Fumiao Zhang

Molecular characterization of hepcidin gene in common carp (Cyprinus carpio L.) and its expression pattern responding to bacterial challenge.

Hepcidin is a cysteine-rich cationic antimicrobial peptide (AMP), which plays an important role in host innate immune system and iron regulation. A great many of hepcidin genes have been identified from vertebrates, including various fish species. Here we report the cloning and identification of a hepcidin cDNA from the liver of common carp (Cyprinus carpio L.). The full-length cDNA of the common carp hepcidin was 647 bp, which contained an ORF of 276 bp encoding a prepropeptide of 91 amino acid residues. The predicted preprohepcidin consisted of three domains: a signal peptide of 24 amino acids, a prodomain of 42 amino acids and a mature peptide of 25 amino acids, which containd eight cysteine residues at the identical conserved position. The genomic organization of common carp hepcidin was identified, which contained three exons and two introns, similarly to corresponding genes in mammals and other fish species. Sequence alignment and phylogenetic analysis showed that hepcidins were conserved in different species, and common carp hepcidin was type 1 hepcidin according to the phylogenetic tree, which had the highest identity with mud loach and zebrafish. Real-time PCR assay showed that the hepcidin gene was mostly expressed in liver, and expressed in all the other tested tissues of common carp in different levels. When challenged with pathogenic bacterium, Vibrio anguillarum, the expression level of common carp hepcidin was quickly up-regulated in liver, spleen, head kidney and hindgut, implying that hepcidin may be an important component of the innate immune system of common carp and involved in mucosal immune response against invading pathogens.

Identification and expression analysis of irak1 gene in common carp Cyprinus carpio L.: indications for a role of antibacterial and antiviral immunity

In this study, the full-length complementary (c)DNA of interleukin-1 receptor-associated kinase 1 gene (irak1) was cloned from common carp Cyprinus carpio. The complete open reading frame of irak1 contained 2109 bp encoding a protein of 702 amino acid residues that comprised a death domain, a ProST region, a serine-threonine-specific protein kinase catalytic domain and a C-terminal domain. The amino-acid sequence of C. carpio Irak1 protein shared sequence homology with grass carp Ctenopharyngodon idellus (84.5%). The phylogenetic tree of IRAKs separated the polypeptides into four clades, comprising IRAK1s, IRAK2s, IRAK3s and IRAK4s. Cyprinus carpio Irak1 fell into the cluster with previously reported IRAK1s including teleost Irak1s. The irak1 gene was highly expressed in gills, followed by brain, skin, hindgut, buccal epithelium, spleen, foregut, head kidney and liver, and was expressed at lowest levels in gonad and muscle. The irak1 messenger (m)RNA expression was up-regulated in liver, spleen, head kidney, foregut, hindgut, gills and skin after stimulation with Vibrio anguillarum and poly(I:C), and significantly high up-regulated expression was observed in liver and spleen. These results implied that irak1 might participate in antibacterial and antiviral innate immunity. These findings gave the indications that irak1 may participate in antibacterial and antiviral immunity.

Molecular characterization and expression pattern of X box-binding protein-1 (XBP1) in common carp (Cyprinus carpio L.): Indications for a role of XBP1 in antibacterial and antiviral immunity

Abstract X box‐binding protein‐1 (XBP1) is a transcription factor that is essential for the unfolded protein response (UPR) and the differentiation of plasma cells, and some findings have also uncovered its function in innate immunity. XBP1 typically has two different transcripts, un‐spliced (XBP1u) and spliced forms (XBP1s), but XBP1s is an active transcription factor in the regulation of target genes. To date, there is no evidence about the identification and function of XBP1 in common carp. Moreover, no data are currently available regarding the role of fish XBP1 in innate immunity. Thus, to determine whether XBP1 is involved in innate immune response in common carp, we cloned CcXBP1s and examined the expression of XBP1s and a XBP1s stimulated gene (IL‐6) after Aeromonas hydrophila (A. hydrophila) and polyinosinic‐polycytidylic acid (polyI:C) challenges. The results imply that CcXBP1s, as an active transcription factor, might play regulation roles in the antibacterial and antiviral innate immune responses of common carp. This allows us to gain new insights into the immunological function of XBP1 in fish innate immunity and the evolution of this important class of genes across vertebrates. HighlightsThe XBP1 cDNA and genomic DNA was cloned and characterized in common carp.XBP1s mRNA was up‐regulated after A. hydrophila or polyI:C challenge.IL‐6 was correlated with the up‐regulation of XBP1s following pathogen challenges.

