Molecular characterization and expression profiles in response to bacterial infection of Chinese soft-shelled turtle interleukin-8 (IL-8), the first reptilian chemokine gene

In this study, an IL-8 homologue has been cloned and identified from a reptile, Chinese soft-shelled turtle for the first time. The full-length cDNA of turtle IL-8 was 1188 bp and contained a 312 bp open reading frame (ORF) coding for a protein of 104 amino acids. The chemokine CXC domain, which contained Glu-Leu-Arg (ELR) motif and four cysteine residues, was well conserved in turtle IL-8. The 4924 bp genomic DNA of turtle IL-8 contained four exons and three introns. Phylogenetic analysis showed that the amino acid sequence of turtle IL-8 clustered together with birds. RT-PCR analysis showed that turtle IL-8 mRNA was constitutively expressed liver, spleen, kidney, heart, blood and intestine tissues of control turtles. Real-time quantitative PCR analysis further indicated that the turtle IL-8 mRNA expression was apparent in various tissues at 8 h and up-regulated significantly during 8 h–7 d after Aeromonas hydrophila infection. The present studies will help us to understand the evolution of IL-8 molecule and the inflammatory response mechanism in reptiles.     

Molecular cloning and expression analysis of major histocompatibility complex class I,IIA and IIB genes of blunt snout bream (Megalobrama amblycephala)

Major histocompatibility complex (MHC) plays an important role in the immune response of vertebrates. In this study, we isolated MHC class IIA and IIB genes from blunt snout bream (Megalobrama amblycephala) by rapid amplification of cDNA ends polymerase chain reaction (RACE-PCR). In order to study the function of the MHC genes in M. amblycephala, tissue distribution and immune response of the MHC genes to bacterial challenge were analyzed. All the characteristic features of MHC class II chain structure could be identified in the deduced amino sequences of MHC IIA and IIB, including the leader peptide, α1/β1 and α2/β2 domains, connecting peptide and transmembrane and cytoplasmic regions, as well as conserved cysteines and N-glycosylation site. The deduced amino acid sequence of the MHC IIA and IIB molecules shared from 48% to 88% and from 65% to 77% similarity with those of other teleosts, respectively. Quantitative real-time PCR (qRT-PCR) demonstrated that MHC I and II genes were ubiquitously expressed in ten tissues, with high level in immune related tissues, including kidney, intestine, gill and spleen. Challenge of M. amblycephala with the extracellular pathogen, Aeromonas hydrophila, resulted in a significant increase in the expression of MHC I, MHC IIA and IIB mRNA within 72 h after infection in gill, kidney, intestine and liver, followed by a recovery to normal level after 120 h. The changes of expression levels for MHC IIA and IIB in most tissues were significantly higher than that of MHC I in the corresponding tissues at most time points (P < 0.05). These results demonstrated the MHC genes played an important role in response to bacterial infection in M. amblycephala; however, MHC class I and II genes showed different functional activity, which need be further investigated in teleost.     

Evaluation of a loop-mediated isothermal amplification assay for rapid diagnosis of soft-shelled turtle iridovirus

Softshelled turtle iridovirus (STIV) is the first Asian iridovirus isolated from reptiles, which infects soft-shelled turtles severely and leads to “Red neck disease” associated with high mortality. A set of four specific primers was designed by targeting the STIV Thymidine kinase (TK) gene and amplified STIV DNA specifically under optimized amplification conditions at 63 °C for 60 min. The sensitivity of the loop-mediated isothermal amplification (LAMP) assay was found to be 20 copies/μl of STIV DNA. To evaluate the application of the LAMP assay for detection of STIV in clinical samples, 223 samples suspected of STIV infection from turtle tissues were tested by the LAMP assay and by cell-based virus isolation. A 78.5% concordance was observed between the results of the two methods. In this study, a robust and simple LAMP assay for rapid detection of STIV was developed and evaluated, which is the first suitable for potential diagnosis and helping to monitor STIV infections in the aquaculture industry.     

