Molecular characterization, structural and expression analysis of twelve CXC chemokines and eight CXC chemokine receptors in largemouth bass (Micropterus salmoides).

The chemokine-receptor system plays important roles in the leukocyte trafficking, inflammation, immune cell differentiation, cancer and other biological processes. In the present study, the sequence features, structures and expression patterns of twelve CXC chemokine ligands (CXCL8a.1, CXCL8a.2, CXCL8b.1, CXCL8b.2, CXCL12a, CXCL12b, CXCL13.1, CXCL13.2, CXCL14, CXCL18a, CXCL18b and CXCL19) and eight CXC chemokine receptors (CXCR1, CXCR2, CXCR3.1, CXCR3.2, CXCR3.3, CXCR4a, CXCR4b and CXCR5) of largemouth bass (Micropterus salmoides) were analyzed. All the CXCLs and CXCRs of largemouth bass shared high sequence identities with their teleost counterparts and possessed conserved motifs and structures of CXCLs and CXCRs family. Realtime qPCR revealed that these CXCLs and CXCRs were ubiquitously expressed in all examined tissues, with high expression levels in the immune-related tissues (spleen, head kidney, and gill). Following lipopolysaccharide (LPS) and polyinosinic-polycytidylic acid (polyI:C) stimulations, most of these CXCLs and CXCRs were significantly up-regulated in spleen. In addition, the potential interacted molecules of these CXCLs and CXCRs were analyzed by protein-protein interaction network analysis. To the best of our knowledge, this is the first study that in detail analyzes the CXCLs and CXCRs of largemouth bass. Our results provide valuable basis for study the function and mechanism of chemokine-receptor system in largemouth bass.

Transcriptome analysis of largemouth bass (Micropterus salmoides) challenged with LPS and polyI:C.

Largemouth bass (Micropterus salmoides) is a worldwide commercially important aquatic species. In recent years, pathogenic diseases cause great economic losses and hinder the industry of largemouth bass. To further understand the immune response against pathogens in largemouth bass, splenic transcriptome libraries of largemouth bass were respectively constructed at 12 h post-challenged with phosphate-buffered saline (PBS), lipopolysaccharide (LPS) and polyinosinic-polycytidylic acid (polyI:C) by using RNA sequencing technology (RNA-seq). RNA libraries were constructed using 9 RNA splenic samples isolated from three biological replicates of the three groups and sequenced on the DNBSEQ platform. A total number of 86,306 unigenes were obtained. Through pairwise comparisons among the three groups, we identified 11,295 different expression genes (DEGs) exhibiting significant differences at the transcript level. There were 7, 7, and 13 signal pathways were significantly enriched in LPS-PBS comparison, polyI:C-PBS comparison, and LPS-polyI:C comparison, respectively, indicating that the immune response to different pathogens was distinct in largemouth bass. To the best of our knowledge, this is the first report on the immune response of largemouth bass against different pathogen-associated molecular patterns (PAMPs) stimuli using transcriptomic analysis. Our results provide a valuable resource and new insights to understanding the immune characteristics of largemouth bass against different pathogens.

Molecular characterization of the evolutionary conserved signaling intermediate in Toll pathways (ECSIT) of soiny mullet (Liza haematocheila).

