Environmental viromes reveal the global distribution signatures of deep-sea DNA viruses.

INTRODUCTION: Viruses are abundant and ecologically significant in marine ecosystems. However, the virome of deep-sea sediments is not extensively investigated. OBJECTIVES: To explore the distribution pattern of deep-sea viruses on a global scale, the viromes of DNA viruses isolated from 138 sediments of 5 deep-sea ecosystems were characterized. METHODS: The viral particles were purified from each sediment sample. Then the viral DNAs were extracted and subjected to viral metagenomic analysis. RESULTS: Here, we constructed a global deep-sea environmental virome dataset by analyzing the viral DNA of 138 sediment samples. A total of 347,737 viral operational taxonomic units (vOTUs) were identified, of which 84.94% were hitherto unknown, indicating that deep sea was a reservoir of novel DNA viruses. Furthermore, circular viral genome analysis revealed 98,581 complete genomes. The classified vOTUs included eukaryotic (44.55%) and prokaryotic (25.75%) viruses, and were taxonomically assigned to 63 viral families. The composition and abundance of the deep-sea sediment viromes were dependent on the deep-sea ecosystem as opposed to geographical region. Further analysis revealed that the viral community differentiation in different deep-sea ecosystems was driven by the virus-mediated energy metabolism. CONCLUSION: Our findings showed that deep-sea ecosystems are a reservoir of novel DNA viruses and the viral community is shaped by the environmental characteristics of deep-sea ecosystems, thus presenting critical information for determining the ecological significance of viruses in global deep-sea ecosystems.

Lactobacillus plantarum E2 regulates intestinal microbiota and alleviates Pseudomonas plecoglossicida induced inflammation and apoptosis in zebrafish (Danio rerio).

Pseudomonas plecoglossicida infection is a highly contagious epidemic in aquaculture, causing significant mortality among teleost. Our previous research has demonstrated that Lactobacillus plantarum E2 is beneficial for large yellow croaker in resisting infections caused by P. plecoglossicida. However, the relevant mechanisms remain largely unclear. In the present study, we used zebrafish (Danio rerio) to further explore the function of L. plantarum E2 and its mechanisms for resisting P. plecoglossicida infection. E2 supplementation diet significantly improved the growth rates and α-amylase and trypsin activities of the liver in zebrafish. After challenge with P. plecoglossicida strain PQLYC4, the survival rates of zebrafish were improved, and immune-related genes expression (IL-1ß, TNF-α, IL-8, Ig-Z, TLR-22 and IL-12α) were down-regulated. Histological analysis showed that E2 group had a longer intestinal villus and thicker intestinal walls after 30 days of feeding and healthier intestinal structure after challenge with P. plecoglossicida strain PQLYC4. Furthermore, co-incubation of zebrafish embryo fibroblast (ZF-4 cells) with L. plantarum E2 reduced apoptosis of ZF-4 cells after exposed to P. plecoglossicida. Intestinal microbiota analysis showed that E2 strain significantly increased the relative abundance of Lactobacillus and Pseudomonas, and PCoA analysis revealed a noticeable divergence in the intestinal microbial communities after E2 supplement. Together, our results suggested that E2 strain may promote zebrafish survival against P. plecoglossicida infection by regulating the intestinal microbiota and alleviating inflammatory response and apoptosis, thus exhibiting the potential as a probiotic.

Isolation and Identification of a New Isolate of Anguillid Herpesvirus 1 from Farmed American Eels (Anguilla rostrata) in China.

