Gene expression of leucocytes in vaccinated Japanese flounder (Paralichthys olivaceus) during the course of experimental infection with Edwardsiella tarda

In this paper, we focused on the detection of differentially expressed genes in peripheral blood leucocytes (PBL) during the course of Edwardsiella tarda infection in vaccinated and non-vaccinated Japanese flounder (Paralichthys olivaceus). cDNA microarray analysis was performed to compare the gene expression patterns of the PBL between the vaccinated and non-vaccinated fish in response to E. tarda inoculation. Fish were vaccinated twice, at a two-week interval and experimentally challenged with E. tarda two weeks after the second vaccination. Among the 1187 analyzed genes, 42 genes were up-regulated during the course of infection either in vaccinated or non-vaccinated fish. These genes included immune-related genes, such as MMP-9, MMP-13, CXC chemokine, CD20 receptor and hepcidin. Some immune-related genes were down-regulated after the E. tarda challenge, i.e. interferon inducible Mx protein, MHC class II-associated invariant chain, MHC class II alpha and MHC class II beta encoding genes, immunoglobulin light chain precursor, immunoglobulin light chain and IgM. These responses are thought to be a common reaction of Japanese flounder PBL in the course of edwardsiellosis, irrespective of immunized condition. Ten genes were significantly up-regulated only in vaccinated fish, and 11 genes were significantly up-regulated only in non-vaccinated fish. These genes may have a correlation with the efficacy of vaccination, although we have no evidence to link the different gene expression patterns and the efficacy of vaccination at present.     

Generation of two auxotrophic genes knock-out Edwardsiella tarda and assessment of its potential as a combined vaccine in olive flounder (Paralichthys olivaceus)

Two auxotrophic genes that play essential roles in bacterial cell wall biosynthesis--alanine racemase (alr) gene and aspartate semialdehyde dehydrogenase (asd) gene--knock-out Edwardsiella tarda (Δalr Δasd E. tarda) was generated by the allelic exchange method to develop a combined vaccine system. Green fluorescent protein (GFP) was used as a model foreign protein, and was expressed by transformation of the mutant E. tarda with antibiotic resistant gene-free plasmids harboring cassettes for GFP and asd expression (pG02-ASD-EtPR-GFP). In vitro growth of the mutant E. tarda was similar to wild-type E. tarda when D-alanine and diaminopimelic acid (DAP) were supplemented to growth medium. However, without d-alanine and/or DAP supplementation, the mutant showed very limited growth. The Δalr Δasd E. tarda transformed with pG02-ASD-EtPR-GFP showed a similar growth pattern of wild-type E. tarda when D-alanine was supplemented in the medium, and the expression of GFP could be observed even with naked eyes. The virulence of the auxotrophic mutant E. tarda was decreased, which was demonstrated by approximately 10? fold increase of LD?? dose compared to wild-type E. tarda. To assess vaccine potential of the present combined vaccine system, olive flounder (Paralichthys olivaceus) were immunized with the GFP expressing mutant E. tarda, and analyzed protection efficacy against E. tarda challenge and antibody titers against E. tarda and GFP. Groups of fish immunized with 10? CFU of the Δalr Δasd E. tarda harboring pG02-ASD-EtPR-GFP showed no mortality, which was irrespective to boost immunization. The cumulative mortality rates of fish immunized with 10? or 10? CFU of the mutant bacteria were lowered by a boost immunization. Fish immunized with the mutant E. tarda at doses of 10?-10? CFU/fish showed significantly higher serum agglutination activities against formalin-killed E. tarda than PBS-injected control fish. Furthermore, fish immunized with 10?-10? CFU/fish of the mutant E. tarda showed significantly higher ELISA titer against GFP antigen than fish in other groups. These results indicate that the present double auxotrophic genes knock-out E. tarda coupled with a heterologous antigen expression has a great strategic potential to be used as combined vaccines against various fish diseases.     

Edwardsiella tarda DnaK: expression, activity, and the basis for the construction of a bivalent live vaccine against E. tarda and Streptococcus iniae

Edwardsiella tarda and Streptococcus iniae are important aquaculture pathogens that affect many species of farmed fish. In this study, we analyzed the expression, activity, and immunoprotective potential of E. tarda heat shock protein DnaK. We found that dnaK expression was upregulated under conditions of heat shock, oxidative stress, and infection of host cells. Recombinant DnaK (rDnaK) purified from Escherichia coli exhibited ATPase activity and induced protection in Japanese flounder (Paralichthys olivaceus) against lethal E. tarda challenge. On the basis of these results and our previous observation that a protective S. iniae antigen Sia10 which, when expressed heterogeneously in E. coli DH5α, is secreted into the extracellular milieu, we constructed a chimeric antigen by fusing DnaK to Sia10. The resulting fusion protein Sia10-DnaK was expressed in DH5α via the plasmid pTDK. Western blot analysis indicated that Sia10-DnaK was detected in the culture supernatant of DH5α/pTDK. When flounder were vaccinated with live DH5α/pTDK, strong protection was observed against both E. tarda and S. iniae. ELISA analysis detected specific serum antibody production in fish vaccinated with rDnaK and DH5α/pTDK. Taken together, these results indicate that rDnaK is an intrinsic ATPase with immunoprotective property and that Sia10-DnaK delivered by a live bacterial host is an effective bivalent vaccine candidate against E. tarda and S. iniae infection.     

