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

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

Transcriptome Analysis Reveals Comprehensive Insights into the Early Immune Response of Large Yellow Croaker (<i>Larimichthys crocea</i>) Induced by Trivalent Bacterial Vaccine

<div><p>Vaccination is an effective and safe strategy for combating bacterial diseases in fish, but the mechanisms underlying the early immune response after vaccination remain to be elucidated. In the present study, we used RNA-seq technology to perform transcriptome analysis of spleens from large yellow croaker (<i>Larimichthys crocea</i>) induced by inactivated trivalent bacterial vaccine (<i>Vibrio parahaemolyticus</i>, <i>Vibrio alginolyticus</i> and <i>Aeromonas hydrophila</i>). A total of 2,789 or 1,511 differentially expressed genes (DEGs) were obtained at 24 or 72 h after vaccination, including 1,132 or 842 remarkably up-regulated genes and 1,657 or 669 remarkably down-regulated genes, respectively. Gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichments revealed that numerous DEGs belong to immune-relevant genes, involved in many immune-relevant pathways. Most of the strongly up-regulated DEGs are innate defense molecules, such as antimicrobial peptides, complement components, lectins, and transferrins. Trivalent bacterial vaccine affected the expressions of many components associated with bacterial ligand–depending Toll-like receptor signaling pathways and inflammasome formation, indicating that multiple innate immune processes were activated at the early period of vaccination in large yellow croaker. Moreover, the expression levels of genes involved in antigen processing were also up-regulated by bacterial vaccine. However, the expression levels of several T cell receptors and related CD molecules and signal transducers were down-regulated, suggesting that the T cell receptor signaling pathway was rapidly suppressed after vaccination. These results provide the comprehensive insights into the early immune response of large yellow croaker to vaccination and valuable information for developing a highly immunogenic vaccine against bacterial infection in teleosts.</p></div

Immune-relevant KEGG pathways.

<p>Immune-relevant KEGG pathways.</p

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

Summary of the data for transcriptomes.

<p>Summary of the data for transcriptomes.</p

Putative antigen processing and presentation pathway.

<p>The predicted antigen processing and presentation pathway of large yellow croaker was constructed based on the knowledge of mammalian species. Red background indicates significantly up-regulated expression, and yellow background indicates no significant differential expression. The arrows represent promotion, the solid lines indicate direct relationships between genes, and the dashed lines indicate that more than one step is involved in the process. Bacterial antigens are internalized into host cells via phagosomes or endosomes. Specifically, the antigenic peptides are degraded in the cytoplasm by proteasome, and then transported to the endoplasmic reticulum by TAP and loaded onto MHC-I molecules with the help of ERAP, TAP, TAPBP, and CLAR. Antigenic peptides can also be captured and degraded in endosome and lysosome with the help of CTS, HSP70, and PPT2, and then loaded onto MHC-II molecules.</p

Comparative Transcriptomics Reveals the microRNA-Mediated Immune Response of Large Yellow Croaker (<i>Larimichthys crocea</i>) to <i>Pseudomonas plecoglossicida</i> Infection

Visceral white nodules disease (VWND), caused by Pseudomonas plecoglossicida, is a common disease among cage-farmed large yellow croaker (Larimichthys crocea) in China. However, comprehensive investigations of the molecular defensive mechanisms used by L. crocea in response to P. plecoglossicida infection remain relatively rare. Here, we constructed transcriptomes of the L. crocea spleen at 12 h and 24 h after P. plecoglossicida challenge. We identified 518 novel miRNAs and 823 known miRNAs in the spleen of L. crocea. Between the challenge and control groups, 32 differentially expressed miRNAs (DEmiRNAs), predicted to target 356 genes, and 1152 differentially expressed mRNAs (DEmRNAs) were identified at 12 h post-infection, while 33 DEmiRNAs, predicted to target 278 genes, and 1067 DEmRNAs were identified at 24 h post-infection. Gene ontology (GO) analysis showed that 146 and 126 GO terms were significantly enriched in the target genes at 12 h and 24 h, respectively. Twenty-eight and four immune-associated Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were significantly enriched in the target genes at 12 h and 24 h, respectively. Three immune-associated pathways were among those most enriched in the target genes: Toll-like receptor signaling, endocytosis, and C-type lectin receptor signaling. Network analysis identified 47 DEmRNA-DEmiRNA pairs. In particular, the immune-related genes TLR5S and PIGR were targeted by the miRNAs lcr-miR-7132c and dre-miR-183-5p, respectively. Dual-luciferase assays verified that lcr-miR-7132c downregulated TLR5S, suggesting that this miRNA may participate in regulating the immune response of L. crocea to P. plecoglossicida infection through the TLR5S-mediated signaling pathway. Our results help to clarify the miRNA-mediated immune response of L. crocea to P. plecoglossicida infection