Production of monoclonal antibody against grouper (Epinephelus coioides) CD4-1 and the distribution of CD4-1+ cells.

CD4-a transmembrane glycoprotein molecule expressed on the surface of helper T (Th) cells-plays a central role in adaptive immune protection. In the current study, we developed a monoclonal antibody (mAb) against the grouper CD4-1. Western blotting and immunohistochemistry results revealed that the CD4-1 mAb could recognize the recombinant and natural protein of grouper CD4-1 as well as the CD4-1+ cells in the various tissues from grouper. Tissue distribution analyses revealed that the grouper CD4-1+ cells were expressed in all tissues tested in the healthy grouper, with greater localization in the thymus, head kidney, and spleen tissues. In addition, we tested the changes in the proportion of CD4-1+ cells in the thymus, head kidney, and the gills of grouper post the infection by C. irritans. Our data suggest that the CD4-1 mAb produced against grouper in the current study can be used as a tool to characterize CD4-1+ cells and to investigate the functions of the grouper CD4-1+ cells in the host response against pathogens infection.

Transcriptomic analysis reveals innate immune mechanisms of an underlying parasite-resistant grouper hybrid (Epinephelus fuscogutatus × Epinephelus lanceolatus).

Hybridization is an artificial breeding strategy for generating potentially desirable offspring. Recently, a novel Hulong grouper hybrid (Epinephelus fuscogutatus × Epinephelus lanceolatus) yielded significant growth superiority over its parent. Improved innate immunity is considered as another desirable feature during hybridization. However, whether this Hulong grouper achieved disease resistance has not yet been revealed. In this study, we first examine the infection intensity of C. irritans in the Hulong grouper, and found that the Hulong grouper is less susceptible to C. irritans primary infection. A higher immobilization titer was found in the infected Hulong grouper at Day 2 when compared with the control grouper. Furthermore, severe hyperplasia was observed in the orange-spotted grouper, but not in the Hulong grouper's skin epidermis. To further understand the innate immune mechanism against C. irritans, we conducted a comparative transcriptome analysis of the Hulong grouper during the infection. There are 6464 differentially expressed genes (DEGs) identified in the skin between the control and infected Hulong grouper. This indicates that the innate immune components, such as the complement system, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, Interleukin 17 (IL-17) signaling pathway, and Toll-like receptor (TLR) signaling pathway were up-regulated during the infection. These results show that the C. irritans infection can induce a remarkable inflammatory response in the Hulong grouper. Moreover, a total of 75 pairs of orthologs with the ratio of nonsynonymous (Ka) to synonymous (Ks) substitutions ＞1, considered rapidly evolving genes (REGs), was identified between the Hulong and orange-spotted grouper. More critically, most REGs were enriched in the immune system, suggesting that rapid evolution of the immune system might occur in the Hulong grouper. These results provide a more comprehensive understanding of the innate immunity mechanism of the hybrid Hulong grouper.

Identification and characterization of c-raf from orange-spotted grouper (Epinephelus coioides).

C-Raf proto-oncogene serine/threonine kinase is a mitogen-activated protein kinase (MAP) kinase kinase, which can initiate a mitogen-activated protein kinase (MAPK) cascade by phosphorylating the dual-specific MAP kinase kinases (MEK1/2), and in turn activate the extracellular signal-regulated kinases (ERK1/2). To study the function of c-Raf in teleost fish, a c-Raf cDNA sequence from orange-spotted grouper (Epinephelus coioides) was cloned. Ecc-Raf shared 81%-99% amino acid identity with other vertebrate c-Raf molecules, and shared the highest amino acid identity (99%) with Lates calcarifer c-Raf. Genomic structure analysis revealed that grouper c-Raf shared a conserved exon structure with other vertebrates. Tissue distribution showed that Ecc-Raf was mainly transcribed in systemic immune organs. Ecc-Raf was distributed throughout the cytoplasm of transfected GS cells and the overexpression of Ecc-Raf only slightly enhanced the activation of Activator protein 1. The phosphorylation levels of Ecc-Raf can be induced by PMA and H2O2 treatment, in contrast to DMSO or untreated HKLs. Moreover, the phosphorylation level of the Raf-MEK-ERK axis was downregulated after 24 h of SGIV infection. On the other hand, the total level and phosphorylation level of c-Raf significantly increased post C. irritans infection and showed an enhanced level post immunization. The results of this study suggested that the Raf-MEK-ERK cascade was involved in the response to viral or parasitic infections.

