Xiao-Chun Luo

Immune-related genes expression profile in orange-spotted grouper during exposure to Cryptocaryon irritans.

Cryptocaryon irritans is one of the most important ectoparasites of marine fish. To identify the potential role of immune‐related genes in antiparasitic immune responses in fish, we monitored the expression change of IL‐8, COX‐2, C‐type lectin and transferrin in local and systemic immune organs of orange‐spotted grouper post‐C. irritans infection. IL‐8 expression was up‐regulated during the course of infection in the skin, while COX‐2 and transferrin expression was up‐regulated in the gill. COX‐2 expression was significantly down‐regulated in the spleen (0·7–5% of its control) and head kidney (0·5–4% of its control) post‐primary infection. Transferrin expression was also down‐regulated in the spleen and head kidney from 6 h to 5 days post‐primary infection. However, C‐type lectin expression was up‐regulated in all tested organs post‐infection, with the exception of day 7 in the spleen post‐primary infection where the expression level was slightly down‐regulated (44% of its control). These results suggest that these four immune‐related genes play an important role in grouper anti‐C. irritans infection and that local immune organs as the active organs contribute more than systemic immune organs to this course.

Molecular characterization and functional analysis of TRAF6 in orange-spotted grouper (Epinephelus coioides)

Tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) is a crucial signal transducer in both the TNFR superfamily and Toll-like receptor/interleukin 1R family. Although significant progress has been made in clarifying the role of TRAF6 in mammals, the function of TRAF6 in fish is still poorly understood. In this study, we cloned the orange-spotted grouper (Epinephelus coioides) TRAF6 (EcTRAF6) cDNA, with an open reading frame of 1713bp encoding 570 amino acids. Sequence analysis indicated that EcTRAF6 contains the four characteristic domains conserved in the TRAF family, including an N-terminal RING finger, two zinc fingers, a coiled-coil domain, and a C-terminal MATH domain. Homology alignment and phylogenetic analysis demonstrated that EcTRAF6 shares high sequence identity with TRAF6 of other fish species. The EcTRAF6 gene contains seven exons and six introns, which is similar to the organization in ayu, but not in the common carp, human, or mouse (six exons and five introns). EcTRAF6 transcripts were broadly expressed in all tissues tested, and increased after infection with Cryptocaryon irritans. Intracellular localization showed EcTRAF6 was distributed mainly in the cytoplasm. Over-expression of wild type (WT) EcTRAF6, truncated forms of EcTRAF6, including ΔZinc finger 2 and ΔMATH, and a mutant of C78A activated NF-κB strongly in HEK293T cells; whereas truncations, including ΔRING, ΔZinc finger 1 and Δcoiled-coiled, and a mutant of K132R induced the activity of NF-κB slightly compared to WT EcTRAF6, implying the latter has a more crucial role in downstream signal transduction. Together, these results suggested EcTRAF6 functions like that of mammals to activate NF-κB, and it might have an important role in host defense against parasitic infections.

Identification of IRAK-4 in grouper (Epinephelus coioides) that impairs MyD88-dependent NF-κB activation

Interleukin 1 (IL-1) receptor-associated kinase (IRAK) family members are crucial signal transducer in the Toll-like receptor/IL-1R signal pathway, which mediates downstream signal cascades involved in the innate and adaptive immune responses. In this study, we identified an IRAK-4 protein (EcIRAK-4) in the orange-spotted grouper (Epinephelus coioides), with an N-terminal death domain, a proST domain, and a central kinase domain, similar to that of other fishes and mammals. A sequence alignment and phylogenic analysis demonstrated that full-length EcIRAK-4 shares a high degree of sequence identity with those of other fishes, especially the roughskin sculpin, and their death domains and kinase domains share greater identity than their proST domains. A conservation analysis indicated that most of the functional sites in mammalian IRAK-4 are conserved in IRAK-4 of the grouper and other fishes, with the exception of the sites of interaction with IRAK-2 and one autophosphorylation site within the activation loop. EcIRAK-4 is broadly expressed in all the tissues examined, with highest expression in the head kidney and liver. After infection with Cryptocaryon irritans, EcIRAK-4 expression was significantly upregulated, especially in the skin, which suggests that this molecule is involved in the hosts defense against parasitic infection. Surprisingly, after cotransfection with grouper MyD88, EcIRAK-4 significantly impaired the NF-κB activity induced by MyD88. EcIRAK-4 was uniformly distributed throughout the cytoplasm in HeLa cells. These findings suggest that although IRAK-4 is evolutionarily conserved between fish and mammals, its signal transduction function is markedly different.

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.

