Time-resolved RNA-seq provided a new understanding of intestinal immune response of European eel (Anguilla anguilla) following infection with Aeromonas hydrophila.

No studies systematically examined the intestinal immune response for yellow stage of European eel (Anguilla anguilla) with Aeromonas hydrophila infection by time-resolved RNA-seq. Here, we examined transcriptional profiles of the intestines at three-time points following infection with A. hydrophila. Intraperitoneal injections caused mortalities within 48 h post-injection (hpi), with the survival rate 87.5% at 24 hpi and 83.9% at 48 hpi. The result from KEGG pathway enrichment analysis showed that the immune related "cytosolic DNA-sensing pathway" was significantly enriched at the first and second time points (6 hpi and 18 hpi), with the up-regulated expression of irf3, il1b, tnfaip3, cxcl8a, ap1-2, c-fos, polr3d, polr3g and polr3k both at 6 hpi and 18 hpi, but not at the third time point (36 hpi). According to the KEGG annotation, 326 immune and inflammation-related DEGs were found. The co-expression network of those 326 DEGs revealed the existence of three modules, and tlr1 was found to be in the center of the biggest module which contained massive DEGs from "signal transduction" and "transport and catabolism". The c3 isoforms showed different expression pattern among the three time points, indicating a unique activation of complement systems at 18 hpi. Furthermore, two cathelicidins (aaCATH_1 and aaCATH_2) were highly up-regulated at the first two time points, and the bacterial growth inhibition assay revealed their antibacterial properties against A. hydrophila. Our data indicated the important roles of cytosolic DNA-sensing pathway, as well as transcripts including tlr1, c3, polr and cathelicidins in the intestine of A. anguilla in response to A. hydrophila infection. The present study will provide leads for functional studies of host-pathogen interactions.

A Closed-Tube Loop-Mediated Isothermal Amplification Assay for the Visual Detection of Staphylococcus aureus.

Food-borne diseases induced by Staphylococcus aureus contamination seriously affect human health and food safety. Therefore, a closed-tube loop-mediated isothermal amplification (LAMP) assay for the visual detection of S. aureus was developed in this study. Firstly, two pairs of outer and inner primers were designed targeting on a conserved fragment of gyrB gene in different S. aureus strains. Secondly, the weakly buffered gyrB-LAMP assays were optimized under various pH values and other conditions, followed by the visual evaluation of five pH-sensitive indicators, and the cresol-red was chosen as the best dye for the best visual performance. Thirdly, the cresol-red-based LAMP assay showed good sensitivity with the detection limit of 5.4 copies/µL for purified DNAs, and good specificity with no cross-reaction with other related species. The specificity of the amplified products was further confirmed by XbaI restriction enzyme digestion analysis. Finally, the cresol-red-based LAMP assay was validated by the clinical-dried fish samples inoculated with known numbers of S. aureus and further validated by 20 blind samples. To our knowledge, this is the first report of a closed-tube LAMP assay based on pH-sensitive indicators for the visual detection of the food-borne S. aureus by the gyrB gene.

Molecular cloning and expression analysis of a fish specific interferon regulatory factor, IRF11, in orange spotted grouper, Epinephelus coioides.

Interferon regulatory factors (IRFs) are transcription mediators which play vital roles in multiple biological processes, such as antiviral defense, immune response, cell growth regulation and apoptosis. A fish specific IRF, termed IRF11, has been identified in previous study through searching fish genome databases. Herein, a transcript of IRF11, EcIRF11 was cloned from orange-spotted grouper, Epinephelus coioides. The EcIRF11 cDNA sequence has 1573 bp in length, encoding a putative protein of 261 amino acids, with a high degree of similarity found between EcIRF11 and its teleost counterparts. Comparative analyses in teleost genomes revealed that IRF11 may have an ancient origin at least 450 million years ago, and the locus harbouring IRF11 might have experienced chromosomal rearrangement and/or inversion during evolution. Expression analysis revealed that the other two members, IRF1 and IRF2 also in the IRF1 subgroup (SG) as IRF11, exhibited high expression levels in early experimental infection phase in response to viral stimulation of poly I:C and to bacterial stimulation of Vibrio parahaemolyticus infections in the fish, while EcIRF11 is not transcriptionally modulated at the examined time points except in kidney at 6 h following poly I:C stimulation. Taken together, the results obtained in this study indicate that IRF11 might have been originated from the same ancestor as IRF1 and IRF2, but exhibits distinct basal and induced expression, implying its different function which needs further characterization.