Non-mammalian Toll-like receptor 18 (Tlr18) recognizes bacterial pathogens in common carp ( Cyprinus carpio L.): Indications for a role of participation in the NF-κB signaling pathway

ABSTRACT Toll‐like receptors are important pattern recognition receptors that can recognize pathogen‐associated molecular patterns (PAMPs) and play a critical role in innate immunity. In the present study, tlr18 was identified from common carp (Cyprinus carpio L.) (named Cctlr18). The deduced amino acid sequence contained only a signal peptide, eight LRR (leucine‐rich repeat) motifs, a transmembrane region and a TIR (Toll/IL‐1 receptor) domain. Phylogenetic analysis showed that CcTlr18 was most closely related to Ctenopharyngodon idella Tlr18. Quantitative real‐time PCR analysis showed that Cctlr18 was constitutively expressed in all investigated tissues with the highest expression level in the skin and lowest expression in the gonad. After injection with inactivated Aeromonas hydrophila, Cctlr18 expression was significantly up‐regulated in the head kidney, foregut, hindgut and skin. Moreover, significant up‐regulation of Cctlr8 was observed in the spleen, head kidney, hindgut and skin after immersion with live A. hydrophila. In addition, the expression of Cctlr18 was up‐regulated in PGN or flagellin‐stimulated HKLs. Luciferase reporter assays showed that Cctlr18 activated NF‐&kgr;B in 293 T cells and that NF‐&kgr;B activity was enhanced in Cctlr18 and Ccmyd88 co‐transfected cells. Furthermore, Cctlr18 could induce the expression of cytokines genes, including ifn, il‐1&bgr; and il‐10, in EPC cells. The results suggested that Cctlr18 plays an important role in the immune response and provides basic information for investigating the mechanisms of fish tlr18. HighlightsCctlr18 was cloned and characterized from the common carp (Cyprinus carpio L.).The expression of Cctlr18 was up‐regulated after challenge with Aeromonas hydrophila.Cctlr18 could activate NF‐&kgr;B in 293 T cells.Cctlr18 could induce the expression of ifn, il‐1&bgr; and il‐10 in EPC cells.

Characterization of IgM-binding protein: A pIgR-like molecule expressed by intestinal epithelial cells in the common carp (Cyprinus carpio L.)

The adaptive mucosal immune system seems to be an important defence mechanism for fish, but the binding of immunoglobulin M (IgM) in mucosal organs has yet to be clarified in fish. The present study was designed to search for the protein that binds IgM in the intestinal epithelium and determine its distribution in mucosa-associated lymphoid tissues of the common carp (Cyprinus carpio L.). The serum-derived carp IgM fraction was isolated by Sephadex G-200 and assessed for purity by SDS-PAGE under reducing conditions. Serum IgM was subsequently used in affinity chromatography of IgM-sepharose for isolation of a specific binding protein from the intestinal epithelium. The resultant adsorbed protein (IgM-binding protein) demonstrated a single band using SDS-PAGE, with a relative molecular mass of 43.5 kDa. These results demonstrate for the first time that IgM-sepharose can be used as affinity chromatography to purify membrane proteins that bind IgM in fish. Using immunohistochemistry, we found that the distribution of IgM-binding protein in intestinal tissues was abundant, while that of splenic leukocytes were undetectable. Our study indicates that IgM-binding protein might be involved in transportation of IgM in intestine tissues, which is distinct from the IgM receptor on splenocytes.

