Se Pyeong Im

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.

Enhancement of glycoprotein-based DNA vaccine for viral hemorrhagic septicemia virus (VHSV) via addition of the molecular adjuvant, DDX41

ABSTRACT The use of molecular adjuvants to improve the immunogenicity of DNA vaccines has been thoroughly studied in recent years. Glycoprotein (G)‐based DNA vaccines had been proven to be effective in combating infection against Rhabdovirus (especially infectious hematopoietic necrosis virus, IHNV) in salmonids. DDX41 is a helicase known to induce antiviral and inflammatory responses by inducing a type I IFN innate immune response. To gain more information regarding G‐based DNA vaccines in olive flounder (Paralicthys olivaceus), we tried to develop a more efficient G‐based DNA vaccine by adding a molecular adjuvant, DDX41. We designed a DNA vaccine in which the VHSV glycoprotein (G‐protein) and DDX41 were driven by the EF‐1&agr; and CMV promoters, respectively. Olive flounders were intramuscularly immunized with 1 &mgr;g of plasmids encoding the G‐based DNA vaccine alone (pEF‐G), the molecular adjuvant alone (pEF‐D), or the vaccine‐adjuvant construct (pEF‐GD). At two different time points, 15 and 30 days later, the fish were intraperitoneally infected with VHSV (100 &mgr;L; 1 × 106 TCID50/mL). Our assays revealed that the plasmid constructs showed up‐regulated expression of IFN‐1 and its associated genes at day 3 post‐vaccination in both kidney and spleen samples. Specifically, pEF‐GD showed statistically higher expression of immune response genes than pEF‐G and pEF‐D treated group (p < 0.05/p < 0.001). After VHSV challenge, the fish group treated with pEF‐GD showed higher survival rate than the pEF‐G treated group, though difference was not statistically significant in the 15 dpv challenged group however in the 30 dpv challenged group, the difference was statistically significant (p < 0.05). Together, these results clearly demonstrate that DDX41 is an effective adjuvant for the G‐based DNA vaccine in olive flounder. Our novel findings could facilitate the development of more effective DNA vaccines for the aquaculture industry. HighlightsThe adjuvant effect of DDX41 was assessed in this study.Simultaneous expression of VHSV glycoprotein and DDX41 showed enhanced IFN‐mediated immune response.The vaccine‐adjuvant construct showed enhanced regulation of type I interferon and IFN‐related genes.

Investigation of variable lymphocyte receptors in the alternative adaptive immune response of hagfish

Jawless vertebrates have an alternative adaptive immune system mediated by variable lymphocyte receptors (VLRs), VLRA, VLRC and VLRB. In investigation on the adaptive immunity of hagfish, avian influenza virus hemagglutinin (H9N2-HA1) was used as a model antigen, with mRNA expression levels of VLRA, VLRC and Ikaros were up-regulated in the first week post-immunization. CD45 was up-regulated after the first week; and expression of VLRB progressively increased over the course of the trial. The transcriptional/translational activation of VLRB in blood was verified. The VLRBs cloned from these transcripts showed diversity in their leucine-rich repeats (LRRs). The production of specific VLRB increased in a time- and dose-dependent manner, detected by an anti-VLRB antibody (11G5). The plasma VLRB could distinguish H9N2-HA1 from unrelated proteins, but not from other HA1 subtypes. Together, our findings show that VLRs play a major role in the alternative adaptive immune system of hagfish by responding to specific foreign substances, such as H9N2-HA1.

Comparison of proteome typing and serotyping of Streptococcus parauberis isolates from olive flounder (Paralichthys olivaceus)

The olive flounder (Paralichthys olivaceus) is a cultivated marine species that is economically important in Korea and Japan. Several bacterial pathogens have caused severe mortalities in farmed olive flounder, especially Streptococcus parauberis. We collected 145 S. parauberis isolates from diseased olive flounders from 2003 to 2008 in Jeju Island, South Korea and characterized them by Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI TOF MS) and by serology. The serological analysis divided the isolates into serotype I (62.1%) and serotype II (36.6%) and the proteome analysis divided the isolates into cluster 1 (43.4%) and cluster 2 (56.6%). All cluster 1 isolates had serotype I, but cluster 2 consisted of serotype I (32.9%), serotype II (64.6%), and others (2.5%). Further detailed analysis of the mass spectra led to identification of several specific m/z peaks that enabled discrimination between cluster 1 and 2 and between serotype I and II within cluster 2. Our results suggest that MALDI TOF MS analysis has potential as an alternative method for the rapid and reliable identification of the fish pathogen S. parauberis.