Mammalian evolutionary conserved signaling intermediate in Toll pathways (ECSIT) is an important intracellular protein that involves in innate immunity, embryogenesis, and assembly or stability of the mitochondrial complex I. In the present study, the ECSIT was characterized in soiny mullet (Liza haematocheila). The full-length cDNA of mullet ECSIT was 1860 bp, encoding 449 amino acids. Mullet ECSIT shared 60.4%∼78.2% sequence identities with its teleost counterparts. Two conserved protein domains, ECSIT domain and C-terminal domain, were found in mullet ECSIT. Realtime qPCR analysis revealed that mullet ECSIT was distributed in all examined tissues with high expressions in spleen, head kidney (HK) and gill. Further analysis showed that mullet ECSIT in spleen was up-regulated from 6 h to 48 h after Streptococcus dysgalactiae infection. In addition, the co-immunoprecipitation (co-IP) assay confirmed that mullet ECSIT could interact with tumor necrosis factor receptor-associated factor 6 (TRAF6). Molecular docking revealed that the polar interaction and hydrophobic interaction play crucial roles in the forming of ECSIT-TRAF6 complex. The resides of mullet ECSIT that involved in the interaction between ECSIT and TRAF6 were Arg107, Glu113, Phe114, Glu124, Lys120 and Lys121, which mainly located in the ECSIT domain. Our results demonstrated that mullet ECSIT involved in the immune defense against bacterial and regulation of TLRs signaling pathway by interaction with TRAF6. To the best of our knowledge, this is the first report on ECSIT of soiny mullet, which deepen the understanding of ECSIT and its functions in the immune response of teleosts.

Molecular characterization and functional analysis of DIGIRR from golden pompano (Trachinotus ovatus).

Mammalian single immunoglobulin (Ig) interleukin-1 receptor related molecule (SIGIRR), an important member of the Toll/interleukin-1 receptor (TIR) family, plays important balancing roles in the inflammatory responses. In the present study, the double Ig interleukin-1 receptor related molecule (DIGIRR), the homologous of SIGIRR, was characterized in golden pompano (Trachinotus ovatus) (termed as trDIGIRR). The full-length cDNA of trDIGIRR was 2,167 bp with an open reading frame (ORF) of 1,572 bp encoding 523 amino acids. The trDIGIRR contained several conserved domains including a signal peptide, two Ig domains, a transmembrane domain and a TIR domain, and shared high sequence identities with its teleost counterparts. Realtime qPCR analysis revealed that the trDIGIRR was distributed in all tissues examined, with high expressions in intestine, liver and head kidney. The expressions of trDIGIRR were induced by Vibrio alginolyticus, lipopolysaccharide (LPS) and polyinosinic-polycytidylic acid (poly I:C) challenge. Further analysis revealed that trDIGIRR was mainly located in the cytoplasm. In addition, the co-immunoprecipitation (co-IP) assay identified that trDIGIRR could interact with myeloid differentiation factor 88 (MyD88), but not interact with TIR domain containing adaptor protein inducing interferon-ß (TRIF). Our results provide basis for studying the immune role of fish DIGIRR.

Molecular characterization and expression analysis of TRIF, TRAF6, and TBK1 of golden pompano (Trachinotus ovatus).

Toll/IL-1R domain-containing adaptor-inducing IFN-ß (TRIF), tumor necrosis factor receptor-associated factor 6 (TRAF6) and TANK-binding kinase 1 (TBK1) are critical signal transducers in toll-like receptors (TLRs) signaling pathway. In the present study, TRIF, TRAF6 and TBK1 were characterized from golden pompano (Trachinotus ovatus), named as TroTRIF, TroTRAF6 and TroTBK1, respectively. The full cDNA length of TroTRIF, TroTRAF6 and TroTBK1 was 2297 bp, 2293 bp, and 2482 bp, which respectively encoded 589, 573 and 723 amino acids. The deduced amino acids sequences of TroTRIF, TroTRAF6 and TroTBK1 contained conserved motifs, similar to their counterparts in other vertebrates. Phylogenetic tree analysis revealed that TroTRIF, TroTRAF6 and TroTBK1 were well clustered with their counterparts in other fish species. Quantitative Real-Time PCR (qPCR) analysis showed that TroTRIF, TroTBK1 and TroTRAF6 were detected in all examined tissues of healthy fish, but shared distinct transcript levels. Moreover, the expressions of TroTRIF, TroTBK1 and TroTRAF6 were generally induced by polyriboinosinic-polyribocytidylic acid (polyI:C), lipopolysaccharide (LPS), and Vibrio alginolyticus stimulation in vivo, indicating their critical roles in the immune defense of golden pompano against pathogen invasion. Our results provide valuable information for understanding the functions of these genes in golden pompano.