Orange-spotted grouper (Epinephelus coioides) NADPH oxidase: Cloning and expression analysis after Cryptocaryon irritans infection.

Phagocytic cells are activated to produce a large amount of reactive oxygen species (ROS) that kill pathogens quickly and efficiently through oxidation. NADPH oxidase is the main source of intracellular ROS. In the present study, five subunits of the phagocytic NADPH oxidase complex were identified in orange-spotted grouper (Epinephelus coioides). The open reading frame of grouper gp91phox, p22phox, p67phox, p47phox, and p40phox were 1,698 bp, 564 bp, 1,497 bp, 1,290 bp, and 1,050 bp, respectively, and encoded 565, 187, 498, 429, and 349 amino acids. Evolutionary analysis indicated that these proteins are evolutionarily homologous to the corresponding proteins of other fish and mammals, and contain conserved functional domains and sites that are important in mammals. In addition, real-time polymerase chain reaction analysis showed that the expression of these five genes was higher in immune-related tissues in normal grouper, and that these genes were up-regulated in gill and spleen after C. irritans infection, which suggests that these genes may be involved in the defense against C. irritans infection.

Immunomodulatory Effects of N-Acetyl Chitooligosaccharides on RAW264.7 Macrophages.

The ongoing development of new production methods may lead to the commercialization of N-acetyl chitooligosaccharides (NACOS), such as chitosan oligosaccharides (COS). The bioactivity of NACOS, although not well detailed, differs from that of COS, as they have more acetyl groups than COS. We used two enzymatically produced NACOS with different molecular compositions and six NACOS (NACOS1-6) with a single degree of polymerization to verify their immunomodulatory effects on the RAW264.7 macrophage cell line. We aimed to identify any differences between COS and various NACOS with a single degree of polymerization. The results showed that NACOS had similar immune enhancement effects on RAW264.7 cells as COS, including the generation of reactive oxygen species (ROS), phagocytotic activity, and the production of pro-inflammation cytokines (IL-1ß, IL-6, and TNF-α). However, unlike COS and lipopolysaccharide (LPS), NACOS1 and NACOS6 significantly inhibited nitric oxide (NO) production. Besides their immune enhancement effects, NACOS also significantly inhibited the LPS-induced RAW264.7 inflammatory response with some differences between various polymerization degrees. We confirmed that the NF-κB pathway is associated with the immunomodulatory effects of NACOS on RAW264.7 cells. This study could inform the application of NACOS with varying different degrees of polymerization in human health.

Role of major histocompatibility complex II antigen-presentation pathway genes in orange-spotted grouper infected with Cryptocaryon irritans.

Cryptocaryon irritans, a pathogen model for fish mucosal immunity, causes skin mucosal and systematic humoral immune response. Where and how MHC II antigen presentation occurs in fish infected with C. irritans remain unknown. In this study, the full-length cDNA of the grouper cysteine protease CTSS was cloned. The expression distributions of six genes (CTSB, CTSL, CTSS, GILT, MHC IIA and MHC IIB) involved in MHC II antigen presentation pathway were tested. These genes were highly expressed in systematic immune tissues and skin and gill mucosal-associated immune tissues. All six genes were upregulated in skin at most time points. Five genes expected CTSS was upregulated in spleen at most time points. CTSB, CTSL and MHC IIA were upregulated in the gill and head kidney at some time points. These results indicate that the presentation of MHC II antigen intensively occurred in local infected skin and gill. Spleen, not head kidney, had the most extensive systematic antigen presentation. In skin, six genes most likely peaked at day 2, earlier than in spleen (5-7 days), marking an earlier skin antibody peak than any recorded in serum previously. This significant and earlier mucosal antigen presentation indicates that specific immune response occurs in local mucosal tissues.

Characterization and functional analysis of grouper (Epinephelus coioides) MEK1 and MEK2.