The inhibitory effect of environmental ammonia on Danio rerio LPS induced acute phase response

Ammonia is a toxic by-product of amino acid catabolism and a common environmental pollutant that has been associated with increased disease susceptibility in fish although the mechanism is not well understood. We addressed the hypothesis that elevated environmental ammonia acts by impairing the acute phase response (APR). Specifically, we determined the impact of sub-lethal acute (24 h) and chronic (14 d) ammonia exposure on acute phase protein gene expression in zebrafish (Danio rerio) in response to a challenge with bacterial lipopolysaccharide (LPS: i.p. 10 μg/g after 24 h). A panel of LPS-responsive genes (SAA, HAMP, LECT2, Hp and IL1β) were identified and evaluated by real-time quantitative PCR. Ammonia was found to impair induction of SAA, HAMP and LECT2 by 50-90%. Both short (15 min, 1 h and 24 h) and long-term (14 days) exposure to high environmental ammonia concentrations significantly elevated whole-body cortisol levels compared with control fish. Our results reveal for the first time that exposure to high environmental levels of ammonia suppresses the innate immune response in fish. We hypothesize that high environmental ammonia-mediated elevation of cortisol levels in zebrafish may be playing a key role in this immunosuppression, while the mechanisms involved remains to be elucidated.     

Molecular characterization of nucleotide binding and oligomerization domain (NOD)-2, analysis of its inductive expression and down-stream signaling following ligands exposure and bacterial infection in rohu (Labeo rohita)

Nucleotide-binding and oligomerization domain (NOD)-2 is a cytoplasmic pattern recognition receptor (PRR) and is a member of NOD like receptor (NLR) family. It senses a wide range of bacteria and viruses or their products and is involved in innate immune responses. In this report, NOD-2 gene was cloned and characterized from rohu (Labeo rohita) which is highly commercially important fish species in the Indian subcontinent. The full length rohu NOD-2 (rNOD-2) cDNA comprised of 3176 bp with a single open reading frame (ORF) of 2949 bp encoding a polypeptide of 982 amino acids (aa) with an estimated molecular mass of 109.65 kDa. The rNOD-2 comprised two N-terminal CARD domains (at 4-91 aa and 111-200 aa), one NACHT domain (at 271-441 aa) and seven C-terminal leucine rich repeat (LRR) regions. Phylogenetically, rNOD-2 was closely related to grass carp NOD-2 (gcNOD2) and exhibited significant similarity (94.2%) and identity (88.6%) in their amino acids. Ontogeny analysis of rNOD-2 showed its constitutive expression across the developmental stages, and highlighted the embryonic innate defense system in fish. Tissue specific analysis of rNOD-2 by quantitative real-time PCR (qRT-PCR) revealed its wide distribution; highest expression was in liver followed by blood. In response to PGN and LTA stimulation, Aeromonas hydrophila and Edwardsiella tarda infection, and poly I:C treatment, expression of rNOD-2 and its associated downstream molecules RICK and IFN-γ were significantly enhanced in the treated fish compared to control. These findings suggested the key role of NOD-2 in augmenting innate immunity in fish in response to bacterial and viral infection. This study may be helpful for the development of preventive measures against infectious diseases in fish.     

Virulence plasmid of Aeromonas hydrophila induces macrophage apoptosis and helps in developing systemic infection in mice

Pathogenic Aeromonas hydrophila Strain AO1 bears a 21 kb plasmid encoding several virulence determinants. Infection studies revealed that this isolate induced cytotoxicity in BALB/c mice splenic macrophages involving reactive oxygen species generation. DNA gel, Hoechst 33342, annexin-V and TUNEL assay documented macrophage death induced by 21 kb plasmid bearing isolates to be apoptotic in nature. Apoptosis induced by the plasmid bearing isolates involved initiator caspase-8 and caspase-9 and executed by effector caspase-3. ELISA revealed the wild-type isolate as weak inducer of pro-inflammatory cytokine IL-1β. Oral infection with wild-type isolates caused systemic infection in BALB/c mice. With plasmid curing the isolate looses several virulence attributes including cytotoxic potential. The cured isolate induced significant amounts of IL-1β from infected macrophages, disseminated into Peyer's patches, spleen and liver but never attained the bacterial loads recorded with wild-type isolates and were rapidly cleared. Transformation of 21 kb plasmid helped the cured bacteria regain wild-type virulence attributes, apoptotic potential and ability to cause systemic infection in mice. Thus the 21 kb plasmid is a virulence factor in mice. It helps in suppressing the production of pro-inflammatory cytokine IL-1β and induced apoptosis of host macrophages enabling A. hydrophila to evade host immune responses and establish systemic infection in mice.     