Evolution of IFN-λ in tetrapod vertebrates and its functional characterization in green anole lizard (Anolis carolinensis).

IFN-λ (IFNL), i.e. type III IFN genes were found in a conserved gene locus in tetrapod vertebrates. But, a unique locus containing IFNL was found in avian. In turtle and crocodile, IFNL genes were distributed in these two separate loci. As revealed in phylogenetic trees, IFN-λs in these two different loci and other amniotes were grouped into two different clades. The conservation in gene presence and gene locus was also observed for the receptors of IFN-λ, IFN-λR1 and IL-10RB in tetrapods. It is further revealed that in North American green anole lizard Anolis carolinensis, a single IFNL gene was situated collinearly in the conserved locus as in other tetrapods, together with its receptors IFN-λR1 and IL-10RB also identified in this study. The IFN-λ and its receptors were expressed in all examined organs/tissues, and their expression was stimulated following the injection of polyI:polyC. The ISREs in promoter of IFN-λ in lizard were responsible to IRF3 as demonstrated using luciferase report system, and IFN-λ in lizard functioned through the receptors, IFN-λR1 and IL-10RB, as the up-regulation of ISGs was observed in ligand-receptor transfected, and also in recombinant IFN-λ stimulated, cell lines. Taken together, it is concluded that the mechanisms involved in type III IFN ligand-receptor system, and in its signalling pathway and its down-stream genes may be conserved in green anole lizard, and may even be so in tetrapods from xenopus to human.

A new antimicrobial peptide SCY2 identified in Scylla Paramamosain exerting a potential role of reproductive immunity.

A new antimicrobial peptide named SCY2 with 65.08% identity in amino acid sequence to the known scygonadin (SCY1) was first characterized in Scylla paramamosain based on its cloned full-length cDNA and genomic DNA sequences. The SCY2 gene was dominantly expressed in the ejaculatory duct of male crabs and its mRNA transcripts were discerned mainly in the glandular epithelium of the inner wall and the secretion inside the ejaculatory duct. Although the SCY2 gene could not be induced with the challenge of the bacteria and fungi tested, its induction reached the highest level at the peak period of mating in mature male crabs either in June or November, suggesting its induction was likely related to seasonal reproduction changes. Moreover, it was interesting to note that, from analysis of its transcripts and protein, SCY2 was significantly expressed only in the ejaculatory duct of pre-copulatory males before mating, however it was clearly detected in the spermatheca of post-copulatory females after mating accompanied by the decreased level of SCY2 expression in the ejaculatory duct. These results suggested that the SCY2 was probably transferred from the male during mating action with the female for the purpose of protecting fertilization. The recombinant SCY2 was more active against the Gram-positive than the Gram-negative bacteria tested. It was further observed that the SCY2 transcripts were significantly increased with addition of exogenous progesterone in tissue cultures whereas the several other hormones tested had no any effect on SCY2 expression, indicating that there might be a relationship between the SCY2 expression and the induction of hormones in vivo. In summary, this study demonstrated that one role of SCY2 was likely to be involved in crab reproduction and it exerted its reproductive immune function through the mating action and the maintenance of inner sterility in the spermatheca of the female, thus leading to successful fertilization of S. paramamosain.

Macrophage migration inhibitory factor (MIF) family in arthropods: Cloning and expression analysis of two MIF and one D-dopachrome tautomerase (DDT) homologues in mud crabs, Scylla paramamosain.