Anguillid herpesvirus 1 (AngHV-1) is a pathogen that causes hemorrhagic disease in various farmed and wild freshwater eel species, resulting in significant economic losses. Although AngHV-1 has been detected in the American eel (Anguilla rostrata), its pathogenicity has not been well characterized. In this study, an AngHV-1 isolate, tentatively named AngHV-1-FC, was isolated from diseased American eels with similar symptoms as those observed in AngHV-1-infected European eels and Japanese eels. AngHV-1-FC induced severe cytopathic effects in the European eel spleen cell line (EES), and numerous concentric circular virions were observed in the infected EES cells by transmission electron microscopy. Moreover, AngHV-1-FC caused the same symptoms as the naturally diseased European eels and Japanese eels through experimental infection, resulting in a 100% morbidity rate and 13.3% mortality rate. The whole genome sequence analyses showed that the average nucleotide identity value between AngHV-1-FC and other AngHV-1 isolates ranged from 99.28% to 99.55%. However, phylogenetic analysis revealed that there was a genetic divergence between AngHV-1-FC and other AngHV-1 isolates, suggesting that AngHV-1-FC was a new isolate of AngHV-1. Thus, our results indicated that AngHV-1-FC can infect farmed American eels, with a high pathogenicity, providing new knowledge in regard to the prevalence and prevention of AngHV-1.

The genome of a hadal sea cucumber reveals novel adaptive strategies to deep-sea environments.

How organisms cope with coldness and high pressure in the hadal zone remains poorly understood. Here, we sequenced and assembled the genome of hadal sea cucumber Paelopatides sp. Yap with high quality and explored its potential mechanisms for deep-sea adaptation. First, the expansion of ACOX1 for rate-limiting enzyme in the DHA synthesis pathway, increased DHA content in the phospholipid bilayer, and positive selection of EPT1 may maintain cell membrane fluidity. Second, three genes for translation initiation factors and two for ribosomal proteins underwent expansion, and three ribosomal protein genes were positively selected, which may ameliorate the protein synthesis inhibition or ribosome dissociation in the hadal zone. Third, expansion and positive selection of genes associated with stalled replication fork recovery and DNA repair suggest improvements in DNA protection. This is the first genome sequence of a hadal invertebrate. Our results provide insights into the genetic adaptations used by invertebrate in deep oceans.

Identification of a New Antifungal Peptide W1 From a Marine Bacillus amyloliquefaciens Reveals Its Potential in Controlling Fungal Plant Diseases.

A bacterium, Bacillus amyloliquefaciens W0101, isolated from the Arctic Ocean, showed potent antifungal activity against several plant pathogenic fungi. An antifungal peptide W1, with a molecular weight of approximately 2.4 kDa, was purified from the culture supernatant of the strain W0101 using ion-exchange chromatography and high-performance liquid chromatography. By analysis of Liquid Chromatograph-Mass Spectrometer, the peptide W1 was identified as a new antifungal peptide derived from the fragment of preprotein translocase subunit YajC. Further analysis revealed that W1 could disrupt the hyphae and spores of Sclerotinia sclerotiorum and inhibit its growth. W1 suppressed S. sclerotiorum and Fusarium oxysporum at a minimum inhibitory concentration of 140 and 58 µg/ml, respectively. The antifungal activity of W1 remained stable at 20-80°C or pH 6-11, with reduced activity at 100-110°C and pH 4-5, and under three protease treatments. Additionally, W1 also had a certain extent of metal ion resistance. These results therefore suggest that the peptide W1 from marine B. amyloliquefaciens W0101 may represent a new antifungal peptide with potential application in the biocontrol of plant diseases.

Whole genome sequencing of a snailfish from the Yap Trench (~7,000 m) clarifies the molecular mechanisms underlying adaptation to the deep sea.