Sexually dimorphic expression and regulatory sequence of dnali1 in the olive flounder Paralichthys olivaceus

Dynein axonemal light intermediate chain 1 (dnali1) is an important part of axonemal dyneins and plays an important role in the growth and development of animals. However, there is little information about dnali1 in fish. Herein, we cloned dnali1 gene from the genome of olive flounder (Paralichthys olivaceus), a commercially important maricultured fish in China, Japan, and Korea, and analyzed its expression patterns in different gender fish. The flounder dnali1 DNA sequence contained a 771 bp open reading frame (ORF), two different sizes of 5′ untranslated region (5′UTR), and a 1499 bp 3′ untranslated region (3′UTR). Two duplicated 922 nt fragments were found in dnali1 mRNA. The first fragment contained the downstream coding region and the front portion of 3′UTR, and the second fragment was entirely located in 3′UTR. Multiple alignments indicated that the flounder Dnali1 protein contained the putative conserved coiled-coil domain. Its expression showed sexually dimorphic with predominant expression in the flounder testis, and lower expression in other tissues. The gene with the longer 5′UTR was specifically expressed in the testis. The highest expression level in the testis was detected at stages IV and V. Transient expression analysis showed that the 922 bp repeated sequence 3′UTR of dnali1 down-regulated the expression of GFP at the early stage in zebrafish. The flounder dnali1 might play an important role in the testis, especially in the period of spermatogenesis, and the 5′UTR and the repetitive sequences in 3′UTR might contain some regulatory elements for the cilia.

     

Purification and characterization of an antimicrobial histone H1-like protein and its gene from the testes of olive flounder, Paralichthys olivaceus

An approximately 21?kDa antimicrobial protein was purified from an acidified testis extract of olive flounder, Paralichthys olivaceus, by ion-exchange and C(18) reversed-phase HPLC. A comparison of the N-terminal amino acid sequence with those of other known antimicrobial polypeptides revealed high homology between this antimicrobial protein and other histone H1 molecules; thus, it was designated flounder histone H1-like protein (fH1LP). fH1LP showed potent antimicrobial activity against Gram-positive bacteria, including Bacillus subtilis, Staphylococcus aureus, and Streptococcus iniae (minimal effective concentrations [MECs], 2.8-30.0?μg/ml), Gram-negative bacteria, including Aeromonas hydrophila, Escherichia coli D31, Vibrio parahaemolyticus (MECs, 1.4-12.0?μg/ml), and Candida albicans (MEC, 2.0?μg/ml). cDNA cloning and tissue distribution studies of fH1LP indicated that it is constitutively expressed in testis and ovary. The fH1LP expression level was significantly dependent on developmental stage, and decreased dramatically after hatching. However, lipopolysaccharide stimulation did not induce fH1LP mRNA in other immune organs, including the kidney and spleen. These results suggest that fH1LP plays an important role in innate immunity in fish during reproduction, including mating, fertilization, and hatching.     

Redox regulation of protein expression in Saccharomyces cerevisiae mitochondria: possible role of VDAC

Using Saccharomyces cerevisiae mutants depleted of either isoform of VDAC (voltage dependent anion selective channel) we studied the role of the cytosol and mitochondria redox states in regulation of the expression levels of some mitochondrial proteins. The studied proteins are MnSOD and subunits of the protein import machinery of the mitochondrial outer membrane, i.e. Tom70, Tom40 and Tob55 (Sam50). We have shown that both the cytosol and mitochondria redox states depend on the presence of a given VDAC isoform. The cytosol redox state is mediated by VDAC1, although VDAC2 has a quantitative effect, whereas the mitochondria redox state depends on the presence of both VDAC isoforms. Moreover, we have shown that the cytosol redox status but not the mitochondrial one is decisive for the expression levels of the studied mitochondrial proteins. Thus, expression levels of some mitochondrial proteins is influenced by VDAC and this regulatory process at least partially does not require its channel activity as VDAC2 does not form a channel. Thus, VDAC can be regarded as a participant of signaling pathways in S. cerevisiae cells.     