The macrophage migration inhibitory factor (MIF) family, consisting of MIF and D-dopachrome tautomerase (DDT) in vertebrates, is evolutionarily ancient and has been found across Kingdoms including vertebrates, invertebrates, plants and bacteria. The mammalian MIF family are chemokines at the top of the inflammatory cascade in combating infections. They also possess enzymatic activities, e.g. DDT catalysis results in the production of 5,6-dihydroxyindole (DHI), a precursor of eumelanin. MIF-like genes are widely distributed, but DDT-like genes have only been described in vertebrates and a nematode. In this report, we cloned a DDT-like gene, for the first time in arthropods, and a second MIF in mud crab. The mud crab MIF family have a three exon/two intron structure as seen in vertebrates. The identification of a DDT-like gene in mud crab and other arthropods suggests that the separation of MIF and DDT preceded the divergence of protostomes and deuterostomes. The MIF family is differentially expressed in tissues of adults and during embryonic development and early life. The high level expression of the MIF family in immune tissues, such as intestine and hepatopancreas, suggests an important role in mud crab innate immunity. Mud crab DDT is highly expressed in early embryos, in megalops and crablets and this coincides with the requirement for melanisation in egg chorion tanning and cuticular hardening in arthropods, suggesting a potential novel role of DDT in melanogenesis via its tautomerase activity to produce DHI in mud crab. The clarification of the presence of both MIF and DDT in this report paves the way for further investigation of their functional roles in immunity and in melanogenesis in mud crab and other arthropods.

Interferon regulatory factor 10 (IRF10): Cloning in orange spotted grouper, Epinephelus coioides, and evolutionary analysis in vertebrates.

IRF10 gene was cloned in orange spotted grouper, Epinephelus coioides, and its expression was examined following poly(I:C) stimulation and bacterial infection. The cDNA sequence of grouper IRF10 contains an open reading frame of 1197 bp, flanked by 99 bp 5'-untranslated region and 480 bp 3'- untranslated region. Multiple alignments showed that the grouper IRF10 has a highly conserved DNA binding domain in the N terminus with characteristic motif containing five tryptophan residues. Quantitative real-time PCR analysis revealed that the expression of IRF10 was responsive to both poly(I:C) stimulation and Vibrio parahemolyticus infection, with a higher increase to poly(I:C), indicating an important role of IRF10 in host immune response during infection. A phyletic distribution of IRF members was also examined in vertebrates, and IRF10 was found in most lineages of vertebrates, not in modern primates and rodents. It is suggested that the first divergence of IRF members might have occurred before the evolutionary split of vertebrate and cephalochordates, producing ancestors of IRF (1/2/11) and IRF (4/8/9/10)[(3/7) (5/6)], and that the second and/or third divergence of IRF members occurred following the split, thus leading to the subsets of the IRF family in vertebrates.

Identification of three IFN-γ inducible lysosomal thiol reductase (GILT)-like genes in mud crab Scylla paramamosain with distinct gene organizations and patterns of expression.

Vertebrate gamma-interferon inducible lysosomal thiol reductase (GILT) is an IFN-γ-inducible protein and is involved in MHCII-restricted antigen processing and cross-presentation of MHCI-restricted antigens in adaptive immunity. Outside of the endocytic MHC pathway, GILT regulates the cellular redox state, inhibits T cell activation, neutralizes extracellular pathogens and is also a host factor of some bacterial pathogens. In this report, we isolated and characterized three divergent GILT-like genes, GILT1, GILT2 and GILT3, which share only 30.9-40.4% identities in a crustacean mud crab Scylla paramamosain. Whilst the crab GILT1 and GILT3 possess four and five exons, respectively, the GILT2 is intronless, suggesting that GILT2 may arise from a recent retroposition event. The invertebrate GILT-like genes have diverse gene organizations and may be evolved in a species/lineage-specific manner as suggested by phylogenetic tree analysis. The amino acid sequences equivalent to human mature GILT are well conserved, including the GILT signature and nine of the ten cysteine residues that potentially form 5 disulfide bonds in human GILT, across the animal kingdom. However, most invertebrate GILT-like molecules lack the human-type N-terminal propeptide, as well as the human-type C-terminal with a conserved cysteine residue, suggesting differences in post translational processing and mode of action. All the three GILT-like genes are highly expressed in the hepatopancreas and up-regulated by pathogenic bacterial infection suggesting a role in immune defense against bacterial diseases. This study may provide the basis for further investigation of the expanding functions of GILT-like molecules in immunity and other physiological processes in mud crabs and other animals.