Pathogen invasion changes the intestinal microbiota composition and induces innate immune responses in the zebrafish intestine

Abstract Numerous bacteria are harbored in the animal digestive tract and are impacted by several factors. Intestinal microbiota homeostasis is critical for maintaining the health of an organism. However, how pathogen invasion affects the microbiota composition has not been fully clarified. The mechanisms for preventing invasion by pathogenic microorganisms are yet to be elucidated. Zebrafish is a useful model for developmental biology, and studies in this organism have gradually become focused on intestinal immunity. In this study, we analyzed the microbiota of normal cultivated and infected zebrafish intestines, the aquarium water and feed samples. We found that the predominant bacteria in the zebrafish intestine belonged to Gammaproteobacteria (67%) and that feed and environment merely influenced intestinal microbiota composition only partially. Intestinal microbiota changed after a pathogenic bacterial challenge. At the genus level, the abundance of some pathogenic intestinal bacteria increased, and these genera included Halomonas (50%), Pelagibacterium (3.6%), Aeromonas (2.6%), Nesterenkonia (1%), Chryseobacterium (3.4‰), Mesorhizobium (1.4‰), Vibrio (1‰), Mycoplasma (0.7‰) and Methylobacterium (0.6‰) in IAh group. However, the abundance of some beneficial intestinal bacteria decreased, and these genera included Nitratireductor (0.8‰), Enterococcus (0.8‰), Brevundimonas (0.7‰), Lactococcus (0.7‰) and Lactobacillus (0.4‰). Additionally, we investigated the innate immune responses after infection. ROS levels in intestine increased in the early stages after a challenge and recovered subsequently. The mRNA levels of antimicrobial peptide genes lectin, hepcidin and defensin1, were upregulated in the intestine after pathogen infection. These results suggested that the invasion of pathogen could change the intestinal microbiota composition and induce intestinal innate immune responses in zebrafish. HighlightsGammaproteobacteria had the highest abundance level of zebrafish intestine.Aeromonas hydrophila challenge changed intestinal microbiota abundance.Reactive oxygen species level of intestine increased after A. hydrophila infection.Antimicrobial peptides participated in intestinal antibacterial response.

Cytochemical characterization of peripheral blood cell populations of two Cyprinidae, Carassius auratus and Ctenopharyngodon idellus

Fish are the most diverse species of all vertebrate groups, and their blood cells have shown variable characteristics in terms of morphology. Cytochemical staining for enzyme activity in blood leukocytes will help assess the immune function of fish. We characterize blood cells from crucian carp (Carassius auratus) and grass carp (Ctenopharyngodon idellus) by using a Diff‐Quick stain as well as different cytochemical methods. Blood specimens obtained from crucian carp and grass carp were evaluated after cytochemical staining for acid phosphatase (ACP), alkaline phosphatase (ALP), naphthol AS chloroacetate esterase (AS‐DNCE), naphthyl acetate esterase (NAE), α‐naphthyl butyrate esterase (NBE), peroxidase (MPO) and periodic acid–Schiffs reaction (PAS) using commercial kits. Blood cell types were evaluated based on their morphological characteristics and the presence or absence of specific chromogen. The expression pattern of enzymes was similar between the two Cyprinidae and was also broadly consistent with other fish species. However, there were some interesting differences detected between crucian carp and grass carp, including naphthol AS chloroacetate esterase activity in monocytes, peroxidase activity and location in thrombocytes. The ACP, ALP and MPO expressions of different leukocytes of the two Cyprinidae were evaluated by Image Pro Plus and were analysed for statistical significant differences. This investigation provides basic haematology and enzyme activity analyses for crucian carp and grass carp and serves as an approach to evaluating the immune response of fish.