Integrated transcriptome analysis of immune-related mRNAs and microRNAs in Macrobrachium rosenbergii infected with Spiroplasma eriocheiris.

Macrobrachium rosenbergii (M. rosenbergii), is a major aquaculture species in China and Southeast Asia. However, infection with Spiroplasma eriocheiris (S. eriocheiris) has caused huge economic losses to the cultivation of M. rosenbergii. Currently, there are few reports on the immune response mechanism of M. rosenbergii that are infected with S. eriocheiris. To clarify the immune response mechanism of M. rosenbergii infected with S. eriocheiris, the key immune genes which respond to the infection with the pathogen and the regulation of related microRNAs (miRNAs) on them were identified. In this study, the mRNA and miRNA transcriptome of hepatopancreas of M. rosenbergii at different infection stages were analyzed using high-throughput sequencing and qRT-PCR. In the mRNA transcriptome, 27,703 and 33,402 genes were expressed in healthy and susceptible M. rosenbergii, respectively. By digital gene-expression profiling analysis, 23,929 and 24,325 genes were expressed, and 223 and 373 genes were significantly up-regulated and down-regulated, respectively. A total of 145 key genes related to Toll, IMD, JAK/STAT and MAPK were excavated from the transcriptome. In the miRNA transcriptome, 549 miRNAs (Conserved: 41, PN-type: 83, PC-type: 425) were sequenced, of which 87 were significantly up-regulated and 23 were significantly down-regulated. Among the related immune pathways, there are 259 miRNAs involved in the regulation of target genes in the Toll and IMD pathways, 231 JAK/STAT pathways and 122 MAPK pathways. qRT-PCR differential detection of immune-related miRNAs and mRNAs showed that 22 miRNAs with significant differences (P < 0.05) such as mro-miR-100, PC-mro-3p-27 and PN-mro-miR-316 had corresponding regulatory relationships with 22 important immune genes such as TLR2, TLR3, TLR4, TLR5, MyD88, Pelle and Relish in different stages after infection. In this study, the immune genes and related regulatory miRNAs of M. rosenbergii in response to S. eriocheiris infection were obtained. The results can provide basic data to further reveal the immune defense mechanism of M. rosenbergii against S. eriocheiris infection.

Structural analysis of toll-like receptor 18 from soiny mullet (Liza haematocheila): Giving insights on the ligand binding mechanism of fish specific TLRs.

Toll-like receptors (TLRs) are important pattern recognition receptors (PRRs) of innate immune system, playing crucial roles in immune defense against pathogens. TLR18, a member of TLR1 family, is fish-specific TLR and involves in the immune response against bacterial infection. Currently, the structural biology of fish TLR18 is poorly elaborated. In this study, the structure and ligand binding of TLR18 (smTLR18) of soiny mullet (Liza haematocheila), an economically valuable aquaculture mugilid species, were analyzed. The extracellular domain (ECD) of smTLR18 formed an open-loop horseshoe-shaped structure with the concave surfaces made up of 19 parallel ß-strands (LRR1-LRR19), lacking Z-loop that seen in human TLR9. The intracellular Toll/interleukin (IL)-1 (TIR) domain contained a central 4-parallel ß-sheet (ßA-ßD) surrounded by 5 α-helices (αA-αE). Molecular docking analysis revealed that both ECD domain and TIR domain of smTLR18 could form homodimers. For the ECD homodimer, the main residues involved in dimer formation were located from LRR10 to LRR14. For the TIR homodimer, the residues involved in dimer formation were located in BB loop, αB helix, αC helix and DD loop. Ligand binding analyses revealed that peptidoglycans (PGNs) and lipopolysaccharides (LPS), two main bacterial pathogen-associated molecular patterns (PAMPs), were the potential ligands of smTLR18. The van der Waals and Coulombic interactions contributed to the interactions between smTLR18 and PGNs, while only van der Waals dominated the interactions between smTLR18 and LPS. The residues involved in ligands binding were located from LRR9 to LRR13. Our results provided the structural bases for elucidate the ligand binding of fish TLR18.