Cloning and expression analyses of interferon regulatory factor (IRF) 3 and 7 genes in European eel, Anguilla anguilla with the identification of genes involved in IFN production.

Interferon regulatory factor (IRF) 3 and IRF7 have been identified as regulators of type I interferon (IFN) gene expression in mammals. In the present study, the two genes were cloned and characterized in the European eel, Anguilla anguilla. The full-length cDNA sequence of IRF3 and IRF7 in the European eel, named as AaIRF3 and AaIRF7 consists of 2879 and 2419 bp respectively. Multiple alignments showed that the two IRFs have a highly conserved DNA binding domain (DBD) in the N terminus, with the characteristic motif containing five tryptophan residues, which is a feature present in their mammalian homologues. But, IRF7 has only four of the five residues in other species of fish. The expression of AaIRF3 and AaIRF7 both displayed an obvious dose-dependent manner following polyinosinic:polycytidylic acid (PolyI:C) challenge. In vivo expression analysis showed that the mRNA level of AaIRF3 and AaIRF7 was significantly up-regulated in response to PolyI:C stimulation in all examined tissues/organs except in muscle, with a lower level of increase observed in response to lipopolysaccharide (LPS) challenge and Edwardsiella tarda infection, indicating that AaIRF3 and AaIRF7 may be more likely involved in antiviral immune response. In addition, some pattern recognition receptors genes related with the production of type I IFNs and those genes in response to type I IFNs were identified in the European eel genome database, indicating a relatively conserved system in the production of type I IFN and its signalling in the European eel.

Characterization of four Mx isoforms in the European eel, Anguilla anguilla.

Mx protein is known to play an important role in vertebrate immune response to viral infection. In this study, cDNA sequences of four Mx isoforms, designated as MxA, B, C and D were characterized in the European eel, Anguilla anguilla. These sequences contained an open reading frame of 1899, 1896, 1866, 1779 bp, flanked by 95, 53, 138, 69 bp of 5' untranslated region and 389, 241, 136, 124 bp of 3' untranslated region, respectively. A phylogenetic tree constructed with Mx peptide sequences from vertebrates revealed that MxA, C and D in the European eel formed into a clade containing zebrafish MxA and MxB and Mx proteins in other teleosts, whereas MxB in the eel was clustered together with zebrafish MxD, MxG and MxF. The transcription level of all Mx isoforms increased in a poly I:C dose-dependent manner in peripheral blood leukocytes of eels, as revealed by real-time PCR. A further experiment was conducted to reveal the temporal change in expression of these isoforms in various organs/tissues following poly I:C stimulation, and significant increase in expression was observed at various degrees in different organs or in different sampling occasions within the 12 h experimental period. In particular, MxA had the highest level of increase, while MxB had the lowest; and three isoforms, MxA, MxB and MxD had the highest increase in intestine, while the highest increase of MxC expression was observed in liver. These four isoforms of eel Mx are thus expressed differentially, and further work is certainly required to clarify the activity of promoter elements and antiviral activity of these Mx isoforms.

Isolation and characterization of a novel antibacterial peptide derived from hemoglobin alpha in the liver of Japanese eel, Anguilla japonica.