Development of Rapid Immunochromatographic Test for Hemagglutinin Antigen of H7 Subtype in Patients Infected with Novel Avian Influenza A (H7N9) Virus

Background Since human infection with the novel H7N9 avian influenza virus was identified in China in March 2013, the relatively high mortality rate and possibility of human-to-human transmission have highlighted the urgent need for sensitive and specific assays for diagnosis of H7N9 infection. Methodology/Principal Findings We developed a rapid diagnostic test for the novel avian influenza A (H7N9) virus using anti-hemagglutinin (HA) monoclonal antibodies specifically targeting H7 in an immunochromatographic assay system. The assay limit of detection was 103.5 pfu/ml or 103TCID50 of H7N9 virus. The assay specifically detected H7N9 viral isolates and recombinant HA proteins of H7 subtypes including H7N7 and H7N9, but did not react with non-H7 subtypes including H1N1, H3N2, H5N1, H5N9, and H9N2. The detection sensitivity was 59.4% (19/32) for H7N9 patients confirmed by RT-PCR. Moreover, the highest sensitivity of 61.5% (16/26) was obtained when testing H7N9 positive sputum samples while 35.7% (5/14) of nasopharyngeal swabs and 20% (2/10) of fecal samples tested positive. No false positive detection was found when testing 180 H7N9 negative samples. Conclusions/Significance Our novel rapid assay can specifically detect H7 HA antigen, facilitating rapid diagnosis for prevention and control of the on-going H7N9 epidemic.

Enzymatic characteristics of a recombinant neutral protease I (rNpI) from Aspergillus oryzae expressed in Pichia pastoris.

A truncated neutral protease I (NpI) from Aspergillus oryzae 3.042 was expressed in Pichia pastoris with a high enzyme yield of 43101 U/mL. Its optimum pH was about 8.0, and it was stable in the pH range of 5.0-9.0. Its optimum temperature was about 55 °C and retained >90% activity at 50 °C for 120 min. Recombinant NpI (rNpI) was inhibited by Cu(2+) and EDTA. Eight cleavage sites of rNpI in oxidized insulin B-chain were determined by mass spectrometry, and five of them had high hydrophobic amino acid affinity, which makes it efficient in producing antihypertensive peptide IPP from β-casein and a potential debittering agent. The high degree of hydrolysis (DH) of rNpI to soybean protein (8.8%) and peanut protein (11.1%) compared to papain and alcalase makes it a good candidate in the processing of oil industry byproducts. The mutagenesis of H(429), H(433), and E(453) in the deduced zinc-binding motif confirmed rNpI as a gluzincin. All of these results show the great potential of rNpI to be used in the protein hydrolysis industry.

Comparative transcriptional profile of the fish parasite Cryptocaryon irritans

BackgroundCryptocaryon irritans is an obligate ectoparasitic ciliate pathogen of marine fishes. It can infect most marine teleosts and cause heavy economic losses in aquaculture. There is currently no effective method of controlling this disease, and little information is available regarding the genes involved in its development and virulence. We aimed to investigate the distinct features of the three major life-cycle stages of C. irritans in terms of gene transcription level, and identify candidate vaccines/drug targets. We established a reference transcriptome of C. irritans by RNA-seq.MethodsThree cDNA libraries using total poly(A)+ mRNA isolated from trophonts, tomonts, and theronts was constructed and sequenced, respectively. Clean reads from the three stages were de novo assembled to generated unigene. Annotation of unigenes and transcriptomic comparison of three stages was performed.ResultsTotals of 73.15, 62.23, and 109.57 million clean reads were generated from trophont, tomont, and theront libraries, respectively. After de novo assembly, 49,104 unigenes were obtained, including 9,253 unigenes with significant similarities to proteins from other ciliates. Transcriptomic comparisons revealed that 2,470 genes were differentially expressed among the three stages, including 2,011, 1,404, and 1,797 genes that were significantly differentially expressed in tomont/theront, tomont/trophont, and theront/trophont pairwise comparisons, respectively. Based on the results of hierarchical clustering, all differentially expressed genes (DEGs) were located in five major clusters. DEGs in clusters 1 and 2 were more highly expressed in tomonts than in other stages, DEGs in cluster 3 were dominant in the tomont and trophont stages, whereas clusters 4 and 5 included genes upregulated in the theront stage. In addition, Immobilization antigens (I-antigens) and proteases have long been considered major targets for vaccine development and potential drug targets in parasites, respectively. In the present study, nine putative I-antigens transcripts and 161 protease transcripts were found in the transcriptome of C. irritans.ConclusionIt was concluded that DEGs enriched in tomonts were involved in cell division, to increase the number of theronts and ensure parasite continuity. DEGs enriched in theronts were associated with response to stimuli, whereas genes enriched in trophonts were related to nutrient accumulation and cell growth. In addition, the I-antigen and protease transcripts in our transcriptome could contribute to the development of vaccines or targeted drugs. Together, the results of the present study provide novel insights into the physiological processes of a marine parasitic ciliate.

