Transcriptome and expression profiling analysis revealed changes of multiple signaling pathways involved in immunity in the large yellow croaker during Aeromonas hydrophila infection

<p>Abstract</p> <p>Background</p> <p>The large yellow croaker (<it>Pseudosciaena crocea</it>) is an economically important marine fish in China suffering from severe outbreaks of infectious disease caused by marine bacteria such as <it>Aeromonas hydrophila </it>(<it>A. hydrophila</it>), resulting in great economic losses. However, the mechanisms involved in the immune response of this fish to bacterial infection are not fully understood. To understand the molecular mechanisms underlying the immune response to such pathogenic bacteria, we used high-throughput deep sequencing technology to investigate the transcriptome and comparative expression profiles of the large yellow croaker infected with <it>A. hydrophila</it>.</p> <p>Results</p> <p>A total of 13,611,340 reads were obtained and assembled into 26,313 scaffolds in transcriptional responses of the <it>A. hydrophila</it>-infected large yellow croaker. Via annotation to the NCBI database, we obtained 8216 identified unigenes. In total, 5590 (68%) unigenes were classified into Gene Ontology, and 3094 unigenes were found in 20 KEGG categories. These genes included representatives from almost all functional categories. By using Solexa/Illumina's DeepSAGE, 1996 differentially expressed genes (P value < 0.05) were detected in comparative analysis of the expression profiles between <it>A. hydrophila</it>-infected fish and control fish, including 727 remarkably upregulated genes and 489 remarkably downregulated genes. Dramatic differences were observed in genes involved in the inflammatory response. Bacterial infection affected the gene expression of many components of signaling cascades, including the Toll-like receptor, JAK-STAT, and MAPK pathways. Genes encoding factors involved in T cell receptor (TCR) signaling were also revealed to be regulated by infection in these fish.</p> <p>Conclusion</p> <p>Based on our results, we conclude that the inflammatory response may play an important role in the early stages of infection. The signaling cascades such as the Toll-like receptor, JAK-STAT, and MAPK pathways are regulated by <it>A. hydrophila </it>infection. Interestingly, genes encoding factors involved in TCR signaling were revealed to be downregulated by infection, indicating that TCR signaling was suppressed at this early period. These results revealed changes of multiple signaling pathways involved in immunity during <it>A. hydrophila </it>infection, which will facilitate our comprehensive understanding of the mechanisms involved in the immune response to bacterial infection in the large yellow croaker.</p

Identification of Two Subgroups of Type I IFNs in Perciforme Fish Large Yellow Croaker Larimichthys crocea Provides Novel Insights into Function and Regulation of Fish Type I IFNs

Like mammals, fish possess an interferon regulatory factor 3 (IRF3)/IRF7-dependent type I IFN responses, but the exact mechanism by which IRF3/IRF7 regulate the type I IFNs remains largely unknown. In this study, we identified two type I IFNs in the Perciforme fish large yellow croaker Larimichthys crocea, one of which belongs to the fish IFNd subgroup, and the other is assigned to a novel subgroup of group I IFNs in fish, tentatively termed IFNh. The two IFN genes are constitutively expressed in all examined tissues, but with varied expression levels. Both IFN genes can be rapidly induced in head kidney and spleen tissues by polyinosinic-polycytidylic acid. The recombinant IFNh was shown to be more potent to trigger a rapid induction of the antiviral genes MxA and PKR than the IFNd, suggesting that they may play distinct roles in regulating early antiviral immunity. Strikingly, IFNd, but not IFNh, could induce the gene expression of itself and IFNh through a positive feedback loop mediated by the IFNd-dependent activation of IRF3 and IRF7. Furthermore, our data demonstrate that the induction of IFNd can be enhanced by the dimeric formation of IRF3 and IRF7, while the IFNh expression mainly involves IRF3. Taken together, our findings demonstrate that the IFN responses are diverse in fish and are likely to be regulated by distinct mechanisms

Molecular characterization and expression analysis of beta(2)-microglobulin in large yellow croaker Pseudosciaena crocea