We isolated and characterized a novel antibacterial peptide, AJHbα, derived from hemoglobin alpha in the liver of Japanese eel, Anguilla japonica. It with concentration of 11.30 µM exhibited stronger antibacterial activity against pathogenic bacterium 1 × 10(6) cell ml(-1)Edwardsiella tarda than other two bacteria. The extraction procedure for AJHbα included extraction with acetate acid, ultrafiltration, cation-exchange chromatography on HiTrap™ CM FF, reverse-phase liquid chromatography on Source 5R RPC and C18 RP-HPLC. MALDI-TOF MS suggested that the peptide had an observed molecular weight of 2388.05 Da. Its amino acid sequence determined by Edman degradation was similar to those of hemoglobin alpha chain in other fish by BLAST analysis. A complete N-terminal amino acid sequence of the AJHbα was FAHWPDLGPGSPSVKKHGKVIM corresponding to the cDNA sequence by RACE amplification. Its synthetic peptide had strong antibacterial activities against ten Gram-positive or negative bacteria. To our knowledge, AJHbα was the first identified fragment of hemoglobin alpha chain with strong antibacterial activity in fish.

Quantitative gene expression and in situ localization of scygonadin potentially associated with reproductive immunity in tissues of male and female mud crabs, Scylla paramamosain.

Scygonadin (Scy) is an important antimicrobial peptide which was first isolated from the seminal plasma of Scylla serrata (now renamed as Scylla paramamosain). Elucidation of the Scy expression pattern in tissues will help in understanding its potential function associated with the reproductive immunity. In our study, Scy mRNA transcripts and its protein were found widely distributed in mature male and female crabs. Scy mRNA transcripts were significantly demonstrated in the ejaculatory duct and hemocytes of males but were much less expressed in the other tissues tested. In addition, Scy mRNA transcripts were discerned in a number of cells in the glandular epithelium of the inner wall and in the secretion inside the ejaculatory duct using the in situ hybridization method. In females, Scy mRNA transcripts were obviously demonstrated in the hemocytes and gills but weakly detected in other tissues tested. The copy number of scygonadin mRNA transcripts in the ejaculatory duct of males was greatly higher than those in other tissues, in particular, was over 60,000 fold that in the hemocytes of females. Using immunohistochemistry, the Scy protein was found at higher levels in male tissues than in female ones, particularly in the reproductive duct of males. It was also interesting to note that Scy gene expression was not significantly induced with lipopolysaccharide challenge. However, it was highly expressed in the ejaculatory duct and the seminal vesicle of pre-copulatory males and in the spermathecae of post-copulatory females under mating conditions. The results suggested that Scy, as an important antimicrobial component, probably performed more functions in males, and was likely to be involved in a function associated with crab fertilization and reproduction in both males and females during mating.

The expression pattern of scygonadin during the ontogenesis of Scylla paramamosain predicting its potential role in reproductive immunity.

The antimicrobial peptide scygonadin (Scy) was first isolated from the gonad of Scylla serrata and its gene is predominantly expressed in the ejaculatory duct of adult males. Thus, its function was predicted to be associated with reproductive immunity, but this is still unclear and needs further investigation. In our study, the expression pattern of Scy at different developmental stages of both male and female S. paramamosain was investigated, so that the potential function of this peptide could be examined. Using real-time quantitative PCR, Scy mRNA transcripts were demonstrated obviously in the vulnerable embryos and larvae-zoea I but very weakly detected in the larvae-zoea III, megalops and juveniles. The gene expression pattern showed a decreasing trend during the early developmental stages. The Scy gene had low expression in the ejaculatory duct of small and medium crabs (100g and 200g in weight) whose gonads were underdeveloped. However, the level of Scy expression was significantly increased in large crabs (300g in weight), which had normally become sexually mature at this size. It was further observed that the numbers of Scy mRNA transcripts in sexually mature crabs were significantly more abundant than in immature ones. In addition, the Scy gene was significantly expressed in the ejaculatory duct of mature male crabs during the mating period (April and May) and reached their highest expression in May. Using immunohistochemistry, the Scy protein was strongly detected in the testis and seminal vesicle of small crabs. However, in large crabs, Scy protein was intensively present in more tissues than in small crabs, including the ejaculatory duct, posterior ejaculatory duct, gill and muscle of males, and also in the spermatheca, gill and muscle of females. It is also interesting to note that Scy mRNA transcripts were detected in other crab species and showed similar expression pattern to those in S. paramamosain. This study extended our knowledge concerning the antimicrobial peptide scygonadin, which has its function principally in the ejaculatory duct of males but which may also play a role at different developmental stages of S. paramamosain from embryogenesis to maturation, and is also widely distributed in other crabs.

