Transcriptional Regulation and Signaling of Type IV IFN with Identification of the ISG Repertoire in an Amphibian Model, Xenopus laevis.

The type IV IFN (IFN-υ) is reported in vertebrates from fish to primary mammals with IFN-υR1 and IL-10R2 as receptor subunits. In this study, the proximal promoter of IFN-υ was identified in the amphibian model, Xenopus laevis, with functional IFN-sensitive responsive element and NF-κB sites, which can be transcriptionally activated by transcription factors, such as IFN regulatory factor (IRF)1, IRF3, IRF7, and p65. It was further found that IFN-υ signals through the classical IFN-stimulated gene (ISG) factor 3 (ISGF3) to induce the expression of ISGs. It seems likely that the promoter elements of the IFN-υ gene in amphibians is similar to type III IFN genes, and that the mechanism involved in IFN-υ induction is very much similar to type I and III IFNs. Using recombinant IFN-υ protein and the X. laevis A6 cell line, >400 ISGs were identified in the transcriptome, including ISGs homologous to humans. However, as many as 268 genes were unrelated to human or zebrafish ISGs, and some of these ISGs were expanded families such as the amphibian novel TRIM protein (AMNTR) family. AMNTR50, a member in the family, was found to be induced by type I, III, and IV IFNs through IFN-sensitive responsive element sites of the proximal promoter, and this molecule has a negative role in regulating the expression of type I, III, and IV IFNs. It is considered that the current study contributes to the understanding of transcription, signaling, and functional aspects of type IV IFN at least in amphibians.

Thioclava litoralis sp. nov., a novel species of alphaproteobacterium, isolated from surface seawater.

A Gram-negative, aerobic, rod-shaped, non-motile bacterium, designated as FTW29T, was isolated from surface seawater sampled in Futian district, Shenzhen, China. Growth of strain FTW29T was observed at 15-42 ℃ (optimum, 28-30 ℃), pH 4.0-9.0 (optimum, pH 5.5-7.5) and in the presence of 0.5-10% NaCl (optimum, 3.0% NaCl). Strain FTW29T showed 95.0-96.8% 16 S rRNA gene sequence similarity to various type strains of the genera Thioclava, Sinirhodobacter, Rhodobacter, Haematobacter and Frigidibacter of the family Paracoccaceae, and its most closely related strains were Thioclava pacifica DSM 10,166T (96.8%) and Thioclava marina 11.10-0-13T (96.7%). The phylogenomic tree constructed on the bac120 gene set showed that strain FTW29T formed a clade with the genus Thioclava, with a bootstrap value of 100%. The evolutionary distance values between FTW29T and type strains of the genus Thioclava were 0.17-0.19, which are below the recommended standard (0.21-0.23) for defining a novel genus in the family Paracoccaceae. In strain FTW29T, the major fatty acids identified were summed feature 8 (C18:1ω7c) and C16:0, and the predominant respiratory quinones were ubiquinone-10 and ubiquinone-9. The composition of polar lipids in strain FTW29T included diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, an unidentified phospholipid, an unidentified aminolipid, two unidentified glycolipids and an unidentified lipid. The genome of strain FTW29T comprised one circle chromosome and six plasmids, with a G + C content of 61.4%. The average nucleotide identity, average amino acid identity, and digital DNA-DNA hybridization values between strain FTW29T and seven type strains of the genus Thioclava were 76.6-78.4%, 53.2-56.4% and 19.3-20.4%, respectively. Altogether, the phenotypic, phylogenetic and chemotaxonomic evidence illustrated in this study suggested that strain FTW29T represents a novel species of the genus Thioclava, with the proposed name Thioclava litoralis sp. nov. The type strain is FTW29T (= KCTC 82,841T = MCCC 1K08523T).

Microbulbifer bruguierae sp. nov., isolated from sediment of mangrove plant Bruguiera sexangula, and comparative genomic analyses of the genus Microbulbifer.