MEK dual-specificity protein kinases are a group of mitogen-activated protein kinase kinases, which act as an integration point by transferring extracellular signals to the nucleus. To investigate the function of MEK in teleost fish, we cloned MEK1 and MEK2 cDNA sequences from the orange-spotted grouper (Epinephelus coioides). EcMEK1 and EcMEK2 shared 80% amino acid identity with each other. EcMEK1 had 89-99% amino acid identity with teleosts or mammals, whereas EcMEK2 shared 85-97% amino acid identity. The exon structures of the grouper MEK1/2 genes were conserved with zebrafish and human MEK1/2. Tissue distribution analysis showed that EcMEK1 and EcMEK2 had a similar expression pattern in grouper tissues and was mainly transcribe in systemic immune organs. Both EcMEK1 and EcMEK2 were distributed throughout the cytoplasm of transfected GS or HEK293T cells. Overexpression of EcMEK1 or EcMEK2 activated Activator protein 1 dependent luciferase. The phosphorylation levels of EcMEK1/2 and EcERK1/2 were significantly increased in head kidney leukocytes by stimulation with PMA treatment. The grouper MEK1/2-ERK1/2 axis was activated in Cryptocaryon irritans infection and showed an enhanced phosphorylation after immunization.

A novel MKK gene (EcMKK6) in Epinephelus coioides: Identification, characterization and its response to Vibrio alginolyticus and SGIV infection.

Mitogen-activated protein kinase 6 (MKK6) is one of the major important central regulatory proteins response to environmental and physiological stimuli. In this study, a novel MKK6, EcMKK6, was isolated from Epinephelus coioides, an economically important cultured fish in China and Southeast Asian counties. The open reading frame (ORF) of EcMKK6 is 1077 bp encoding 358 amino acids. EcMKK6 contains a serine/threonine protein kinase (S_TKc) domain, a tyrosine kinase catalytic domain, a conserved dual phosphorylation site in the SVAKT motif and a conserved DVD domain. By in situ hybridization (ISH) with Digoxigenin-labeled probe, EcMKK6 mainly located at the cytoplasm of cells, and a little appears in the nucleus. EcMKK6 mRNA can be detected in all eleven tissues examined, but the expression level is different in these tissues. After challenge with Vibrio alginolyticus and Singapore grouper iridovirus (SGIV), the transcription level of EcMKK6 was apparently up-regulated in the tissues examined. The data demonstrated that the sequence and the characters of EcMKK6 were conserved, EcMKK6 showed tissue-specific expression profiles in healthy grouper, and the expression was significantly varied after pathogen infection, indicating that EcMKK6 may play important roles in E. coioides during pathogen-caused inflammation.

Molecular characterization and function analysis of grouper (Epinephelus coioides) Bruton's tyrosine kinase BTK.

Bruton's tyrosine kinase (BTK) is a Tec-family tyrosine kinase and plays a crucial role in B cell antigen receptor (BCR) signal pathway. Mutations in humans and mice BTK gene results in X-linked agammaglobulinemia (XLA) and X-linked immunodeficiency (XLD), respectively. To study the function of BTK in teleost, we cloned a BTK gene from orange-spotted grouper. Homology analysis showed that the grouper BTK (EcBTK) had a high amino acid identity with other vertebrates (63%-92%) and shared the highest amino acid identity with ballan wrasse Labrus bergylta BTK. EcBTK comprises a Bruton's tyrosine kinase pleckstrin homology (PH) domain, a Tec homology (TH) domain, a Src homology 3 (SH3) domain, a Src homology 2 (SH2) domain and a Protein Kinases, catalytic (PKc) domain. Tissue distribution analysis showed that EcBTK was mainly expressed in immune organs. EcBTK was uniform distributed throughout the cytoplasm of transfected HEK293T cells and overexpression of EcBTK slightly down-regulates NF-κB activity. Ibrutinib treatment can reduce the phosphorylation level of grouper's BTK. In groupers infected with Cryptocaryon irritans, up-regulation of EcBTK were not seen in the early stage of infected skin and gill until days 14-21. The phosphorylation level of grouper BTK was significantly increased in infected skin and gill.

Identification and functional analysis of grouper (Epinephelus coioides) B-cell linker protein BLNK.