Hadal environments (depths below 6,000 m) are characterized by extremely high hydrostatic pressures, low temperatures, a scarce food supply, and little light. The evolutionary adaptations that allow vertebrates to survive in this extreme environment are poorly understood. Here, we constructed a high-quality reference genome for Yap hadal snailfish (YHS), which was captured at a depth of ~7,000 m in the Yap Trench. The final YHS genome assembly was 731.75 Mb, with a contig N50 of 0.75 Mb and a scaffold N50 of 1.26 Mb. We predicted 24,329 protein-coding genes in the YHS genome, and 24,265 of these genes were successfully functionally annotated. Phylogenetic analyses suggested that YHS diverged from a Mariana Trench snailfish approximately 0.92 million years ago. Many genes associated with DNA repair show evidence of positive selection and have expanded copy numbers in the YHS genome, possibly helping to maintain the integrity of DNA under increased hydrostatic pressure. The levels of trimethylamine N-oxide (TMAO), a potent protein stabilizer, are much higher in the muscles of YHS than in those of shallow-water fish. This difference is perhaps due to the five copies of the TMAO-generating enzyme flavin-containing monooxygenase-3 gene (fmo3) in the YHS genome and the abundance of trimethylamine (TMA)-generating bacteria in the YHS gut. Thus, the high TMAO content might help YHS adapt to high hydrostatic pressure by improving protein stability. Additionally, the evolutionary features of the YHS genes encoding sensory-related proteins are consistent with the scarce food supply and darkness in the hadal environments. These results clarify the molecular mechanisms underlying the adaptation of hadal organisms to the deep-sea environment and provide valuable genomic resources for in-depth investigations of hadal biology.

Development of monoclonal antibody against IgT of a perciform fish, large yellow croaker (Larimichthys crocea) and characterization of IgT+ B cells.

Teleost immunoglobulin T (IgT) is considered to be a primitive immunoglobulin class specialized in mucosal immunity. In the present study, a recombinant protein containing the CH2 region of large yellow croaker (Larimichthys crocea) IgT heavy chain was expressed, purified, and used as an immunogen to produce a monoclonal antibody (mAb) against large yellow croaker IgT. Western blotting results indicated that the obtained mouse anti-IgT mAb could specifically recognize a 45 kDa protein in the skin mucus of large yellow croaker, which was identified as the IgT heavy chain by mass spectrometric analysis. Immunofluorescence assay (IFA) analysis further demonstrated that this mouse anti-IgT mAb could recognize membrane-bound IgT (mIgT) molecules on large yellow croaker IgT+ leukocytes. This mAb also could be used for sorting of large yellow croaker IgT+ B cells by flow cytometry sorting technology. Then, flow cytometric immunofluorescence analysis (FCIA) results showed that the percentages of IgT+ B cells in skin, gills, gut, spleen, head kidney and peripheral blood lymphocytes were 27.553% ± 3.312%, 12.588% ± 3.538%, 12.355% ± 3.352%, 13.075 ± 2.258%, 5.552 ± 3.275%, and 2.600 ± 0.521%, respectively, indicating that mucosal tissues (skin, gills, and gut) contained a high ratio of IgT+ B cells. Accordingly, the high protein levels of IgT were also detected in these mucosal tissues, suggesting that IgT may play a role in mucosal immunity in large yellow croaker. Taken together, our data demonstrated that the mouse anti-IgT mAb developed in this study could be used for characterizing IgT+ B cells and studying the functions of IgT in large yellow croaker.

An outbreak of visceral white nodules disease caused by Pseudomonas plecoglossicida at a water temperature of 12°C in cultured large yellow croaker (Larimichthys crocea) in China.

Visceral white nodules disease (VWND) caused by Pseudomonas plecoglossicida is a common disease in cage-farmed large yellow croaker (Larimichthys crocea) in China. VWND usually occurred at water temperature of 16-19℃, resulting in high mortality in farmed large yellow croaker. Now, P. plecoglossicida as its pathogen has been considered nonpathogenic at 7-12℃. During February 2019, an infectious disease outbreak was observed in cage-farmed large yellow croaker at a water temperature of 12℃ in Ningde, China. This disease is characterized by white granulomatous lesions in internal organs of the diseased fish, which was similar with the symptoms of the VWND in large yellow croaker. Then, we isolated a bacterial strain named PQLYC4 from visceral lesions of the diseased fish. The experimental infection studies demonstrated that the strain PQLYC4 was the pathogen of the disease, which was further identified as P. plecoglossicida by the analysis of morphology, 16s rRNA gene homology and average nucleotide identity based on the whole genome sequence. Our results revealed that P. plecoglossicida strain PQLYC4 could cause the outbreak of the VWND at 12℃, a water temperature lower than that reported previously, thus providing new knowledges of prevalence and prevention of the VWND in large yellow croaker.