Nation '863' Project [2006AA100310]; National Natural Science Foundation of China [30571439, 30871925]; Scientific Research Foundation of Third Institute of Oceanography. SOA [2009001]beta(2)-Microglobulin (beta(2)m), a protein necessary for proper folding, peptide binding, and surface display of class I antigens plays an important role in immune response. The full-length cDNA containing beta(2)m was cloned from the spleen cDNA library of large yellow croaker Pseudosciaena crocea (Pscr-beta (2) m) by expressed sequence tag (EST) analysis. The Pscr-beta (2) m is 926 nucleotides (nt) long, including an open reading frame (ORF) of 348 nt encoding a polypeptide of 116 amino acids (aa). The deduced Pscr-beta (2) m possessed all characteristic domains of beta(2)m in other species, including a 16-aa leader peptide and a typical immunoglobulin (Ig) and major histocompatibility complex protein (MHC) signature YSCRVTH at residues 81-87. Homology modeling showed that the 3D structure of Pscr-beta (2) m protein is similar to that of human beta(2)m, except for a beta-strand (G) being lost in Pscr-beta (2) m due to amino acid deletion (positions 94-95). Tissue expression profile analysis revealed that the Pscr-beta (2) m was constitutively expressed in all tissues examined, such as kidney, spleen, liver, gills, heart, intestine, brain, and muscle, although at different levels. Upon stimulation with poly(I:C) or inactivated trivalent bacterial vaccine, the expression of Pscr-beta (2) m was significantly up-regulated in intestine, kidney and spleen at 24 h post-induction, and increase of Pscr-beta (2) m transcripts was also observed in liver post-induction with poly(I:C). Real-time PCR further revealed that the expression of Pscr-beta (2) m in intestine, kidney and spleen tissues was differentially regulated by poly(I:C) and bacterial vaccine during 72 h of induction. These results suggested that Pscr-beta (2) m might be involved in both antiviral and antibacterial mechanisms in large yellow croaker

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). Ca2+ 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 Ca2+-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

Expression analysis of immune-relevant genes in the spleen of large yellow croaker (Pseudosciaena crocea) stimulated with poly I : C

A SMART cDNA library from spleen of large yellow croaker (Pseudosciaena crocea) stimulated by poly I:C was constructed. A total of 1039 clones from the library were single-pass sequenced and compared with known sequences in the GenBank database. Of those expressed sequence tags (ESTs), 607 were identified as orthologs of known genes in the GenBank databases by Blast X search. Four hundred and thirty-two did not show significant homology with any known sequences in the public databases. These identified ESTs represented at least 252 different genes, which were categorised into nine groups according to their function. Of the identified genes, 159 genes (63.1%) shared homology with fish genes while 93 (36.9%) showed the highest homology to the genes from other species. Forty-six genes were identified to be involved in immune functions, including complement system components, immunoglobulins, antigen processing and presentation proteins, interferon system proteins, cytokines, and some innate defence molecules. The most frequently occurring genes in this spleen cDNA library were hepcidin precursors represented by 46 ESTs, which were divided into five groups based on their putative amino acid sequences. The expression analysis of selected genes during polyI:C induction was performed by reverse transcription-PCR (RT-PCR), including Mx protein, beta2-microglobulin (beta(2)m), CD2 binding protein 1(CD2BP1), placenta-specific 8 genes, MHC class II associated invariant chain (li) and cytochrome b-245 alpha peptide (Cyba). The results revealed that expression levels of Mx protein, beta(2)m, placenta-specific 8 genes, and Cyba were significantly upregulated at 30 h after induction with poly I:C, and the CD2BP1 expression was also induced by polyl:C, suggesting that these genes may be involved in an immune response induced by poly LC in large yellow croaker. (c) 2006 Elsevier Ltd. All rights reserved

Molecular and functional characterization of a novel stefin analogue in large yellow croaker (Pseudosciaena crocea)

Scientific Research Foundation of Third Institute of Oceanography, SOA [2009001]; Nation '863' Project [2006AA100306, 2007AA091406]; Key Project of Science and Technology of Fujian province [2009N0039]In this paper, we report the molecular cloning of a novel stefin analogue from the spleen of large yellow croaker Pseudosciaena crocea (Lycstefin). The open reading frame (ORF) of 297 nucleotides (nt) of Lycstefin encodes a protein of 99 amino acids (aa) with a putative molecular weight of 11 kDa, in which no signal peptide and potential N-glycoslation site are predicted. The deduced Lycstefin possesses the structural features of the mammalian stefins, including two conserved motifs known to interact with the active sites of family C1 cysteine peptidases: one glycine in the N-terminal region (G(6)) and Gln-Xaa-Val-Xaa-Gly motif (Q(48)LVAG(52)). It shares 32-47.5% aa sequence identity to the sequences found in mammals and other fish species and is rich in cysteine residues (seven cysteines). Genomic analysis revealed that Lyccys gene, 757 nt long, consisted of three exons and two introns. The Lycstefin gene was constitutively expressed in various tissues examined although at different levels. Upon stimulation with poly(I:C) or inactivated trivalent bacterial vaccine, Lycstefin transcript was significantly tip-regulated in spleen and head kidney while down-regulated in blood. Immuno-electron microscopy showed that Lycstefin was mainly localized in the cytoplasm of spleen cells of large yellow croaker, and also in the nucleus. Recombinant Lycstefin protein fused with glutathione S-transferase (rLycstefin) was shown to have strong inhibitory activity against papain with a K(i) of 1.3 x 10(-13) M. The in vivo experiments revealed that Lycstefin could not modulate the expression levels of large yellow croaker tumor necrosis factor-alpha 2 (TNF-alpha 2) and interleukin-10 in spleen and head kidney. To our knowledge, this is the first report on the molecular and functional identification of a stefin analogue in bony fish. (C) 2009 Elsevier Ltd. All rights reserved