Soluble expression and purification of a crab antimicrobial peptide scygonadin in different expression plasmids and analysis of its antimicrobial activity.

Scygonadin is an anionic antimicrobial peptide recently identified from the seminal plasma of Scylla serrata. To gain more detailed information on its antimicrobial activity, scygonadin mature peptide was expressed in Escherichia coli in order to obtain a large quantity of biologically active product. An approximately 43 kDa fusion protein CKS-scygonadin was obtained in a highly stable and soluble form. The soluble component of the fusion CKS-scygonadin was purified by immobilized metal affinity chromatography (IMAC). A single 11 kDa recombinant scygonadin was cleaved from CKS-scygonadin and purified from the cleavage mixture using an affinity chromatography column with a yield of 10.6 mg/L. Alternatively, a recombinant scygonadin was purified from pET28-scygonadin by one-step Ni(2+) affinity chromatography and 65.9 mg/L pure recombinant scygonadin was obtained which was higher than that purified from pTrc-CKS/scygonadin in bacteria culture. The recombinant scygonadin was confirmed using SDS-PAGE analysis and MS-fingerprinting. Both recombinant products of scygonadin from different expressed plasmids showed the activity against both Gram-positive and Gram-negative bacteria, but no activity against yeast and fungi tested. The kinetic studies showed that the recombinant scygonadin was strong active against Staphylococcus aureus and the killing of S. aureus appeared time and dose dependent. Considering the quantity of recombinant product and the applicability of purification, the pET28-scygonadin expression system is a better choice to produce large quantities of recombinant scygonadin for commercial use in future. This is the first report on the heterologous expression of antimicrobial peptide scygonadin in E. coli.

A male-specific expression gene, encodes a novel anionic antimicrobial peptide, scygonadin, in Scylla serrata.

Scygonadin is a novel antimicrobial peptide, which was originally isolated from the seminal plasma of the mud crab, Scylla serrata. Based on the partial 20-residue NH(2)-terminal sequence of the peptide, H-Gly-Gln-Ala-Leu-Asn-Lys-Leu-Met-Pro-Lys-Ile-Val-Ser-Ala-Ile-Ile-Tyr-Met-Val-Gly-OH, scygonadin was cloned from the gonads of S. serrata using a degenerated reverse transcriptase (RT)-PCR and rapid amplification of cDNA ends (RACE). The full-length cDNA sequence contains an open reading frame of 539 bases (excluding polyA) with a coding capacity of 126 amino acids, which constitutes a putative NH(2)-terminal signal sequence (1-24) and a mature peptide (25-126). Analysis of the genomic DNA sequence revealed that scygonadin consists of 2300 bp containing two introns (1569 and 120 bp) and three exons (187, 131 and 218 bp) and this sequence is different from any other reported antimicrobial peptide. The theoretical pI of the mature peptide is 6.09, which suggests that it is an anionic molecule. The sex and tissue-specific expression of the scygonadin gene was revealed using RT-PCR and Northern-blot analysis of multiple tissues of S. serrata males and females and this demonstrated that the scygonadin gene was predominantly expressed in the male reproductive tract of S. serrata and was restricted to the ejaculatory duct. This suggests that scygonadin might be one of the antibacterial peptides responsible for protection of the male crab reproductive tract from invading pathogenic microorganisms, so as to maintain a sterile environment leading to successful fertilization.