An ancient molecule with novel function: Alanine aminotransferase as a lipopolysaccharide binding protein with bacteriocidal activity

Alanine aminotransaminase (ALT) has been identified from bacteria to plants to animals including humans. The increase in serum ALT is regarded as an index for clinical diagnosis of liver function in humans. However, ALT elevation is also reported in non-liver injury conditions and in apparently healthy people, suggesting it may play a fundamental role physiologically. Herein we isolated an alt homolog, Amphialt, from Branchiostoma japonicus, an intermediatary species from invertebrates to vertebrates, which encoded a polypeptide of 500 amino acids with more than 62 and 52% sequence identity to vertebrate and invertebrate ALT isoenzymes, respectively. It was constitutively expressed in many tissues including the hepatic caecum, the precursor of liver, and its expression in the caecum was significantly up-regulated by challenge with lipopolysaccharides (LPS). Strikingly, recombinant AmphiALT, with a specific activity of 0.114 ± 0.02 U/mg, was capable of specifically binding to the Gram-negative bacteria Escherichia coli and Aeromonas hydrophila and to their conserved molecule LPS, as well as inhibiting the growth of E. coli and causing its lysis. In contrast, AmphiALT did not bind to the Gram-positive bacteria Staphyloccocus aureus and Bacillus subtilis as well as their conserved molecule LTA. In addition, a high homology noted between amphioxus and mammalian ALT sequences suggested a functional conservation of ALT evolutionarily, hinting at the clue that mammalian ALT may also play an antibacterial role similar to that of AmphiALT. Taken together, it is proposed that AmphiALT is an immune-relevant molecule capable of identifying LPS and causing damage to Gram-negative bacteria like E. coli and A. hydrophila. It also bolsters the notion that the hepatic caecum of amphioxus is the precursor of vertebrate liver, acting as a major tissue in acute phase response.     

Molecular cloning,expression analysis and functional characterization of interleukin-22 in So-iny mullet, Liza haematocheila

In the present study, interleukin-22 (IL-22) from So-iny mullet (Liza haematocheila) was identified, and its tissue expression in both healthy and Streptococcus dysgalactiae-infected fish was examined. The full length cDNA sequence of mullet IL-22 was 1070 bp, containing an open reading frame of 555 bp. The deduced amino acid sequence shared high similarity (45.1–67.9%) with IL-22 from other fish species. Mullet IL-22 also contained an IL-10 family signature and four cysteine residues that were well conserved in other vertebrate IL-22 molecules. Mullet IL-22 mRNA was highly expressed in kidney, moderately expressed in liver and gut, and relatively weakly expressed in spleen, and its expression was significantly up-regulated in all the examined tissues following S. dysgalactiae infection. Furthermore, recombinant mullet IL-22 protein was shown to promote the expression of β-defensin in the four tissues and to increase the survival rate of the fish infected with S. dysgalactiae. Our results suggest mullet IL-22 plays an important role in the immune defense against bacterial infection and has the potential to be used to treat bacterial diseases in fish.     

Identification and analysis of an intracellular Cu/Zn superoxide dismutase from Sepiella maindroni under stress of Vibrio harveyi and Cd

Superoxide dismutases (SODs) are ubiquitous family of metalloenzymes involved in protecting organisms from excess reactive oxygen species damage. In this paper, a novel intracellular Cu/ZnSOD from Sepiella maindroni (designated as SmSOD) was identified and characterized. The full-length cDNA sequence of SmSOD (GenBank accession No. KF908850) was 709 bp containing an open reading frame (ORF) of 459 bp, encoding 153 amino acid residues peptide with predicted pI/MW (6.02/15.75 kDa), a 131 bp-5′- and 116 bp-3′- untranslated region (UTR). BLASTn analysis and phylogenetic relationship strongly suggested that the sequence shared high similarity with known Cu/Zn SODs. Several highly conserved motifs, including two typical Cu/Zn SOD family domains, two conserved Cu-/Zn-binding sites (H-47, H-49, H-64, H-120 for Cu binding, and H-64, H-72, H-81, D-84 for Zn binding) and intracellular disulfide bond (C-58 and C-146), were also identified in SmSOD. Time-dependent mRNA expression of SmSOD in hepatopancreas was recorded by quantitative real-time RT-PCR after Vibrio harveyi injection and Cd²⁺ exposure. The results indicated that SmSOD was an acute-phase protein involved in the immune responses against pathogens and biological indicator for metal contaminants in aquatic environment.     