TLR13, TLR22, TRAF6, and TAK1 in the soiny mullet (Liza haematocheila): Molecular characterization and expression profiling analysis.

Toll-like receptors (TLRs) and their associated signaling pathways play pivotal roles in the immune response to invading pathogens. Here, TLR13, TLR22, tumor necrosis factor receptor-associated factor 6 (TRAF6), and transforming growth factor-ß-activated kinase1 (TAK1) were characterized in the soiny mullet (Liza haematocheila), representative mugilid species that is widely cultured in Asia. The four mullet genes, which shared characteristic features with their counterparts in other teleosts, were ubiquitously expressed in all of the examined tissues, albeit with different expression patterns. Following Streptococcus dysgalactiae infection, the four genes were upregulated to different degrees in various mullet tissues. These results indicated that the four genes were involved in the mullet immune response to bacterial infection. To the best of our knowledge, this is the first characterization of these four genes in mullet. Our results provide a basis for future studies of TLR signaling pathways in mullet, as well as for similar studies in other mugilids.

Molecular characterization of three toll-like receptors (TLR21, TLR22, and TLR25) from a primitive ray-finned fish Dabry's sturgeon (Acipenser dabryanus).

Dabry's sturgeon (Acipenser dabryanus) is a useful model for the study of fish evolution, as it is one of the most primitive actinopterygian species. However, studies of the immune system of this fish are limited. Here, we identified three toll-like receptors (adaTLR21, adaTLR22, and adaTLR25) from Dabry's sturgeon. The three sturgeon TLRs had characteristic TLR features, including a signal peptide, several leucine rich repeat (LRR) domains, a transmembrane domain, and a Toll/interleukin-1 receptor (TIR) domain. Although the predicted amino acid sequences encoded by the sturgeon adaTLR21, adaTLR22, and adaTLR25 had somewhat low levels of sequence identity and similarity with TLRs from other fish species, the three sturgeon TLRs fell in well-supported clades with other teleost TLRs in our neighbor-joining phylogenetic tree. Real-time quantitative PCR showed that the three sturgeon TLRs were ubiquitously expressed in all examined tissues from healthy adult sturgeon, but that their expression patterns varied greatly among the different tissues. The three sturgeon TLRs were also expressed across all embryonic developmental stages that were examined, but their expression levels differed between developmental stages. All three TLRs were upregulated in head-kidney primary leucocytes following lipopolysaccharide (LPS) and polyinosinic: polycytidylic acid (polyI:C) stimulation. To the best of our knowledge, this is the first characterization of these three TLRs in Darby's sturgeon. Our results provide a framework for further studies of TLR ligand specificity and signaling pathways in sturgeon, and increase our understanding of the functional evolution of TLRs in vertebrates.

Molecular characterization and expression analysis of TLR1 and TLR4 from the endangered fish Dabry's sturgeon (Acipenser dabryanus).

Toll-like receptors (TLRs) are important pattern recognition receptors (PRRs) of teleost innate immune system. However, information about TLRs is absent in Dabry's sturgeon (Acipenser dabryanus), one of the most primitive actinopterygii species. In the present study, the full lengths of adaTLR1 and adaTLR4 were cloned from Dabry's sturgeon using RT-PCR and RACE-PCR. The obtained adaTLR1 was 2957 bp in length, encoding a putative protein of 767 amino acids and adaTLR4 cDNA was 2902 bp in length, encoding a putative protein of 830 aa. Both adaTLR1 and adaTLR4 possessed several typical TLRs motifs, including signal peptides, leucine-rich repeat (LRR) motifs, a transmembrane domain and a TIR motifs. In addition, adaTLR4 contained three conserved boxes in its TIR motif, involving in TLRs signal transduction. A proline, important for LPS recognition of mammalian TLR4, was also found in adaTLR4. Physicochemical features of adaTLR1 and adaTRL4 were also analyzed. Quantitative realtime PCR showed that both transcripts were ubiquitously expressed in all 11 normal tissues selected, but they exhibited different expression patterns, with adaTLR1 highly expression in heart and adaTLR4 highly in skin. Further, adaTLR1 and adaTLR4 were up-regulated in the primary head-kidney leucocytes following LPS and polyI:C stimulation, indicating that both genes involved in the sturgeon immune response to LPS and polyI:C. To our best knowledge, this was the first report of these genes in sturgeon and these results provided the basis for further elucidating the ligand specificity and signaling pathway of fish TLRs.