Expression and regulation of miR-1, -133a, -206a, and MRFs by thyroid hormone during larval development in Paralichthys olivaceus

MiR-1, miR-133a, and miR-206a have been identified as muscle-specific miRNAs. They play multiple crucial roles in the regulation of muscle development. Here, we show that these miRNAs were differentially expressed during the larval development of flounder, and specifically expressed in skeletal muscle and heart in adult tissues/organs. The expression levels of these miRNAs were significantly changed by thyroid hormone (TH) or thiourea (TU) treatment during metamorphosis from 17 dph (days post hatching) to 42 dph. In addition, the expression levels of MyoD and Myf5 mRNAs markedly increased at 14 dph (pre-metamorphosis) compared to metamorphic stages, and their expression levels are far above the myogenin during larval development. Moreover, these MRFs (myogenic regulatory factors) expression were directly or indirectly regulated by thyroid hormone or thiourea during metamorphosis. All the results suggest that miRNAs and MRFs might be involved in signaling pathway of TH or TU-mediated flounder metamorphosis.     

Molecular cloning and characterization of PLCB1 (phospholipase C, beta 1) gene from the olive flounder, Paralichthys olivaceus

PLCB1 (phospholipase C, beta 1) cDNA was cloned from olive flounder (Paralichthys olivaceus) cDNA via rapid amplification of cDNA ends (RACE). The cDNA for olive flounder PLCB1 (PoPLCB1) encodes for a polypeptide of 1,244 amino acids in length containing a well-conserved PH domain, catalytic X and Y domains, a C2 domain. From the sequence information of the BAC library, we assembled a contig containing the whole flounder PLCB1 cDNA sequences, and determined the exon/intron structure of the gene spanning > 110,743 bp DNA. PoPLCB1 gDNA sequences demonstrated the new sequence (exon 15), which has only been observed in the fish, is located between the X and Y domain of the PLCB1, and that PoPLCB1 exists as two splice variants-PoPLCB1a (1,244 amino acids) and PoPLCB1b (1,210 amino acids). Phylogenic analysis and sequence comparison of PoPLCB1 with other PLC isozymes showed a close relationship with the PLCB1 isozyme. Tissue-specific mRNA of PoPLCB1 was expressed predominantly in the brain and heart tissues. Between the two splicing variants of PoPLB1 in RT-PCR by tissue, PoPLCB1a showed a major expression pattern in more diverse types of tissues than the PoPLCB1b. PoPLCB1 gene expression was compared with that of the inflammatory cytokines IL-1β and TNF-α in infected spleen and kidney tissues via real-time RT-PCR assays following stimulation with LPS. After the stimulation, the expression of PoPLCB1 increased significantly prior to IL-1B and TNF-α expression. This provided direct evidence suggesting that PoPLCB1 may perform a crucial role in immune responses against pathogens and in inflammation.     

Cloning of a calcitonin gene-related peptide from genomic DNA and its mRNA expression in flounder, Paralichthys olivaceus

A part of genomic DNA including the calcitonin gene-related peptide (CGRP) gene was cloned from flounder by the genome-walking method. The intron/exon boundary was predicted to occur exactly at the same position as in salmon. The 37-amino acid molecule coded by the region from the intron/exon boundary to the stop codon was preceded by a typical Lys-Arg cleavage signal and included a cleavage/amidation site common to the CGRP of other vertebrates. The predicted amino acid sequence of flounder CGRP had 78%, 78%, 78%, 81%, and 73-78% identity to that of salmon, cod, frog, chicken, and mammalian CGRPs, respectively. Among vertebrates, CGRP is more conserved than calcitonin (CT) because the identity of flounder CT to mammalian CTs is 31-50%. Expression analysis indicated that this hormone is synthesized in the brain, heart, intestine, testis, and ovary. Since we have previously shown that the CGRP receptor is expressed in these tissues, it is suggested that CGRP secreted from each tissue functions in a paracrine or autocrine manner.     

mRNA expression of pancreatic enzyme precursors and estimation of protein digestibility in first feeding larvae of the Japanese flounder,Paralichthys olivaceus

An understanding of digestibility in marine fish larvae is required to formulate a diet to replace zooplankton. Using flounder, this study was aimed at determining which digestive enzymes are synthesized in the larval pancreas, and how the proteins are cleaved in the digestive canal. Whole mount in situ hybridization indicated that the mRNA of all digestive enzyme precursors examined, including trypsin, chymotrypsin, elastase, carboxypeptidase A and B, and lipase, was expressed in the pancreas of first feeding larvae at 3 days post-fertilization. In the larvae before differentiation of the stomach, protein digestion in the digestive canal mainly depends on pancreatic proteases. So, to evaluate protein digestibility in the larval digestive canal, the digestion of proteins by pancreatic extract was monitored by gel electrophoresis. It was indicated that thyroglobulin, albumin and lactate dehydrogenase were rapidly cleaved to polypeptide fragments, but ferritin and catalase exerted resistance to proteolysis, suggesting that digestibility in the larval digestive canal differs depending on protein species.