B-cell linker protein (BLNK) is an adaptor protein that plays a crucial role in the B cell antigen receptor (BCR) signal pathway. To investigate the function of BLNK in teleost fish, we cloned a BLNK ortholog gene from the orange-spotted grouper (Epinephelus coioides). Homology analysis showed that the grouper BLNK (EcBLNK) had a 34%-77% amino acid identity in comparison to other vertebrates and shared the highest amino acid identity with BLNK from the Asian seabass Lates calcarifer. EcBLNK comprises an N-terminal SAM domain and a C-terminal B-cell linker SH2 domain. Ten tyrosine residues were well conserved between teleost fish and mammals. Tissue distribution analysis showed that EcBLNK was expressed mainly in immune organs and expression was at the highest level in head kidney. Co-localization of EcBLNK and EcCD79a was observed in transfected HEK293T cells. Overexpression of EcBLNK did not activate nuclear factor kappa-light-chain-enhancer of activated B cells. The protein level of EcBLNK in grouper head kidney leukocytes was increased by stimulation with lipopolysaccharide. In groupers infected with Cryptocaryon irritans, EcBLNK was regulated in the infected sites and the systemic organ which suggests that EcBLNK was activated in the immune response to parasite infection.

Characterization and expression patterns of ERK1 and ERK2 from Epinephelus coioides against Cryptocaryon irritans infection.

Mitogen-activated protein kinases (MAPKs), a group of serine-threonine protein kinases, play a crucial role in immunoreaction response to extra environmental stresses. In this study, two novel MAPKs, Ec-ERK1 and Ec-ERK2, were identified from Epinephelus coioides. Both Ec-ERK1 and Ec-ERK2 sequences contain a highly conserved Thr-Glu-Tyr (TEY) motif, an HRD domain, and an ATP binding loop containing GXGXXG. An analysis of phylogenetic relationships demonstrated that ERK amino acid sequences were conserved between different species indicating that the functions may be similar. Ec-ERK1 and Ec-ERK2 mRNA can be detected in all thirteen tissues examined, but the expression level is different in these tissues. The expression patterns of these two genes in E. coioides were also detected against Cryptocaryon irritans infection, which is capable of killing large numbers of fish in a short time and has a serious impact on aquaculture. The expression was up-regulated in most of the tissues examined, with the highest expressions of Ec-ERK1 (3.9 times) occurring in the head kidney and Ec-ERK2 (3.5 times) occurring in the spleen. There was no significant correlation between the expression of Ec-ERK1/Ec-ERK2 and the expression of nuclear factor kappaB (NF-kB). The results indicated the sequences and the characters of Ec-ERK1/ERK2 were conserved, Ec-ERK1/ERK2 showed tissue-specific expression patterns in healthy grouper, and their expressions were significantly varied post C. irritans infection, suggesting Ec-ERK1/ERK2 may play important roles in these tissues during pathogen-caused inflammation.

Grouper (Epinephelus coioides) TCR signaling pathway was involved in response against Cryptocaryon irritans infection.

T cell activation is a complicated process accompanying with the activation of T cell receptor (TCR) signaling pathway, which is not well described in teleost fish. The initiation of this pathway depends on the interaction of membrane TCR co-receptors (e.g. CD4/8, CD3 and CD45) and a series of cytoplasmic protein tyrosine kinases (e.g. Lck, Fyn and ZAP70). Cyptocaryon irritans is a ciliate pathogen of marine fish white spot disease causing huge economic lost in marine aquaculture. This parasite can infect fish gill and skin and is considered to be a good pathogen model for fish gill and skin mucosal immunity. Our previous studies showed the locally mucosal antibody response was important for fish defense against this parasite. While how TCR signaling pathway involved in T cell activation to help B cell activation in C. irritans infected fish is still not known. In the present study, we cloned a grouper TCR co-receptor gene EcCD3ε (537 bp) and its three kinase genes, including EcLck (1512 bp), EcFyn (1605 bp) and EcZAP70 (1893 bp). Homology analysis showed that they all shared the highest identity with corresponding genes from Takifugu rubripes (EcCD3ε 41%, EcLck 88%, EcFyn 98% and EcZAP70 93%), and their conserved motifs involved in the signaling transduction were analyzed. The tissue distribution analysis showed these four genes were high expressed in thymus, and it is interesting to find their comparative high expression in skin, gill and midgut mucosal immune tissues. In C. irritans infected grouper, the expression of three TCR co-receptors (EcCD4-1, EcCD3ε and EcCD45) and three kinases (EcLck, EcFyn and EcZAP70) was tested in skin, gill, head kidney and spleen at 0, 12 h, 24 h, 2 d, 3 d, 5 d and 7 d. All six genes were significantly up-regulated in skin at most tested time points, which indicate the possibility of skin local T cell activation to support the local antibody response. Compared to three TCR co-receptors, significantly up-regulation of three kinases were seen in the spleen, and the spleen fold changes of these three kinases were much higher than head kidney, which indicates spleen maybe the major systematic immune organs for T cell activation in C. irritans infected fish.

