reticulum of epithelioma papulosum cyprini (EPC) cells, which is similar to TLR7/TLR8 in mammals. The two TLRs were constitutively expressed in all tissues tested, especially in immune-related tissues such as spleen, head kidney and gills. An increased expression of LycTLR7 and LycTLR8 was observed in head kidney and spleen of large yellow croakers stimulated by poly (I:C), a viral mimic. In head kidney, their mRNA expression was up-regulated more than 10 times compared to the controls at 12 h after poly (I:C) stimulation. These results suggested that LycTLR7 and LycTLR8 may play a role in the defense against viral infection like their mammalian homologs.

Proteomic Analysis of Differential Protein Expression in Acidithiobacillus ferrooxidans Grown on Ferrous Iron or Elemental Sulfur.

In this study, a proteomic analysis of Acidithiobacillus ferrooxidans by two-dimensional electrophoresis identified 24 proteins that were differentially expressed when the cells were grown on ferrous iron (Fe(2+)) or elemental sulfur (S°). Sixteen of these proteins were upregulated by growth on S° or downregulated by growth on Fe(2+), including four proteins involved in disulfide bond reduction such as pyridine nucleotide-disulfide oxidoreductase, heterodisulfide reductase subunit B, thioredoxin-disulfide reductase, and cysteine desulfurase IscS, and three proteins involved in saccharide metabolism. A total of eight proteins were upregulated by growth on Fe(2+) or downregulated by S°. Northern blots further confirmed the differences in transcription for these differentially expressed proteins. We functionally characterized cysteine desulfurase IscS, and found that its overexpression in E. coli promoted the growth of the cells in LB containing 2.5 % sodium thiosulfate. Our results provide new insights into the molecular basis for S° and Fe(2+) oxidation by this extreme acidophile.

Molecular cloning and characterization of caspase-3 in large yellow croaker (Pseudosciaena crocea).

Caspases-3, a member of the cysteine-aspartic acid protease (caspase) family, plays critical roles in the execution of apoptotic pathway. In this study, a caspase-3 homologue was cloned and characterized from large yellow croaker (Pseudosciaena crocea). The full-length cDNA of large yellow croaker caspase-3 (Lyccasp3) is 2222bp with an open reading frame of 858 bp encoding a polypeptide of 285 amino acids (aa). Lyccasp3 exhibited a conserved caspase-3 architecture including a prodomain, a large subunit and a small subunit. Moreover, several residues known to be critical in the caspase-3 catalytic centre and binding pocket, as well as the active-site pentapeptide motif Q(172)ACRG(176) were present in the deduced Lyccasp3. Recombinant Lyccasp3 (rLyccasp3) produced in Escherichia coli exhibited obvious hydrolyzing activity against synthetic peptide substrate Ac-DEVD-pNA. The Lyccasp3 was constitutively expressed in all the tissues examined, although the expression levels varied from tissue to tissue. Real-time PCR analysis revealed that Lyccasp3 transcript in spleen and kidney was quickly increased after stimulation with either poly (I:C) or inactivated trivalent bacterial vaccine. Enzyme activities of Lyccasp3 were also up-regulated in these two tissues post-stimulation when analyzed by hydrolyzing activity assay. Since the activity of large yellow croaker caspase-9 (Lyccasp9) in the spleen and kidney also increased when the fish was stimulated with the poly(I:C) or bacterial vaccine [1], we therefore proposed that the intrinsic apoptotic pathway, which is initiated by caspase-9 and executed by caspase-3, was activated during the immune response induced by poly(I:C) or bacterial vaccine in large yellow croaker.

Identification of differentially expressed genes in Sulfobacillus sp. TPY grown on either elemental sulphur or Fe(2+).

Sulfobacillus sp. TPY is a moderately thermophilic and acidophilic bacterium found in hydrothermal vents in the Pacific Ocean. This bacterium can oxidize ferrous sulfate (Fe(2+)) and elemental sulfur (S(0)) under separate conditions. We used random arbitrarily primed polymerase chain reaction (RAP-PCR) to screen and identify differentially expressed genes from bacteria grown on Fe(2+) or S(0) as the energy source. Fifty-five differential cDNA fragments were isolated and subjected to single-pass sequencing. Thirty-five fragments were identified as orthologs of known genes in the GenBank databases, of which 19 were confirmed to be differentially expressed at the transcriptional level by Northern blot analysis. Among these 19 genes, 14 genes, including isocitrate dehydrogenase, formyltetrahydrofolate deformylase, 3-hydroxybutyryl-CoA dehydrogenase, and GTP-binding protein, were upregulated in TPY grown on Fe(2+) or downregulated in TPY grown on S(0), while five genes such as the outer membrane adhesion-like protein, phosphomannomutase, and cysteine desulfurase sufS were upregulated in TPY strain grown on S(0) or downregulated in TPY grown on Fe(2+). These altered genes are involved in metabolism, osmotic stress, cell membrane alterations, oxidative stress, and the regulatory adaptive response. These results will aid our understanding of the molecular basis of Fe(2+) or S(0) oxidation by the moderately thermophilic and acidophilic bacteria.

Molecular characterization and bioactivity of a CXCL13 chemokine in large yellow croaker Pseudosciaena crocea.

A CXCL13-like chemokine cDNA was isolated from large yellow croaker (Pseudosciaena crocea) by expressed sequence tag (EST) analysis (LycCXCL13). The full-length cDNA of LycCXCL13 is 796 nucleotides (nt) encoding a protein of 97 amino acids (aa), with a putative molecular weight of 10.7 kDa. The deduced LycCXCL13 contains a 24-aa signal peptide and a 73-aa mature polypeptide, which possesses the typical arrangement of four cysteines as found in other known CXC chemokines (C(25), C(27), C(52) and C(68)). It shares 35, 36 and 39% aa sequence identities to green puffer CXCL13-like, Atlantic salmon CXCL13 and Japanese flounder CXCL13 chemokines, and 24-29% identities to CXCL13 chemokines in mammals, respectively. Phylogenetic analysis showed that LycCXCL13 is more closely related to the CXCL13 subgroup than to any other CXC chemokine subgroups. LycCXCL13 gene was constitutively expressed in all tissues examined, except for intestine. Upon induction with poly(I:C) or inactivated trivalent bacterial vaccine, LycCXCL13 gene expression was significantly up-regulated in spleen, head kidney, heart and gills at 24 h post-injection. Real-time PCR results showed that LycCXCL13 gene expression reached peak level in spleen and head kidney at 12 h after induction by poly(I:C), while its expression increased to the highest level in head kidney at 24 h or in spleen at 48 h by bacterial vaccine. Recombinant LycCXCL13 protein produced in E. coli BL21 exhibited obvious chemotaxis to the peripheral blood leucocytes (PBLs) from large yellow croaker. These results suggest that LycCXCL13 may be involved in inflammatory responses as well as homeostatic processes in large yellow croaker.