Development of a monoclonal antibody against the CD3ε of olive flounder (Paralichthys olivaceus) and its application in evaluating immune response related to CD3ε

&NA; The T cell receptor (TCR) is the binding site of antigen and is responsible for specifically activating the adaptive immune response. CD3, an essential component of the CD3‐TCR complex, is known to be composed of &ggr;&dgr; and &egr; chains in teleost. However, there are few monoclonal antibodies (mAb) available to identify these molecules on T cells, so we aimed to produce a mAb against CD3&egr; to improve our understanding of T cell immune response in olive flounder (Paralichthys olivaceus). CD3&egr; recombinant protein was expressed in yeast, the expression of which was confirmed by SDS‐PAGE, MALDI‐TOF/TOF MS and Western blot analysis. A CD3&egr;‐specific mAb 4B2 was selected, the specificity of which was examined by confocal microscopy, flow cytometry and RT‐PCR, and the mAb was subsequently used to examine the CD3&egr; lymphocyte population in several different immune organs, with relatively high percentages of these cells seen in trunk‐kidney and spleen, while lower percentages were seen in the liver and peripheral blood of olive flounder. During a viral hemorrhagic septicemia virus (VHSV) infection in olive flounder, the number of CD3&egr; lymphocytes was seen to gradually increase in the liver, spleen and trunk‐kidney of infected fish until 7 days post infection (dpi). In peripheral blood, on the other hand, the increase in CD3&egr; lymphocyte numbers peaked by 3 dpi. These results suggest that CD3&egr; lymphocytes might be involved in the immune response against VHSV. HighlightsRecombinant CD3&egr; was produced using yeast expression system.Monoclonal antibody 4B2 specifically detects the CD3&egr; lymphocyte in olive flounder.CD3&egr; lymphocytes might be involved in immune response related to VHSV.

Rapid MALDI biotyper-based identification and cluster analysis of Streptococcus iniae

Streptococcus iniae causes severe mortalities among cultured marine species, especially in the olive flounder (Paralichthys olivaceus), which is economically important in Korea and Japan. Recently, there has been growing concern regarding the emergence of S. iniae as a zoonotic pathogen. Here, 89 S. iniae isolates obtained from diseased olive flounders collected from 2003 to 2008 in Jeju Island, South Korea, were characterized using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The results were aligned both with the available Bruker Daltonics data-base and with a new set of S. iniae data entries developed in our laboratory, and the results were compared. When we used the Bruker Daltonics database, the 89 isolates yielded either “no reliable identification” or were incorrectly identified as Streptococcus pyogenes at the genus level. When we used the new data entries from our laboratory, in contrast, all of the isolates were correctly identified as S. iniae at the genus (100%) and species (96.6%) levels. We performed proteomic analysis, divided the 89 isolates into cluster I (51.7%), cluster II (20.2%), and cluster III (28.1%), and then used the MALDI Biotyper software to identify specific mass peaks that enabled discrimination between clusters and between Streptococcus species. Our results suggest that the use of MALDI TOF MS could outperform the conventional methods, proving easier, faster, cheaper and more efficient in properly identifying S. iniae. This strategy could facilitate the epidemiological and taxonomical study of this important fish pathogen.

Complete Genomic and Lysis-Cassette Characterization of the Novel Phage, KBNP1315, which Infects Avian Pathogenic Escherichia coli (APEC)

Avian pathogenic Escherichia coli (APEC) is a major pathogen that causes avian colibacillosis and is associated with severe economic losses in the chicken-farming industry. Here, bacteriophage KBNP1315, infecting APEC strain KBP1315, was genomically and functionally characterized. The evolutionary relationships of KBNP1315 were analyzed at the genomic level using gene (protein)-sharing networks, the Markov clustering (MCL) algorithm, and comparative genomics. Our network analysis showed that KBNP1315 was connected to 30 members of the Autographivirinae subfamily, which comprises the SP6-, T7-, P60-, phiKMV-, GAP227- and KP34-related groups. Network decomposition suggested that KBNP1315 belongs to the SP6-like phages, but our comparison of putative encoded proteins revealed that key proteins of KBNP1315, including the tail spike protein and endolysin, had relative low levels of amino acid sequence similarity with other members of the SP6-like phages. Thus KBNP1315 may only be distantly related to the SP6-like phages, and (based on the difference in endolysin) its lysis mechanism may differ from theirs. To characterize the lytic functions of the holin and endolysin proteins from KBNP1315, we expressed these proteins individually or simultaneously in E. coli BL21 (DE3) competent cell. Interestingly, the expressed endolysin was secreted into the periplasm and caused a high degree of host cell lysis that was dose-dependently delayed/blocked by NaN3-mediated inhibition of the SecA pathway. The expressed holin triggered only a moderate inhibition of cell growth, whereas coexpression of holin and endolysin enhanced the lytic effect of endolysin. Together, these results revealed that KBNP1315 appears to use a pin-holin/signal-arrest-release (SAR) endolysin pathway to trigger host cell lysis.