Two novel p38 MAPKs identified from Epinephelus coioides and their expression pattern in response to Cryptocaryon irritans infection.

P38 mitogen-activated protein kinases (MAPKs) are one of the most important central regulatory proteins response to extra environmental stresses. In this study, two novel p38 MAPKs, Ec-P38γ and Ec-P38δ, were identified from Epinephelus coioides, an economically important cultured fish in China and Southeast Asian counties. Both of Ec-p38γ and Ec-p38δ sequences contain a serine/threonine protein kinase (S_TKc) domain and a highly conserved Thr-Gly-Tyr (TGY) motif. Analysis of phylogenetic relationships illustrated that p38 amino acid sequences were conserved between different species indicating that the functions may be similar. The four subtypes of p38 (α, ß, γ, and δ) mRNA can be detected in all thirteen tissues examined, but the expression level is different in these tissues. The expression patterns of the four Ec-p38 subtypes in E. coioides were also detected response to Cryptocaryon irritans infection, one of the most important protozoan pathogens of marine fish. The expression of four p38 subtypes was up-regulated in the tissues examined, with the highest expressions of Ec-p38α (5.2 times) and Ec-p38δ (4.2 times) occurring in the skin, while Ec-p38ß (24.8 times) and γ (16.6 times) occurred in the spleen. There was no significantly correlation between the expression of Ec-p38γ/Ec-p38δ and the expression of nuclear factor kappaB (NF-kB). The results indicated the sequences and the characters of Ec-p38γ and Ec-p38δ were conserved, the p38 subtypes showed tissue-specific expression patterns in healthy grouper, and their expressions were significantly up-regulated post C. irritans infection, suggesting these p38 MAPKs may play important roles in these tissues during pathogen-caused inflammation.

Characterization and expression analysis of grouper (Epinephelus coioides) co-stimulatory molecules CD83 and CD80/86 post Cryptocaryon irritans infection.

Co-stimulatory molecules (CD83, CD80 and CD86), belong to immunoglobulin superfamily, are type I membrane glycoprotein, which express on antigen presenting cells and provide the second signal for the activation of T lymphocytes. In the present study, we cloned the grouper's CD83 (675 bp) and CD80/86 (876 bp). Homology analysis showed that both EcCD83 and EcCD80/86 shares the highest amino acid similarity (51% and 47%) for the overall sequence with puffer fish (Takifugu rubripes). Some conserved features and important functional residues in mammalian CD83, CD80 and CD86 were also identified from these molecules of teleosts including grouper, suggesting the function of both molecules may be conserved among vertebrates. In transfected HEK293T cells, both molecules localized on the membrane surface. Tissue distribution analysis showed both EcCD83 and EcCD80/86 mRNAs were mainly expressed in immune organs, and EcCD80/86 was extremely higher expressed in mucosal immune tissues including skin and gill than systematic immune organs, which indicates these co-stimulatory molecules may prime T cell activation in local mucosal tissues. In Cryptocaryon irritans infected groupers, the expression level of EcCD83 and EcCD80/86 were both seen significant up-regulation in the skin at most tested time points.

Characterization, expression, and functional study of IRAK-1 from grouper, Epinephelus coioides.

As crucial components of the toll-like receptor (TLR) and interleukin-1 (IL-1) receptor (IL-1R) signaling pathways, interleukin-1 receptor associated kinase (IRAK) family members play essential roles in an animal's immune response. In this study, an IRAK family member, designated EcIRAK-1, was identified in the orange-spotted grouper Epinephelus coioides, and its role in signal transduction investigated. The full-length EcIRAK-1 gene is 2822 bp, encoding a 760-amino-acid protein that has the typical characteristics of mammalian IRAK-1, including an N-terminal death domain, a ProST domain, a central kinase domain, and C-terminal C1 and C2 domains. EcIRAK-1 shares 42%-79% sequence identity with other fish IRAK-1 proteins, and the death and kinase domains are more conserved than the other domains. Several important amino acids and motifs of mammalian IRAK-1 are also conserved in the grouper and other piscine IRAK-1s. In healthy grouper, EcIRAK-1 was broadly expressed in all the tissues tested, with the highest expression in the gill and skin. After infection with Cryptocaryon irritans, EcIRAK-1 expression increased in the gill and spleen. After its exogenous expression in HEK293T cells, EcIRAK-1 significantly activated nuclear factor kappaB (NF-κB). The death domain, ProST domain, and some conserved amino acids, such as T58, T207, K237, and T387, in EcIRAK-1 are required for its signaling function. These data demonstrate that piscine IRAK-1 has the same structural characteristics as its mammalian counterpart and that its function is conserved among vertebrates.

