Ho Bin Jang

Outer Membrane Vesicles as a Candidate Vaccine against Edwardsiellosis

Infection with Edwardsiella tarda, a Gram-negative bacterium, causes high morbidity and mortality in both marine and freshwater fish. Outer membrane vesicles (OMVs) released from Gram-negative bacteria are known to play important roles in bacterial pathogenesis and host immune responses, but no such roles for E. tarda OMVs have yet been described. In the present study, we investigated the proteomic composition of OMVs and the immunostimulatory effect of OMVs in a natural host, as well as the efficacy of OMVs when used as a vaccine against E. tarda infection. A total of 74 proteins, from diverse subcellular fractions, were identified in OMVs. These included a variety of important virulence factors, such as hemolysin, OmpA, porin, GAPDH, EseB, EseC, EseD, EvpC, EvpP, lipoprotein, flagellin, and fimbrial protein. When OMVs were administrated to olive flounder, significant induction of mRNAs encoding IL-1β, IL-6, TNFα, and IFNγ was observed, compared with the levels seen in fish injected with formalin-killed E. tarda. In a vaccine trial, olive flounder given OMVs were more effectively protected (p<0.0001) than were control fish. Investigation of OMVs may be useful not only for understanding the pathogenesis of E. tarda but also in development of an effective vaccine against edwardsiellosis.

Molecular cloning and functional analysis of nucleotide-binding oligomerization domain 1 (NOD1) in olive flounder, Paralichthys olivaceus

The gene encoding nucleotide-binding oligomerization domain 1 (NOD1) was cloned from olive flounder (Paralichthys olivaceus) and the role played by NOD1 during Edwardsiella tarda infection was evaluated. The complete open reading frame of NOD1 was 2820 bp in length, encoding a 939-amino acid polypeptide. The NOD1 protein contains three conserved domain structures including C-terminal LRRs, a central NACHT motif, and an N-terminal CARD domain, which show similarities of 49-74% to those of other vertebrate counterpart proteins. NOD1 expression was observed in all fish tissues examined, and the levels increased in olive flounder infected with E. tarda, Streptococcus iniae, or viral hemorrhagic septicemia virus (VHSV). When hirame natural embryo (HINAE) cells over-expressing NOD1 were infected with E. tarda, bacterial growth was inhibited, and the IL-1β transcript level increased compared to that of the control. These findings imply that NOD1 plays an important role in response to E. tarda infection of olive flounder.

Complete Genome Sequence and Immunoproteomic Analyses of the Bacterial Fish Pathogen Streptococcus parauberis

Although Streptococcus parauberis is known as a bacterial pathogen associated with bovine udder mastitis, it has recently become one of the major causative agents of olive flounder (Paralichthys olivaceus) streptococcosis in northeast Asia, causing massive mortality resulting in severe economic losses. S. parauberis contains two serotypes, and it is likely that capsular polysaccharide antigens serve to differentiate the serotypes. In the present study, the complete genome sequence of S. parauberis (serotype I) was determined using the GS-FLX system to investigate its phylogeny, virulence factors, and antigenic proteins. S. parauberis possesses a single chromosome of 2,143,887 bp containing 1,868 predicted coding sequences (CDSs), with an average GC content of 35.6%. Whole-genome dot plot analysis and phylogenetic analysis of a 60-kDa chaperonin-encoding gene and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-encoding gene showed that the strain was evolutionarily closely related to Streptococcus uberis. S. parauberis antigenic proteins were analyzed using an immunoproteomic technique. Twenty-one antigenic protein spots were identified in S. parauberis, by reaction with an antiserum obtained from S. parauberis-challenged olive flounder. This work provides the foundation needed to understand more clearly the relationship between pathogen and host and develops new approaches toward prophylactic and therapeutic strategies to deal with streptococcosis in fish. The work also provides a better understanding of the physiology and evolution of a significant representative of the Streptococcaceae.

Seasonal variation and comparative analysis of non-specific humoral immune substances in the skin mucus of olive flounder (Paralichthys olivaceus)

The epidermal secretion of fish contains various non-specific immune substances that act as the first line of defense against invading pathogens. The present study investigated the level of mucosal antibodies, the activities of hemagglutinin and protease, and other enzymes in the skin mucus of farm reared olive flounder (Paralichthys olivaceus) for 1 year, in order to gain an insight into the relationship between these mucosal immune substances and their seasonal variation. These levels varied significantly during different months of sample collection. The present study showed a positive correlation between water temperature and the level of mucosal antibodies, and an inverse relationship between the level of mucosal antibodies and the activity of mucosal hemagglutinin and protease, but no relationship between lysozyme activity and other innate immune substances. This relationship is thought to be a compensatory response in olive flounder to protect itself against pathogenic microorganisms which are inherently present in the aquatic environment.