Molecular characterization and expression analysis of cathepsin C in Chinese giant salamander (Andrias davidianus) after Aeromonas hydrophila infection

Background: Cathepsin C (CTSC) (dipeptidyl peptidase I, DPPI), is a member of the papain superfamily of cysteine proteases and involves in a variety of host reactions. However, the information of CTST in Chinese giant salamander (Andrias davidianus), an amphibian species with important evolutionary position and economic values, remained unclear. Results: The full-length salamander CTSC cDNA contained a 96 bp of 5'-UTR, a 1392 bp of ORF encoding 463 amino acids, and a 95 bp of 3'-UTR. The salamander CTSC possessed several sequence features similar to other reported CTSCs such as a signal peptide, a propeptide and a mature peptide. The active site triad of Cys, His and Asn were also found existing in salamander CTSC. Salamander CTSC mRNA was constitutively expressed in all the examined tissues with significantly variant expression level. The highest expression of CTSC was in intestine, followed with stomach, spleen, lung and brain. Following Aeromonas hydrophila infection for 12 h, salamander CTSC was significantly up-regulated in several tissues including lung, spleen, brain, kidney, heart, stomach and skin. Conclusion: CTSC plays roles in the immune response to bacterial infection, which provided valuable information for further studying the functions of CTSC in salamander.

Characterization of the ligand binding of PGRP-L in half-smooth tongue sole (Cynoglossus semilaevis) by molecular dynamics and free energy calculation

Background: Peptidoglycan (PGN) recognition proteins (PGRPs) are important pattern recognition receptors of the host innate immune system that are involved in the immune defense against bacterial pathogens. PGRPs have been characterized in several fish species. The PGN-binding ability is important for the function of PGRPs. However, the PGRP-PGN interaction mechanism in fish remains unclear. In the present study, the 3-D model of a long PGRP of half-smooth tongue sole (Cynoglossus semilaevis) (csPGRP-L), a marine teleost with great economic value, was constructed through the comparative modeling method, and the key amino acids involved in the interaction with Lys-type PGNs and Dap-type PGNs were analyzed by molecular dynamics and molecular docking methods. Results: csPGRP-L possessed a typical PGRP structure, consisting of five ß-sheets and four α-helices. Molecular docking showed that the van der Waals forces had a slightly larger contribution than Coulombic interaction in the csPGRP-L-PGN complex. Moreover, the binding energies of csPGRP-L-PGNs computed by MM-PBSA method revealed that csPGRP-L might selectively bind both types of MTP-PGNs and MPP-PGNs. In addition, the binding energy of each residue of csPGRP-L was also calculated, revealing that the residues involved in the interaction with Lys-type PGNs were different from that with Dap-type PGNs. Conclusions: The 3-D structure of csPGRP-L possessed typical PGRP structure and might selectively bind both types of MTP- and MPP-PGNs, which provided useful insights to understanding the functions of fish PGRPs.

Functional characterization of a short peptidoglycan recognition protein from Chinese giant salamander (Andrias davidianus).