A Gram-negative, non-motile, strictly aerobic, rod-shaped bacterium, designated as H12T, was isolated from the sediments of mangrove plant Bruguiera sexangula taken from Dapeng district, Shenzhen, PR China. The pairwise 16S rRNA gene sequence analysis showed that strain H12T shared high identity levels with species of the genus Microbulbifer, with the highest similarity level of 98.5 % to M. pacificus SPO729T, followed by 98.1 % to M. donghaiensis CN85T. Phylogenetic analysis using core-genome sequences showed that strain H12T formed a cluster with type species of M. pacificus SPO729T and M. harenosus HB161719T. The complete genome of strain H12T was 4 481 396 bp in size and its DNA G+C content was 56.7 mol%. The average nucleotide identity and digital DNA-DNA hybridization values among strain H12T and type species of genus Microbulbifer were below the cut-off levels of 95-96 and 70 %, respectively. The predominant cellular fatty acids of strain H12T were iso-C15 : 0 (22.5 %) and C18 : 1 ω7c (13.9 %). Ubiquinone-8 was detected as the major respiratory quinone. The polar lipids of strain H12T comprised one phosphatidylglycerol, one phosphatidylethanolamine, one unidentified aminoglycophospholipid, one unidentified glycophospholipid, three unidentified glycolipids, two unidentified aminolipids, and one unidentified lipid. Based on polyphasic evidence, strain H12T represents a novel species of the genus Microbulbifer, for which the name Microbulbifer bruguierae sp. nov. is proposed. The type strain is H12T (=KCTC 92859T=MCCC 1K08451T). Comparative genomic analyses of strain H12T with strains of the genus Microbulbifer reveal its potential in degradation of pectin.

Identification and functional characterization of interleukin-22 (IL-22) in orange-spotted grouper (Epinephelus coioides).

In mammals, IL-22 is considered as a critical cytokine regulating of immunity and homeostasis at barrier surfaces. Although IL-22 have been functional characterization in different species of fish, the studies about distinct responses of IL-22 in different organs/tissues/cell types is rather limited. Here, we identified and cloned IL-22 gene (named as Ec-IL-22) from grouper (Epinephelus coioides). Ec-IL-22 gene was detected in all orangs/tissues examined, and was induced in intestine, gill, spleen, head kidney, and primary head kidney/intestine leukocytes following the stimulation of LPS and poly (I:C), as well as Vibrio harveyi and Singapore grouper iridovirus infection (SGIV). In addition, the stimulation of DSS could induce the expression of Ec-IL-22 in intestine and primary leukocytes from intestine. Importantly, the treatment of recombinant Ec-IL-22 induced the mRNA level of proinflammatory cytokines in primary intestine/head kidney leukocytes. The present results improve the understanding of expression patterns and functional characteristics of fish IL-22 in different organs/tissues/cell types.

Different activation of STAT1 and STAT2 phosphorylation by IFNc, IFNd, and IFNh in tilapia.

Type I IFNs are a subset of cytokines exerting their antiviral effects mainly through the JAK-STAT signalling. Immunogenetic studies have shown that fish possess key components of IFN-JAK-STAT cascade, but the information about the distinct responses of STAT1 and STAT2 to different IFNs is rather limited in fish. Here, we identified and cloned STAT1 and STAT2 genes (named as On-STAT1 and On-STAT2) from tilapia, Oreochromis niloticus. On-STAT1 and On-STAT2 genes were detected in all orangs/tissues examined, and were rapidly induced in spleen, head kidney, and liver following the stimulation of poly(I:C). In addition, the stimulation of poly(I:C), poly(A:T), and different subgroups of recombinant IFNs could induce the expression of On-STAT1 and On-STAT2 in TA-02 cells with distinct induction levels. Importantly, On-STAT2 was rapidly phosphorylated by all three subgroups of IFNs, but the phosphorylation of On-STAT1 was only observed in IFNc- and IFNh-treated TA-02 cells, reflecting the distinct activation of STAT by different subgroups of fish IFNs. The present results thus contribute to better understanding of the JAK-STAT signalling mediated by different subgroups of IFNs in fish.