Phylogenomic Network and Comparative Genomics Reveal a Diverged Member of the ϕKZ-Related Group, Marine Vibrio Phage ϕJM-2012

ABSTRACT Bacteriophages are the largest reservoir of genetic diversity. Here we describe the novel phage ϕJM-2012. This natural isolate from marine Vibrio cyclitrophicus possesses very few gene contents relevant to other well-studied marine Vibrio phages. To better understand its evolutionary history, we built a mathematical model of pairwise relationships among 1,221 phage genomes, in which the genomes (nodes) are linked by edges representing the normalized number of shared orthologous protein families. This weighted network revealed that ϕJM-2012 was connected to only five members of the Pseudomonas ϕKZ-like phage family in an isolated network, strongly indicating that it belongs to this phage group. However, comparative genomic analyses highlighted an almost complete loss of colinearity with the ϕKZ-related genomes and little conservation of gene order, probably reflecting the action of distinct evolutionary forces on the genome of ϕJM-2012. In this phage, typical conserved core genes, including six RNA polymerase genes, were frequently displaced and the hyperplastic regions were rich in both unique genes and predicted unidirectional promoters with highly correlated orientations. Further, analysis of the ϕJM-2012 genome showed that segments of the conserved N-terminal parts of ϕKZ tail fiber paralogs exhibited evidence of combinatorial assortment, having switched transcriptional orientation, and there was recruitment and/or structural changes among phage endolysins and tail spike protein. Thus, this naturally occurring phage appears to have branched from a common ancestor of the ϕKZ-related groups, showing a distinct genomic architecture and unique genes that most likely reflect adaptation to its chosen host and environment.

Recombinant interferon-γ activates immune responses against Edwardsiella tarda infection in the olive flounder, Paralichthys olivaceus.

Interferon gamma (IFN-γ) is a cytokine that plays a very important role in defining Th1 immune response in all vertebrates. In this study, recombinant IFN-γ (rIFN-γ) from the olive flounder (Paralichthys olivaceus) was produced in an Escherichia coli system using a pET expression vector. Stimulation of whole kidney leukocytes (immune-related cells) in vitro with the resulting rIFN-γ significantly induced the gene expression of interleukin-1β (IL-1β), signal transducer and activator of transcription 1 (STAT1), CXCL13-like chemokine (CXCL13), and IFN-γ. rIFN-γ also weakly induced the expression of IL-1β, tumor necrosis factor-α (TNF-α), CXCL13, and IFN-γ in olive flounder-derived HINAE (non-immune) cells. The effects of rIFN-γ against Edwardsiella tarda infection in vivo were assessed by intraperitoneally injecting a mixture of rIFN-γ (100 ng) and E. tarda (1 × 10(5) CFU/ml) into the olive flounder. The survival rate in the rIFN-γ-injected group was 60% compared to 0% in the group treated with E. tarda only, demonstrating that olive flounder IFN-γ is effective in reinforcing immune responses and preventing against edwardsiellosis.

RNA-seq-based metatranscriptomic and microscopic investigation reveals novel metalloproteases of Neobodo sp. as potential virulence factors for soft tunic syndrome in Halocynthia roretzi.

Bodonids and trypanosomatids are derived from a common ancestor with the bodonids being a more primitive lineage. The Neobodonida, one of the three clades of bodonids, can be free-living, commensal or parasitic. Despite the ecological and evolutionary significance of these organisms, however, many of their biological and pathological features are currently unknown. Here, we employed metatranscriptomics using RNA-seq technology combined with field-emission microscopy to reveal the virulence factors of a recently described genus of Neobodonida that is considered to be responsible for ascidian soft tunic syndrome (AsSTS), but whose pathogenesis is unclear. Our microscopic observation of infected tunic tissues suggested putative virulence factors, enabling us to extract novel candidate transcripts; these included cysteine proteases of the families C1 and C2, serine proteases of S51 and S9 families, and metalloproteases grouped into families M1, M3, M8, M14, M16, M17, M24, M41, and M49. Protease activity/inhibition assays and the estimation of expression levels within gene clusters allowed us to identify metalloprotease-like enzymes as potential virulence attributes for AsSTS. Furthermore, a multimarker-based phylogenetic analysis using 1,184 concatenated amino acid sequences clarified the order Neobodo sp. In sum, we herein used metatranscriptomics to elucidate the in situ expression profiles of uncharacterized putative transcripts of Neobodo sp., combined these results with microscopic observation to select candidate genes relevant to pathogenesis, and used empirical screening to define important virulence factors.