Molecular cloning.

Transcription factor c-Jun is a member of AP-1 transcription complex that can be induced by various pathogens and plays a broad regulatory role in vertebrate immune response. In this study, the complete c-Jun cDNA of large yellow croaker Larimichthys crocea (Lcc-Jun) was cloned, whose open reading frame (ORF) is 984 bp long and encodes a protein of 327 amino acids (aa). The deduced Lcc-Jun protein contains three highly conserved domains, a transactivation domain (TAD, Met1-His118), a DNA binding domain (DBD, Lys218-Arg243), and a Leucine zipper domain (LZD, Leu271-Leu299), as found in other specie c-Jun. Lcc-Jun was constitutively expressed in all examined tissues, with the higher levels in blood, heart, and head kidney. Its transcripts were not only induced in spleen and head kidney by poly (I: C) or LPS, but also up-regulated in primary head kidney leukocytes (PKL), macrophages (PKM), and granulocytes (PKG), suggesting that Lcc-Jun may be involved in immune responses induced by poly (I: C), a viral mimic, and LPS, a Gram-negative bacterial component. Overexpression of Lcc-Jun in PKL increased the expression of cytokines and transcription factors involved in T helper 1 (Th1: TNF-α, IFN-γ, and T-bet) and Th2 (IL-4/13 A/B, IL-6, and GATA3) cell development and differentiation, suggesting that Lcc-Jun may play a role in regulation of Th1/Th2 cell response. These results therefore led us to suggest that the c-Jun-mediated signaling pathways may have an important immune-modulatory function in teleost fish.

The homeostasis-maintaining metabolites from bacterial stress response to bacteriophage infection suppress tumor metastasis.

The antiviral metabolites from bacterial stress response to bacteriophage infection can maintain homeostasis of host cells, while metabolism disorder is a remarkable characteristic of tumorigenesis. In the aspect of metabolic homeostasis, therefore, the antiviral homeostasis-maintaining metabolites of bacteria may possess anti-tumor activity. However, this issue has not been addressed. Here we show that the homeostasis-challenged maintaining metabolites from deep-sea bacteriophage-challenged thermophile can suppress tumor metastasis. The results indicated that the metabolic profiles of the bacteriophage GVE2-infected and virus-free thermophile Geobacillus sp. E263 from a deep-sea hydrothermal vent were remarkably different. Thirteen metabolites were significantly elevated and two metabolites were downregulated in thermophile stress response to GVE2 infection. As an example, the upregulated L-norleucine was characterized. The data showed that L-norleucine had antiviral activity in thermophile. Furthermore, the in vitro and in vivo assays revealed that L-norleucine, as well as its derivative, significantly suppressed metastasis of gastric and breast cancer cells. L-norleucine interacted with hnRNPA2/B1 protein to inhibit the expressions of Twist1 and Snail, two inhibitors of E-cadherin, and promote the E-cadherin expression, leading to the inhibition of tumor metastasis. Therefore, our study presented that antiviral homeostasis-maintaining metabolites of microbes might be a promising source for anti-tumor drugs.

Deep-Sea Hydrothermal Vent Viruses Compensate for Microbial Metabolism in Virus-Host Interactions.