Grouper (Epinephelus coioides) BCR signaling pathway was involved in response against Cryptocaryon irritans infection.

B cell antigen receptor (BCR) plays a crucial role in B cell development and antibody production. It comprises membrane immunoglobulin non-covalently associated with CD79a/CD79b heterodimer. After B cell activation, initial extracellular signals are transduced by BCR complex and amplified by two protein tyrosine kinases, LYN and SYK, which then trigger various pathways. In the present study, we cloned grouper genes for BCR accessory molecules, EcCD79a (669 bp) and EcCD79b (639 bp), as well as two protein tyrosine kinases, EcLYN (1482 bp) and EcSYK (1854 bp). Homology analysis showed that all four molecules had a relatively high amino acid identity compared with those in other animals. Among them, they all shared the highest identity with Takifugu rubripes (EcCD79a 49%, EcCD79b 52%, EcLYN 82% and EcSYK 77%). The conserved features and important functional residues were analyzed. Together with IgM and IgT, tissue distribution analysis showed that all six molecules were mainly expressed in immune organs, particularly systematic immune organs. In groupers infected with Cryptocaryon irritans, up-regulation of EcCD79a and b, EcIgM and EcIgT were not seen in the early stage skin and gill until 14-21 days. Up-regulation of EcCD79a was seen in head kidney at most time points, while EcCD79a and b were only significantly up-regulated in day 14 spleen. Significant up-regulation of EcIgT were seen in day 21 head kidney and day 1, day14 spleen. Significant up-regulation of EcIgM were seen in day 1 head kidney and 12 h spleen. In addition, two protein kinase genes, EcLYN and EcSYK, were up-regulated in the skin at most time points, which suggested that B cells may be activated at the skin local infection site.

Grouper (Epinephelus coioides) IL-34/MCSF2 and MCSFR1/MCSFR2 were involved in mononuclear phagocytes activation against Cryptocaryon irritans infection.

MCSF and its well-known receptor MCSFR had been well studied in humans, regulating the differentiation, proliferation, and survival of the mononuclear phagocyte system. IL-34, which is an alternative ligand of MCSF receptor, was recently identified as a novel cytokine and functionally overlaps with MCSF. However, the functional study of these receptors and their ligands in fish are largely unknown. In the present study, the cDNA of two potential grouper MCSFR ligands have been cloned, EcIL-34 (657 bp) and EcMCSF2 (804 bp), as well as an additional copy of grouper MCSFR, EcMCSFR2 (3141 bp). Sequence analysis showed that these three molecules had higher identities with other fish counterparts compared to mammals and their conserved structures and important functional residues were also analyzed. Tissue distribution analysis showed that EcIL-34 is dominant in brain, gill and spleen compared to EcMCSF2, which is dominant in head kidney, trunk kidney, skin, heart and muscle. EcMCSFR1 was dominant in the most tissues except head kidney and liver compared to EcMCSFR2. The different tissue distribution patterns of these two grouper MCSF receptors and their two ligands indicate the different mononuclear phagocyte differentiation and activation modes in different tissues. In Cryptocaryon irritans infected grouper, EcIL-34 and EcMCSFR2 were the most strongly up-regulated ligand and receptor in the infected sites, gill and skin. Their up-regulation confirmed the proliferation and activation of phagocytes in C. irritans infected sites, which would improve the antigen presentation and elicit the host local specific immune response. In C. irritans infected grouper head kidney, both ligands EcIL-34 and EcMCSF2 (especially EcMCSF2) were up-regulated, but both receptors EcMCSFR1 and EcMCSFR2 were down-regulated, which indicated that the phagocytes differentiation and proliferation may have occurred in this hemopoietic organ, and after that they migrated to the infected cites. The down-regulation of EcIL-34 and EcMCSF2 and no significant change of EcMCSFR1 and EcMCSFR2 in most time point of grouper spleen showed it was less involved in phagocytes response to C. irritans infection.