Evolutionary and functional conservation of IRF7 in the Tibetan frog Nanorana parkeri.

BACKGROUND: The production of interferons (IFNs) is essential for the control of viral infections, and interferon regulatory factor 7 (IRF7) is considered as a vital regulator for the transcription of type I IFNs. Amphibians appear to possess a highly expanded type I IFN repertoire, consisting of intron-containing genes as observed in fish, and intronless genes as in other higher vertebrates. However, the knowledge on transcriptional regulatory mechanism of these two types of type I IFN genes is rather scarce in amphibians. METHODS AND RESULTS: A IRF7 gene named as Np-IRF7 was identified in Tibetan frog (Nanorana parkeri), and bioinformatic analysis revealed that the predicted protein of Np-IRF7 contains several important structural features known in IRF7. Expression analysis showed that Np-IRF7 gene was widely expressed and rapidly induced by poly(I:C) in different organs/tissues. Interestingly, luciferase reporter assay revealed that intronless IFN promoters were more effectively activated than intron-containing IFN promoter in Np-IRF7-transfected cells. Moreover, the overexpression of Np-IRF7 could induce the expression of ISGs and suppress the replication of FV3 in A6 cells. CONCLUSION: Np-IRF7 is indeed the ortholog of known IRF7, and IRF7 is structurally conserved in different lineages of vertebrates. Np-IRF7 played distinct roles in the activation of intron-containing and intronless type I IFN promoters, thus inducing the expression of interferon-stimulated antiviral effectors and providing a protection against ranavirus infection. The present research thus contributes to a better understanding of regulatory function of IRF7 in the IFN-mediated antiviral response of anuran amphibians.

Dextran Sulfate Sodium Salt (DSS) induced enteritis in Orange-spotted grouper, Epinephelus coioides.

The enteritis is a common disease in fish farming, but the pathogenesis is still not fully understood. The aim of the present study was to investigate the inducement of Dextran Sulfate Sodium Salt (DSS) intestinal inflammation on Orange-spotted grouper (Epinephelus coioides). The fish were challenged with 200 µl 3% DSS via oral irrigation and feeding, an appropriate dose based on the disease activity index of inflammation. The results indicated that the inflammatory responses induced by DSS were closely associated with the expression of pro-inflammatory cytokines including interleukin 1ß (IL-1ß), IL-8, IL16, IL-10 and tumor necrosis factor α (TNF-α), as well as NF-κB and myeloperoxidase (MPO) activity. At day5 after DSS treatment, the highest levels of all parameters were observed. Also, the severe intestinal lesions (intestinal villus fusion and shedding), strong inflammatory cell infiltration and microvillus effacement were seen through histological examination and SEM (scanning electronic microscopy) analysis. During the subsequent 18 days of the experimental period, the injured intestinal villi were gradually recovery. These data is beneficial to further investigate the pathogenesis of enteritis in farmed fish, which is helpful for the control of enteritis in aquaculture.

Identification and characterization of two long-type peptidoglycan recognition proteins, PGRP-L1 and PGRP-L2, in the orange-spotted grouper, Epinephelus coioides.