Peptidoglycan (PGN) recognition proteins (PGRPs) are important pattern recognition receptors (PRRs) involved in immune defense against bacterial infections. In this study, a short PGRP (termed AdPGRP-S1) was cloned and functionally characterized from Chinese giant salamander (Andrias davidianus), the largest extant urodela amphibian species. AdPGRP-S1 was 184 aa in length and shared 38.7%-54.9% sequence identities with other vertebrates' short PGRPs. It contained one typical PGRP domain at the C-terminal region and several conserved amino acid (aa) residues involved in amidase and PGN binding. AdPGRP-S1 was constitutively expressed in all tissues examined, with the highest expression level seen in spleen and intestine. It has been shown that AdPGRP-S1 could bind and degrade Lys-PGN and Dap-PGN. Further, AdPGRP-S1 had antibacterial activity against the Gram-negative bacteria, Edwardsiella tarda, and was able to trigger the activation of NF-κB signaling. These results demonstrated that AdPGRP-S1 possesses multiple functions in pathogen recognition, mediating ceullular signaling, and initiating antibacterial response. This is the first functional study of a salamander PGRP, providing insight to further understand the functional evolution of verterbates' PGRPs.

Molecular Cloning, Characterization, and Expression Analysis of Cathepsin A in the Chinese Giant Salamander Andrias davidianus.

Cathepsin A (CTSA) is serine carboxypeptidase, an important protease in the lysosome. In this study, the full complementary DNA (cDNA) sequence of CTSA in Chinese giant salamanders Andrias davidianus was cloned, and its sequence features were analyzed. Tissue expression patterns of CTSA in healthy and Aeromonas hydrophila-infected salamanders were also investigated. The full cDNA sequence of salamander CTSA was 1,620 base pairs in length, encoding 472 amino acids. Salamander CTSA shared high sequence identities with other vertebrates' CTSAs, ranging from 62.7% to 68.9%. In healthy salamanders, CTSA was highly expressed in spleen, followed by brain, intestine, and stomach. After A. hydrophila infection, salamander CTSA was significantly upregulated in lung, heart, muscle, and kidney; was downregulated in liver, spleen, and intestine; and exhibited no significant changes in stomach and skin, indicating that salamander CTSA might play defense roles in multiple tissues during bacterial infection. These results provide a solid basis for further study of the immune function of amphibian CTSA. Received September 18, 2016; accepted June 18, 2017.

Molecular cloning, structural modeling, and expression analysis of MyD88 and IRAK4 of golden pompano (Trachinotus ovatus).

MyD88 and IRAK4 are important components of TLR signaling pathways. However, information about MyD88 and IRAK4 is vacant in golden pompano (Trachinotus ovatus), a marine teleost with great commercial value. Thus, in this study the full lengths of trMyD88 and trIRAK4 were cloned from golden pompano using RT-PCR and RACE-PCR methods. trMyD88 was 1213 bp in length, encoding a putative protein of 288 amino acids (aa), consisting of a 99 aa of death domain at its N-terminal and a 137 aa of the TIR domain at its C-terminal. trIRAK4 was 1606 bp in length, encoding a putative protein of 469 aa, including an N-terminal death domain and a central kinase domain, connected by a ProST domain. Other domains or aa residues needed for their functions were also identified in trMyD88 and trIRAK4. Physicochemical features and 3-D structures of trMyD88 and trIRAK4 were also analyzed. Quantitative real-time PCR revealed that the 2 genes were ubiquitously expressed in tissues from healthy pompano, especially highly in the spleen and head kidney, indicating their roles in the immune response. Further, trMyD88 and trIRAK4 were up-regulated at 12 h after the Vibrio alginilyticus and polyI:C challenge and continued to 48 h post challenge. Our results demonstrated that MyD88 and IRAK4 played important roles in the golden pompano innate immune system, providing the basis for further study of the signaling pathways that these 2 genes are involved in.

Structural insights into ligand binding of PGRP1 splice variants in Chinese giant salamander (Andrias davidianus) from molecular dynamics and free energy calculations.