Molecular and functional characterization of a cystatin analogue in large yellow croaker (Pseudosciaena crocea)

The cDNA of a cystatin analogue was isolated from the spleen Smart cDNA library of large yellow croaker Pseudosciaena crocea (Lyccys). The open reading frame (ORF) of 354 nucleotides (nt) of Lyccys encodes a protein of 118 amino acids (aa), containing a 21-aa signal peptide and a 97-aa mature polypeptide. The deduced Lyccys possessed structural features of the Family II cystatins, including three evolutionally conserved motifs known to interact with the active sites of cysteine peptidases: Gly at the N-terminus (Gly(25)), Gln-X-Val-X-Gly motif (Q(69)LVAG(73)) and Pro-Try pair at the C-terminus (p(106)W(107)). Genomic analysis revealed that Lyccys gene, spanning 2297 nt, consisted of three exons and two introns. The Lyccys gene was constitutively expressed in all eight tissues examined although at different levels. Real-time PCR analysis revealed that Lyccys transcript in spleen and kidney was obviously up-regulated by poly(I:C) or inactivated trivalent bacterial vaccine, while in blood its expression was down-regulated. Immunoelectron microscopy showed that Lyccys was mainly localized to the rough endoplasmic reticulum (rER) or in the vesicular structures in spleen and kidney cells. Recombinant Lyccys protein fused with glutathione S-transferase (rLyccys) was shown to have remarkable protease-inhibitory activity and well affinity binding to papain (with a K-i of 1.3 x 10(-13) M). An in vivo administration of rLyccys could significantly up-regulate the expression levels of large yellow croaker tumor necrosis factor-alpha 2 (TNF-alpha 2) and interieukin-10 in spleen and kidney, but to a lesser extent increase TNF-alpha 1 expression. These results suggest that the Lyccys is a secreted inhibitor of cysteine proteinases, which may have an immunomodulatory function in inflammation response. (C) 2009 Elsevier Ltd. All rights reserved.Scientific Research Foundation of Third Institute of Oceanography, SOA [2009001]; Nation '863' Project [2006AA10A402, 2006AA100310]; National Natural Science Foundation of China [30871925

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

National Natural Science Foundation of China [30871925, 31001131]; Natural Science Foundation of Fujian Province [2009J05076, 2009N0039, 2009N2003]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 2222 bp 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. (C) 2011 Elsevier Ltd. All rights reserved

Pleomorphobacterium xiamenense gen nov, sp nov, a moderate thermophile isolated from a terrestrial hot spring

Scientific Research Project of Marine Public Welfare Industry of China [201205020, 201005032, 200805032]An aerobic, motile, moderately thermophilic rod, designated strain CLWT, was isolated from a terrestrial hot spring in an exposition garden in Xiamen City, Fujian Province, the People's Republic of China. Strain CLWT formed beige, dry colonies on solid 2216E medium and flocks in liquid medium. Cells were Gram-stain-negative, short rods (1.0-3.0 mu m long and 0.4-0.6 mu m wide) with six or more polar flagella. The temperature and pH for growth of strain CLWT were 28-65 degrees C (optimum, 50-58 degrees C) and pH 5.5-9.5 (optimum, pH 6.0-8.0). Growth occurred in the presence of 0.3-6.0% NaCl (optimum 2.5-4.5 %). Phylogenetic analysis based on 16S rRNA gene sequences showed that the closest relative of the isolate was Amaricoccus kaplicensis Ben 101(T) (94.3 % sequence similarity). The DNA G+C content of strain CLWT was 72.2 mol%. The respiratory quinone was ubiquinone 10. The predominant polar lipids consisted of phosphatidylglycerol and phosphatidylethanolamine. The major fatty acids (>10%) were summed feature 8 (consisting of C-18:1 omega 7c and/or C-18.1 omega 6c), C-18:1 omega 7c 11-methyl and C-18:0. Based on phylogenetic, physiological and biochemical data and DNA G+C content, strain CLWT is considered to represent a novel species of a new genus in the family Rhodobacteraceae, for which the name Pleomorphobacterium xiamenense gen. nov., sp. nov. is proposed. The type strain of the type species is CLWT (=LMG 26245(T)=CGMCC 1.10808(T)=MCCC 1A06272(T))