Viruses are believed to be responsible for the mortality of host organisms. However, some recent investigations reveal that viruses may be essential for host survival. To date, it remains unclear whether viruses are beneficial or harmful to their hosts. To reveal the roles of viruses in the virus-host interactions, viromes and microbiomes of sediment samples from three deep-sea hydrothermal vents were explored in this study. To exclude the influence of exogenous DNAs on viromes, the virus particles were purified with nuclease (DNase I and RNase A) treatments and cesium chloride density gradient centrifugation. The metagenomic analysis of viromes without exogenous DNA contamination and microbiomes of vent samples indicated that viruses had compensation effects on the metabolisms of their host microorganisms. Viral genes not only participated in most of the microbial metabolic pathways but also formed branched pathways in microbial metabolisms, including pyrimidine metabolism; alanine, aspartate, and glutamate metabolism; nitrogen metabolism and assimilation pathways of the two-component system; selenocompound metabolism; aminoacyl-tRNA biosynthesis; and amino sugar and nucleotide sugar metabolism. As is well known, deep-sea hydrothermal vent ecosystems exist in relatively isolated environments which are barely influenced by other ecosystems. The metabolic compensation of hosts mediated by viruses might represent a very important aspect of virus-host interactions.IMPORTANCE Viruses are the most abundant biological entities in the oceans and have very important roles in regulating microbial community structure and biogeochemical cycles. The relationship between virus and host microbes is broadly thought to be that of predator and prey. Viruses can lyse host cells to control microbial population sizes and affect community structures of hosts by killing specific microbes. However, viruses also influence their hosts through manipulation of bacterial metabolism. We found that viral genes not only participated in most microbial metabolic pathways but also formed branched pathways in microbial metabolisms. The metabolic compensation of hosts mediated by viruses may help hosts to adapt to extreme environments and may be essential for host survival.

Characterization of Bacterial Communities in Deep-Sea Hydrothermal Vents from Three Oceanic Regions.

Deep-sea hydrothermal vents are considered to be one of the most spectacular ecosystems on Earth. Microorganisms form the basis of the food chain in vents controlling the vent communities. However, the diversity of bacterial communities in deep-sea hydrothermal vents from different oceans remains largely unknown. In this study, the pyrosequencing of 16S rRNA gene was used to characterize the bacterial communities of the venting sulfide, seawater, and tubeworm trophosome from East Pacific Rise, South Atlantic Ridge, and Southwest Indian Ridge, respectively. A total of 23,767 operational taxonomic units (OTUs) were assigned into 42 different phyla. Although Proteobacteria, Actinobacteria, and Bacteroidetes were the predominant phyla in all vents, differences of bacterial diversity were observed among different vents from three oceanic regions. The sulfides of East Pacific Rise possessed the most diverse bacterial communities. The bacterial diversities of venting seawater were much lower than those of vent sulfides. The symbiotic bacteria of tubeworm Ridgeia piscesae were included in the bacterial community of vent sulfides, suggesting their significant ecological functions as the primary producers in the deep-sea hydrothermal vent ecosystems. Therefore, our study presented a comprehensive view of bacterial communities in deep-sea hydrothermal vents from different oceans.

The role of miR-100 in regulating apoptosis of breast cancer cells.

Breast cancer is a serious health problem worldwide. Inhibition of apoptosis plays a major role in breast cancer tumorigenesis. MicroRNAs (miRNAs) play crucial roles in the regulation of apoptosis. However, the regulation of breast cancer apoptosis by miRNAs has not been intensively investigated. To address this issue, the effect of miR-100 on the cell proliferation of different breast cancer cells was characterized in the present study. The results showed that miR-100 was significantly upregulated in SK-BR-3 cells compared with other human breast cancer cells (MCF7, MDA-MB-453, T47D, HCC1954 and SUM149). Silencing miR-100 expression with anti-miRNA-100 oligonucleotide (AMO-miR-100) initiated apoptosis of SK-BR-3 cells in vitro and in vivo. However, the overexpression of miR-100 led to the proliferation inhibition of the miR-100-downregulated breast cancer cells. Antagonism of miR-100 in SK-BR-3 cells increased the expression of MTMR3, a target gene of miR-100, which resulted in the activation of p27 and eventually led to G2/M cell-cycle arrest and apoptosis. The downregulation of miR-100 sensitized SK-BR-3 cells to chemotherapy. Therefore, our finding highlights a novel aspect of the miR-100-MTMR3-p27 pathway in the molecular etiology of breast cancer.