Peptidoglycan (PGN) recognition proteins (PGRPs) are important pattern recognition receptors of the innate immune system. A number of PGRP splicing variants produced by alternative splicing of PGRP genes have been reported. However, several important aspects of interactions between PGRP splice variants and their ligands are still unclear. In the present study, three dimensional models of salamander PGRP1 (adPGRP1) and its splice variant (adPGRP1a) were constructed, and their key amino acids involved in interacting with PGNs were analyzed. The results revealed that adPGRP1a has a typical PGRPs structure containing five ß-sheets and four α-helices, while adPGRP1 contained five ß-sheets and only one α-helix due to the lack of 51 amino acids at its C-terminus. Molecular docking revealed that van der Waals and Coulombic interactions contributed to interactions in the protein-ligand complex. Further binding energy of adPGRP-PGNs computed by the MM-PBSA method revealed that adPGRP1a and adPGRP1 might selectively bind to different PGNs; the former might selectively bind Dap-type PGNs and the latter both types of PGNs. In addition, the binding energy of each residue of adPGRP1a and adPGRP1 was also calculated, revealing that residues involved in the interaction of protein-ligand complexes were different in adPGRP1a and adPGRP1. These results provided a first insight into the potential basis for interaction between PGRPs generated by alternative splicing and PGN derivatives.

Molecular cloning and expression analysis of toll-like receptor genes (TLR7, TLR8 and TLR9) of golden pompano (Trachinotus ovatus).

Toll like receptor (TLR) 7, 8 and 9 are intracellular TLRs which play important roles in host immune defense against bacterial or virus pathogens. In this study, TLR7, 8 and 9 were identified from golden pompano (Trachinotus ovatus), a marine teleost with great economic values. Sequence analysis revealed that the three TLRs contained several conserved characteristic features, including signal peptides, 25 leucine-rich repeat (LRR) motifs, a transmembrane domain and a TIR motif. These three TLRs shared high sequence identity and similarity with their counterparts from other teleosts. The phylogenetic tree analysis showed the three TLRs were clustered well with their piscine counterparts, confirming the correctness of their nomenclatures and closed relationships during evolution. Quantitative real-time PCR revealed that the three TLRs were ubiquitously expressed in all the tested tissues from normal pompano, with high expression in spleen and head kidney, indicating their role in immune reaction. Further, pompano TLR7 and TLR8 was up-regulated in spleen and head kidney from 12 h to 48 h following polyI:C challenge, but remained no changes to Vibrio alginilyticus infection. In contrast, pompano TLR9 could be induced by V. alginilyticus infection but remained apathetic to polyI:C challenge. These results indicated that pompano TLR7, 8 and 9 might have distinct roles in response to bacterial or virus pathogens. Our results provided the basis for further study on ligand specificity and signaling pathways of fish TLRs which are required for elucidating the immune functions of fish TLRs.

Cloning and Expression of ß-Defensin from Soiny Mullet (Liza haematocheila), with Insights of its Antibacterial Mechanism.

Beta-defensins are important part of innate immunity of fish, which are the first defense line against invading pathogens. In this study, the ß-defensin (Lhß-defensin) gene was cloned from spleen tissue of soiny mullet (Liza haematocheila). Lhß-defensin cDNA was 747 bp in length, encoding 63 amino acids. Sequence alignment revealed that Lhß-defensin contained six conserved cysteine residues and shared 97.5% sequence identities with grouper (Epinephelus coioides) ß-defensin. Realtime PCR revealed that Lhß-defensin was highest expressed in the immune related organs, such as spleen, kidney and gut of healthy fish. Following Streptococcus dysgalactiae infection, Lhß-defensin was up-regulated in immune related organs, e.g. 17.6-fold in spleen and 10.87-fold in gut at 24 h post infection (hpi). Lhß-defensin possessed a monomeric structure of a three-stranded anti-parallel ß-sheet and an α-helix stabilized by three disulfide bonds formed by Cys30-Cys58, Cys36-Cys52, and Cys40-Cys59. In addition to the experimental work, computer simulation was also carried out to determine the possible conformation of ß-defensin and its interaction with palmitoyloleoylphosphatidylglycerol (POPG), a model of bacteria membrane. The Lhß-defensin was found to form dimeric structure stabilized by the van der Waals contacts of Leu35 and Cys37 in two anti-parallel ß1-strands and the cation-π interaction between Tyr32 and Arg54 respectively in the two ß1-strands. The most important interactions between ß-defensin and membrane are the electrostatic interactions between Arg residues in ß-defensin and head group of POPG bilayer as well as hydrogen bond interactions between them. Our results were useful for further understanding the potential mechanism of antimicrobial property of fish ß-defensins.