Outer membrane vesicles from β-lactam-resistant Escherichia coli enable the survival of β-lactam-susceptible E . coli in the presence of β-lactam antibiotics

Outer membrane vesicles (OMVs) containing various bacterial compounds are released from mainly gram-negative bacteria. Secreted OMVs play important roles in the ability of a bacterium to defend itself, and thus contribute to the survival of bacteria in a community. In this study, we collected OMVs from β-lactam antibiotic-resistant Escherichia coli established by conjugation assay and the parental β-lactam antibiotic-susceptible strain, and performed comparative proteomic analysis to examine whether these OMVs carried β-lactam-resistant compounds. We also investigated whether both types of OMVs could protect susceptible cells from β-lactam-induced death and/or directly degrade β-lactam antibiotics. Several proteins that can be involved in degrading β-lactam antibiotics were more abundant in OMVs from β-lactam-resistant E. coli, and thus OMVs from β-lactam resistant E. coli could directly and dose-dependently degrade β-lactam antibiotics and fully rescue β-lactam-susceptible E. coli and other bacterial species from β-lactam antibiotic-induced growth inhibition. Taken together, present study demonstrate that OMVs from β-lactam-resistant E. coli play important roles in survival of antibiotic susceptible bacteria against β-lactam antibiotics. This finding may pave the way for new efforts to combat the current global spread of antibiotic resistances, which is considered to be a significant public health threat.

Generation and characterization of hagfish variable lymphocyte receptor B against glycoprotein of viral hemorrhagic septicemia virus (VHSV)

HIGHLIGHTSSuperhydrophobic C‐termini of hagfish VLRB leads to extremely low expression level.C4bp oligomerization domain mediates heptameric VLRB with high binding ability and producttivity.In vitro affinity maturation was efficiently carried out by LRRCT mutagenesis.Fine epitope mapping revealed 37DWDTPL42 is the recognition epitope of selected arVLRBs.The resulting arVLRBs can be used as diagnostic tools or therapeutic agents of VHSV. ABSTRACT Variable lymphocyte receptors B (VLRBs) are non‐immunoglobulin components of the humoral immune system in jawless vertebrates including hagfish (Eptatretus burgeri) and lamprey (Petromyzon marinus). Hagfish VLRBs consist of leucine rich repeat (LRR) modules with a superhydrophobic C‐terminal tail, the latter of which leads to extremely low expression levels in recombinant protein technology. Here, we present an artificially oligomerized VLRB (arVLRB) that conjugates via the C4bp oligomerization domain derived from human C4b‐binding protein (hC4bp) rather than the superhydrophobic tail. The resulting arVLRB had a tightly multimerized form with seven monomeric VLRB arms and showed high expression and secretion levels in a mammalian expression system. To isolate antigen‐specific arVLRB, we constructed large VLRB libraries from hagfish immunized with the fish pathogen, viral hemorrhagic septicemia virus (VHSV). The selected arVLRBs were found to recognize various types of antigens, including the recombinant target protein, purified viruses, and progeny viruses, with high antigen binding abilities and specificities. We also performed in vitro affinity maturation of the arVLRBs through LRRCT mutagenesis, and found that this enhanced their antigen‐binding properties by at least 125‐fold. Our epitope mapping analysis revealed that 37DWDTPL42, which is located in a region conserved among the glycoproteins of all VHSV isolates, is the recognition epitope of the arVLRBs. Thus, our newly developed arVLRB could prove useful in the development of universal diagnostic tools and/or therapeutic agents for the virus. Together, our novel findings provide valuable insights into hagfish VLRB and its potential use as a novel alternative to conventional antibodies for biotechnological applications.

Globular-shaped variable lymphocyte receptors B antibody multimerized by a hydrophobic clustering in hagfish

In hagfish and lampreys, two representative jawless vertebrates, the humoral immunity is directly mediated by variable lymphocyte receptors B (VLRBs). Both monomeric VLRBs are structurally and functionally similar, but their C-terminal tails differ: lamprey VLRB has a Cys-rich tail that forms disulfide-linked pentamers of dimers, contributing to its multivalency, whereas hagfish VLRB has a superhydrophobic tail of unknown structure. Here, we reveal that VLRBs obtained from hagfish plasma have a globular-shaped multimerized form (approximately 0.6 to 1.7 MDa) that is generated by hydrophobic clustering instead of covalent linkage. Electron microscopy (EM) and single-particle analysis showed that the multimerized VLRBs form globular-shaped clusters with an average diameter of 28.7 ± 2.2 nm. The presence of VLRBs in the complex was confirmed by immune-EM analysis using an anti-VLRB antibody. Furthermore, the hydrophobic hagfish C-terminus (HC) was capable of triggering multimerization and directing the cellular surface localization via a glycophosphatidylinositol linkage. Our results strongly suggest that the hagfish VLRB forms a previously unknown globular-shaped antibody. This novel identification of a structurally unusual VLRB complex may suggest that the adaptive immune system of hagfish differs from that of lamprey.