A multidomain galectin involved in innate immune response of pearl oyster Pinctada fucata

Galectins could specifically bind to β-galactoside residues and play crucial roles in innate immune responses of vertebrates and invertebrates. In this study, the cDNA of a galectin with multiple carbohydrate-recognition domains (CRDs) was cloned from pearl oyster Pinctada fucata (designated as PoGal). PoGal cDNA was 2138 bp long and consisted of a 5′-untranslated region (UTR) of 120 bp, a 3′-UTR of 350 bp with two cytokine RNA instability motifs (ATTTA), and an open reading frame (ORF) of 1668 bp encoding a polypeptide of 555 amino acids with an estimated molecular mass of 63.4 kDa and a theoretical isoelectric point of 4.8. PoGal contained four CRDs, each CRD of PoGal all had the conserved carbohydrate-binding motifs H-NPR and WG-ER. PoGal shared 43.7% and 62.9% identity to those of bay scallop and eastern oyster, respectively, which were only two galectins with four CRDs. The phylogenetic analysis revealed that all galectins with four CRDs formed a single clade. PoGal mRNA was constitutively expressed in all detected tissues, and the expression level of PoGal mRNA was significantly up-regulated in digestive gland, mantle, haemocyte, gonad and intestine after Vibrio alginolyticus stimulation. The expression profile analysis showed that the expression level of PoGal mRNA was significantly up-regulated at 4, 8 and 12 h after V. alginolyticus stimulation. These results suggested that PoGal was a constitutive and inducible acute-phase protein that perhaps involved in innate immune response of pearl oyster.     

Study on the immune response to recombinant Hsp70 protein from Megalobrama amblycephala

The expression of heat shock protein 70 (Hsp70) is induced in response to many factors including high temperature, infection, metal pollutants and toxic chemicals. In this study, Megalobrama amblycephala HSP70 promoter was cloned, and characteristic heat shock elements (HSEs) were identified in the promoter region. The recombinant M. amblycephala Hsp70 protein (rMaHsp70) was expressed and purified from Escherichia coli BL21 (DE3). To evaluate in vivo immune response of rMaHsp70, we administered intraperitoneal (IP) injection, and demonstrated that rMaHsp70 stimulated M. amblycephala immune activity by inducing the expression of HSP70, HIF-1α, HSC70, CXCR4b, TNF-α and IL-1β mRNAs in liver, headkidney, spleen and gill, as well as SOD, glutathione, lysozyme and interferon alpha proteins in serum and liver. The effect of rMaHsp70 as adjuvant against Aeromonas hydrophila was assessed by injecting a mixed vaccine of rMaHsp70 and A. hydrophila (A. hydrophila/Hsp70) into M. amblycephala, and the relative percent survival (RPS) in the A. hydrophila/Hsp70 group was 75% compared to 50% in the A. hydrophila/PBS group. Furthermore, rMaHsp70 also promoted the proliferation and suppressed apoptosis in M. amblycephala fin cells (MAF) in a dose-dependent manner. Taken together, these results suggest that rMaHsp70 can induce organic immune response and improve environmental tolerance.     

Molecular cloning,expression and functional analysis of B-cell activating factor (BAFF) in yellow grouper, Epinephelus awoara

B cell activating factor (BAFF), a ligand belonging to the tumor necrosis factor (TNF) family is critical to B cell survival, proliferation, maturation and immunoglobulin secretion. In this study, the yellow grouper (Epinephelus awoara) BAFF (designated EaBAFF) gene was cloned using RT-PCR and RACE (rapid amplification of cDNA ends) techniques. The full-length EaBAFF was 1442 bp and contained an open reading frame of 780 bp encoding a putative protein of 259 amino acids. Amino acids sequence comparison indicated that EaBAFF possessed the TNF signature. The soluble BAFF (EasBAFF) had been cloned into pET28a. SDS-PAGE and Western blotting analysis confirmed that the soluble fusion protein His-EasBAFF was efficiently expressed in Escherichia coli BL21 (DE3). In vitro, the WST-8 assay indicated that EasBAFF was not only able to promote the survival/proliferation of yellow grouper splenic lymphocytes but also able to promote the survival/proliferation of mouse splenic B cells. Our findings may provide valuable information for research into the immune system of E. awoara and EasBAFF may serve as a potential immunologic factor for enhancing immunological efficacy in fish.