Transcriptome analysis of the endangered Chinese giant salamander (Andrias davidianus): Immune modulation in response to Aeromonas hydrophila infection.

The endangered Chinese giant salamander (Andrias davidianus) is the largest extant amphibian species. Disease outbreaks represent one of the major factors threatening A. davidianus populations in the wild and the viability of artificial breeding programmes. Development of future immune therapies to eliminate infectious disease in A. davidianus is dependent on a thorough understanding of the immune mechanisms elicited by pathogen encounters. To this end we have undertaken, for the first time in amphibians, differential transcriptome analysis of the giant salamander response to Aeromonas hydrophila, one of the most devastating pathogens affecting amphibian populations. Out of 87,204 non-redundant consensus unigenes 19,216 were annotated, 6834 of which were upregulated and 906 down-regulated following bacterial infection. 2058 unigenes were involved with immune system processes, including 287 differentially expressed unigenes indicative of the impact of bacterial infection on several innate and adaptive immune pathways in the giant salamander. Other pathways not directly associated with immune-related activity were differentially expressed, including developmental, structural, molecular and growth processes. Overall, this work provides valuable insights into the underlying immune mechanisms elicited during bacterial infection in amphibians that may aid in the future development of disease control measures in protecting the Chinese giant salamander. With the unique position of amphibians in the transition of tetrapods from aquatic to terrestrial habitats, our study will also be invaluable towards the further understanding of the evolution of tetrapod immunity.

Transcriptome analysis of soiny mullet (Liza haematocheila) spleen in response to Streptococcus dysgalactiae.

Soiny mullet (Liza haematocheila) is becoming an economically important aquaculture mugilid species in China and other Asian countries. However, increasing incidences of bacterial pathogenic diseases has greatly hampered the production of the soiny mullet. Deeper understanding of the soiny mullet immune system and its related genes in response to bacterial infections are necessary for disease control in this species. In this study, the transcriptomic profile of spleen from soiny mullet challenged with Streptococcus dysgalactiae was analyzed by Illumina-based paired-end sequencing method. After assembly, 86,884 unique transcript fragments (unigenes) were assembled, with an average length of 991 bp. Approximately 41,795 (48.1%) unigenes were annotated in the nr NCBI database and 57.9% of the unigenes were similar to that of the Nile tilapia. A total of 24,299 unigenes were categorized into three Gene Ontology (GO) categories (molecular function, cellular component and biological process), 13,570 unigenes into 25 functional Clusters of Orthologous Groups of proteins (COG) categories, and 30,547 unigenes were grouped into 258 known pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Following S. dysgalactiae infection, 11,461 differentially expressed unigenes were identified including 4658 up-regulated unigenes and 6803 down-regulated unigenes. Significant enrichment analysis of these differentially expressed unigenes identified major immune related pathways, including the Toll-like receptor, complement and coagulation cascades, T cell receptor signaling pathway and B cell receptor signaling pathway. In addition, 24,813 simple sequence repeats (SSRs) and 127,503 candidate single nucleotide polymorphisms (SNPs) were identified from the mullet spleen transcriptome. To this date, this study has globally analyzed the transcriptome profile from the spleen of L. haematocheila after S. dysgalactiae infection. Therefore, the results of our study contributes to better on the immune system and defense mechanisms of soiny mullet in response to bacterial infection, and provides valuable references for related studies in mugilidae species which currently lack genomic reference.