Transcriptomic variation of locally-infected skin of Epinephelus coioides reveals the mucosal immune mechanism against Cryptocaryon irritans

Abstract Fish skin is the largest immunologically active mucosal organ, providing first‐line defense against external pathogens. However, the skin‐associated immune mechanisms of fish are still unclear. Cryptocaryon irritans is an obligate ectoparasitic ciliated protozoan that infects almost all marine fish, and is believed to be an excellent pathogen model to study fish mucosal immunity. In this study, a de novo transcriptome assembly of Epinephelus coioides skin post C. irritans tail‐infection was performed for the first time using the Illumina HiSeq™ 2500 system. Comparative analyses of infected skin (group Isk) and uninfected skin (group Nsk) from the same challenged fish and control skin (group C) from uninfected control fish were conducted. As a result, a total of 91,082 unigenes with an average length of 2880 base pairs were obtained and among them, 38,704 and 48,617 unigenes were annotated based on homology with matches in the non‐redundant and zebrafish database, respectively. Pairwise comparison resulted in 10,115 differentially‐expressed genes (DEGs) in the Isk/C group comparison (4,983 up‐regulated and 5,132 down‐regulated), 2,275 DEGs in the Isk/Nsk group comparison (1,319 up‐regulated and 956 down‐regulated) and 4,566 DEGs in the Nsk/C group comparison (1,534 up‐regulated and 3,032 down‐regulated). Seven immune‐related categories including 91 differentially‐expressed immune genes (86 up‐regulated and 5 down‐regulated) were scrutinized. Both DEGs and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and immune‐related gene expression analysis were used, and both analyses showed that the genes were more significantly altered in the locally‐infected skin than in the uninfected skin of the same challenged fish. This suggests the skins local immune response is important for host defense against this ectoparasite infection. Innate immune molecules, including hepcidin, C‐type lectin, transferrin, transferrin receptor protein, serum amyloid A, cathepsin and complement components were significantly up‐regulated (fold‐change ranged from 3.3 to 12,944) in infected skin compared with control skin. The up‐regulation of chemokines and chemokine receptors and activation of the leukocyte transendothelial migration pathway suggested that leucocytes intensively migrated to the local infected sites to mount a local immune defense. Toll‐like receptors (TLRs) 1, 2, 5 and 5S were most significantly up‐regulated in the infected skin, suggesting that these TLRs may be involved in parasite pathogen‐associated molecular pattern (PAMPs) recognition. Up‐regulation of the dendritic cell markers CD209 and CD83 and other antigen presentation pathway molecules provided evidence for skin local antigen presentation. Up‐regulation of the T cell markers CD4 and CD48, B cell markers CD22 and CD81 and B cell receptor signaling kinase Lyn, showed the presence and population expansion of T/B cells at locally‐infected sites, which suggested possible activation of a local specific immune response in the skin. Our results will facilitate in‐depth understanding of local immune defense mechanisms in fish skin against ectoparasite infection. HighlightsWe firstly explored Epinephelus coioides skin immune response following local exposure to Cryptocaryon irritans by RNA‐Seq.91082 unigenes with an average length of 2880 base pairs were obtained.DEGs were more significantly altered in the locally‐infected skin than in the uninfected skin of the same challenged fish.Multiple immune‐related pathways were significantly regulated.

Orange-spotted grouper Epinephelus coioides Tak1: molecular identification, expression analysis and functional study

In this study, the complementary (c)DNA sequence encoding orange-spotted grouper Epinephelus coioides Tak1 (ectak1) was cloned, which has an open reading frame of 1728 bp that encodes 575 amino acids (aa). Sequence analysis indicated that Ectak1 contains two characteristic conserved domains, i.e. an N-terminal serine-threonine protein kinase catalytic domain (27-275 aa) and a C-terminal coiled-coil region (499-562 aa). Ectak1 shares high sequence identity with Tak1 from other fish species, especially those of Nile tilapia Oreochromis niloticus (96%) and zebra mbuna Maylandia zebra (96%). ectak1 transcripts were expressed broadly in all of the tissues tested, but ectak1 expression was reduced mainly in the local infection sites (skin and gill) after infection with Cryptocaryon irritans. Intracellular localization analysis showed that Ectak1 was distributed mainly in the cytoplasm. A luciferase reporter assay showed that Ectak1 significantly impaired the NF-κB activity induced by E. coioides Myd88 and Traf6. Overall, these results suggest that Ectak1 functions to reduce the activity of NF-κB induced by toll-like receptor (TLR) signal molecules in HEK-293T cells, and it might have an important role in host defences against parasitic infections.

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

Abstract 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 groupers 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. HighlightsCD83 and CD80/86 genes were identified from the orange‐spotted grouper.EcCD80/86 was extremely high expressed in skin and gill.EcCD83 and EcCD80/86 were significantly up‐regulated in C. irritans infected skin.