Cathepsins in the kidney of olive flounder, Paralichthys olivaceus, and their responses to bacterial infection

Cathepsin activities are responsible for mediating various pathways involved in immune response, including the apoptosis pathway, toll-like receptor (TLR) signaling, cytokine induction and activation of granule serine proteases. In the present study, we investigated cathepsin responses in the kidneys of olive flounder infected with Streptococcus parauberis, analyzing cathepsin expression using a label-free, quantitative proteomic approach in conjunction with quantitative real-time polymerase chain reaction (qRT-PCR). In proteomic analyses, we detected cathepsin B, D, L and S proteins, noting significant decreases and increases in cathepsins B and L, respectively, with infection. Taken together with an evaluation of cathepsin B, D, F, K, L, S and X gene expression in normal and infected kidneys by qRT-PCR, our results indicate that cathepsins B, D, L and S are the dominant lysosomal proteases in the immune system of the teleostei, olive flounder. Cathepsins F, K and X were regarded as minor cathepsins.

Heat shock protein profiles on the protein and gene expression levels in olive flounder kidney infected with Streptococcus parauberis

Heat shock proteins (HSPs) have been observed in cells exposed to a variety of stresses, including infectious pathogens. This study used a label-free, quantitative proteomic approach and transcriptional gene expression analysis to investigate infection-related HSP proteins and their encoding genes in whole kidneys from olive flounder (Paralichthys olivaceus). During Streptococcus parauberis infection in the flounder, the genes encoding Hsp10, Hsp40A4, Hsp40B6, Hsp40B11, Hsp60, Hsp70, glucose regulated protein 78 (Grp78), Hsp90α, Hsp90β and Grp94 were induced, and the protein levels of Hsp60, Hsp70, Hsp90α, Hsp90β and Grp94 were differentially regulated over time. Subsequent results also revealed that Hsp60, Hsp70, Hsp90α, Hsp90β and Grp94 appear to be the dominant and critical HSPs in olive flounder during bacterial infection. This is the first estimation of the differential involvement of HSPs in the immune response of olive flounder exposed to bacterial infection.

Comparative analysis and distribution of pP9014, a novel drug resistance IncP-1 plasmid from Photobacterium damselae subsp. piscicida

Photobacterium damselae subsp. piscicida, a causative agent of pseudotuberculosis, often harbours resistance plasmids (R plasmids) that facilitate horizontal gene transfer of drug resistance genes. R plasmid pP9014 was isolated from P. damselae subsp. piscicida and its complete nucleotide sequence was determined using Next Generation Sequencing technology. A protein network analysis was conducted to determine the relatedness of protein coding sequences, and ClustalW was used for the full nucleotide sequences. The occurrence of pP9014-like plasmids compared with pP99-018-like plasmids in a specific region was determined using probes for their transfer regions. pP9014 is 55851bp long with an overall GC content of 44.4% encoding 61 open reading frames (ORFs) including antimicrobial resistance genes and two conjugative transfer regions (Tra and Trb). The backbone showed highest similarity to Marinobacter adhaerens pHP-42 and Methylophaga sp. JAM7. pP9014 is similar to several IncP plasmids but forms a different subgroup. pP9014 is a unique plasmid in P. damselae subsp. piscicida and was not commonly found in drug-resistant P. damselae subsp. piscicida isolated from different areas and years in Japan. Plasmids similar to the previously reported pP99-018 are more widely distributed. This rarity suggests that plasmids similar to pP99-018 are more compatible with γ-proteobacteria. pP9014 is the first reported IncP-1 plasmid from fish pathogens. Its similarity to other IncP plasmids isolated from soil and human pathogens suggests that plasmids of the IncP-1 incompatibility group are vectors for the transfer of drug resistance genes among diverse environments.