Characterization and expression analysis of two novel CCR6 chemokine receptors and their three potential ligands CCL20Ls of grouper (Epinephelus coioides) post Cryptocaryon irritans infection.

CCR6 have been demonstrated playing an important role in immune cells homing to mucosal tissues, mediating antigen presentation and immune response in mammals. CCR6 in lower vertebrate leukocyte homing has not yet been revealed. Cryptocaryon irritans is believed to be a good pathogen model for skin and gill mucosal immunity. In this study, we identified two CCR6s and their three possible ligands CCL20 like cDNA sequences, designated as grouper EcCCR6A, EcCCR6B, EcCCL20L1, EcCCL20L2 and EcCCL20L3. It is interesting to find that EcCCR6A has a longer second extracellular loop than EcCCR6B, which is more similar to mammalian CCR6. Tissue distribution analysis showed that EcCCR6A pronouncedly dominates in gill and brain while EcCCR6B dominates in head kidney, trunk kidney and thymus. Three chemokine ligands have their own distinct expression pattern in health grouper tissues. EcCCL20L1 dominates in spleen and head kidney, EcCCL20L2 dominates in gill and thymus, whereas EcCCL20L3 dominates in skin and brain. The expression patterns of these chemokines and chemokine receptors were detected in C. irritans infected grouper and the results showed that EcCCR6A, EcCCR6B and EcCCL20L1 were significantly up-regulated in the skin of C. irritans infected fish, which indicated these two chemokine receptors and their ligand may play important role in immune cells' homing to skin mucosal immune tissues under pathogen caused inflammation.

Grouper (Epinephelus coioides) MyD88 and Tollip: intracellular localization and signal transduction function.

Myeloid differentiation factor 88 (MyD88) and Toll-interacting protein (Tollip) are two important regulatory proteins of the Toll-like receptor (TLR) signaling pathways. In this paper, a Tollip sequence of grouper (Epinephelus coioides) was identified and the signal transduction functions of Tollip and MyD88 were studied. The full length of E. coioides Tollip (EcTollip) cDNA with an open reading frame (ORF) of 1734 nucleotides encoded a putative protein of 274 amino acid residues. The EcTollip protein had conservative domains with mammalian homologous proteins, and high identity (78%-95%) with other vertebrates. MyD88 and Tollip were distributed in the HeLa cytoplasm in a highly condensed form. Over-expression of MyD88 could activate nuclear factor-κB (NF-κB) and its function was dependent on the death domain and ID domain on the N-terminal. Some important functional sites of mammalian MyD88 also affected fish MyD88 signal transduction. Tollip impaired NF-κB signals activated by MyD88, and its activity was dependent on the coupling of ubiquitin to the endoplasmic reticulum degradation (CUE) domain on the C-terminal. These results suggest that MyD88 and Tollip of fish and mammals are conservative on function during evolution.

Synergic chitin degradation by Streptomyces sp. SCUT-3 chitinases and their applications in chitinous waste recycling and pathogenic fungi biocontrol.

The genus Streptomyces comprises the most important chitin decomposers in soil and revealing their chitinolytic machinery is beneficial for the conversion of chitinous wastes. Streptomyces sp. SCUT-3, a chitin-hydrolyzing and a robust feather-degrading bacterium, was isolated previously. The potential chitin-degrading enzymes produced by SCUT-3 were analyzed in the present study. Among these enzymes, three chitinases were successfully expressed in Pichia pastoris at comparatively high yields of 4.8 U/mL (SsExoChi18A), 11.2 U/mL (SsExoChi18B), and 17.8 U/mL (SsEndoChi19). Conserved motifs and constructive 3D structures of these three exo- and endochitinases were also analyzed. These chitinases hydrolyzed colloidal chitin to chitin oligomers. SsExoChi18A showed apparent synergic effects with SsEndoChi19 in colloidal chitin and shrimp shell hydrolysis, with an improvement of 29.3 % and 124.9 %, respectively. Compared with SsExoChi18B and SsEndoChi19, SsExoChi18A exhibited the strongest antifungal effects against four plant pathogens by inhibiting mycelial growth and spore germination. This study provided good candidates for chitinous waste-processing enzymes and antifungal biocontrol agents. These synergic chitin-degrading enzymes of SCUT-3 are good targets for its further genetical modification to construct super chitinous waste-degrading bacteria with strong abilities to hydrolyze both protein and chitin, thereby providing a direction for the future path of the chitinous waste recycling industry.