Peptidoglycan recognition proteins (PGRPs) play an important role in innate immunity by recognizing components of pathogenic bacteria (such as peptidoglycan, PGN) and are evolutionarily conserved pattern recognition receptors (PRRs) in both invertebrates and vertebrates. In the present study, two long-type PGRPs (designed as Eco-PGRP-L1 and Eco-PGRP-L2) were identified in orange-spotted grouper (Epinephelus coioides), which is a major economic species cultured in Asia. The predicted protein sequences of both Eco-PGRP-L1 and Eco-PGRP-L2 contain a typical PGRP domain. Eco-PGRP-L1 and Eco-PGRP-L2 exhibited organ/tissue-specific expression patterns. An abundant expression of Eco-PGRP-L1 was observed in pyloric caecum, stomach and gill, whereas a highest expression level of Eco-PGRP-L2 was found in head kidney, spleen, skin and heart. In addition, Eco-PGRP-L1 is distributed in the cytoplasm and nucleus, while Eco-PGRP-L2 is mainly localized in cytoplasm. Both Eco-PGRP-L1 and Eco-PGRP-L2 were induced following the stimulation of PGN and have PGN binding activity. In addition, functional analysis revealed that Eco-PGRP-L1 and Eco-PGRP-L2 possess antibacterial activity against Edwardsiella tarda. These results may contribute to understand the innate immune system of orange-spotted grouper.

Retroposition of the Long Transcript from Multiexon IFN-ß Homologs in Ancestry Vertebrate Gave Rise to the Proximal Transcription Elements of Intronless IFN-ß Promoter in Humans.

IFN-ß is a unique member of type I IFN in humans and contains four positive regulatory domains (PRDs), I-II-III-IV, in its promoter, which are docking sites for transcription factors IFN regulatory factor (IRF) 3/7, NF-κB, IRF3/7, and activating transcription factor 2/Jun proto-oncogene, respectively. In chicken IFN-ß and zebrafish IFNφ1 promoters, a conserved PRD or PRD-like sequences have been reported. In this study, a type I IFN gene, named as xl-IFN1 in the amphibian model Xenopus laevis, was found to contain similar PRD-like sites, IV-III/I-II, in its promoter, and these PRD-like sites were proved to be functionally responsive to activating transcription factor 2/Jun proto-oncogene, IRF3/IRF7, and p65, respectively. The xl-IFN1, as IFNφ1 in zebrafish, was transcribed into a long and a short transcript, with the long transcript containing all of the transcriptional elements, including PRD-like sites and TATA box in its proximal promoter. A retroposition model was then proposed to explain the transcriptional conservation of IFNφ1, xl-IFN1, and IFN-ß in chicken and humans.

Description and genomic characterization of Gallaecimonas kandeliae sp. nov., isolated from the sediments of mangrove plant Kandelia obovate.

The genus Gallaecimonas, proposed by Rodríguez-Blanco et al. (Int J Syst Evol Microbiol 60:504-509, 2010), is mainly isolated from marine environments. So far, only three species have been identified and characterized in this genus. In this study, a new Gallaecimonas strain named Q10T was isolated from the sediments of mangrove plant Kandelia obovate taken from Dapeng district, Shenzhen, China. Strain Q10T was a Gram-stain-negative, non-motile, strictly aerobic, rod-shaped bacterium, and grew with 0-8.0% (w/v) NaCl, at 10-45 °C and at pH 5.5-8.5. Phylogenetic analysis indicated that strain Q10T and the three Gallaecimonas species formed a clade in the tree, with 16S rRNA gene sequence similarities ranging from 96.0 to 97.0%. The major respiratory quinone is Q8. The polar lipids comprised aminolipid, aminophospholipid, diphosphatidylglycerol, glycolipid, phosphatidylethanolamine, phosphatidylglycerol, glycophospholipid and phospholipid. The predominant fatty acids are C16:0, C17:1ω8c, summed feature 3 (C16:1ω7c/C16:1ω6c), and iso-C16:0. The complete genome of strain Q10T is 3,836,841 bp with a G+C content of 62.6 mol%. The orthologous proteins analysis revealed 55 unique proteins in strain Q10T related to important biological processes, especially three frataxins related to iron-sulfur cluster assembly, which may play a pivotal role in environmental adaptability of this species. Based on polyphasic taxonomic data, strain Q10T is considered to represent a novel species within the genus Gallaecimonas, for which the name Gallaecimonas kandelia sp. nov. is proposed. The type strain is Q10T (=KCTC 92860T=MCCC 1K08421T). These results contribute to a better understanding of general features and taxonomy of the genus Gallaecimonas.