Molecular cloning and characterization of Toll-like receptor 14 in Japanese flounder,Paralichthys olivaceus

Toll-like receptors (TLRs) are essential for activation of the innate immune system in response to invading pathogens. TLR14, which is unique to fish, has been identified in several fish species, but its function is unclear. In this study, Japanese flounder (Paralichthys olivaceus) TLR14 gene (JfTLR14) was cloned and its expression profiles were analyzed after infection with viral hemorrhagic septicemia virus, gram-positive Streptococcus iniae and gram-negative Edwardsiella tarda. The coding region of JfTLR14 cDNA was 2,607 bp, encoding 878 amino acid residues. JfTLR14 was highly expressed in head kidney of healthy flounder. In response to infection with VHSV and S. iniae, the JfTLR14 gene was up-regulated at only 1 day post-infection (dpi). However, E. tarda infection increased JfTLR14 gene expression from 1 to 6 dpi. These results imply that JfTLR14 participates more in the immune response against E. tarda infection than in the immune responses to other pathogen infections.     

Sil: A Streptococcus iniae Bacteriocin with Dual Role as an Antimicrobial and an Immunomodulator That Inhibits Innate Immune Response and Promotes S. iniae Infection

Streptococcus iniae is a Gram-positive bacterium and a severe pathogen to a wide range of economically important fish species. In addition, S. iniae is also a zoonotic pathogen and can cause serious infections in humans. In this study, we identified from a pathogenic S. iniae strain a putative bacteriocin, Sil, and examined its biological activity. Sil is composed of 101 amino acid residues and shares 35.6% overall sequence identity with the lactococcin 972 of Lactococcus lactis. Immunoblot analysis showed that Sil was secreted by S. iniae into the extracellular milieu. Purified recombinant Sil (rSil) exhibited a dose-dependent inhibitory effect on the growth of Bacillus subtilis but had no impact on the growths of other 16 Gram-positive bacteria and 10 Gram-negative bacteria representing 23 different bacterial species. Treatment of rSil by heating at 50°C abolished the activity of rSil. rSil bound to the surface of B. subtilis but induced no killing of the target cells. Cellular study revealed that rSil interacted with turbot (Scophthalmus maximus) head kidney monocytes and inhibited the innate immune response of the cells, which led to enhanced cellular infection of S. iniae. Antibody blocking of the extracellular Sil produced by S. iniae significantly attenuated the infectivity of S. iniae. Consistent with these in vitro observations, in vivo study showed that administration of turbot with rSil prior to S. iniae infection significantly increased bacterial dissemination and colonization in fish tissues. Taken together, these results indicate that Sil is a novel virulence-associated bacteriostatic and an immunoregulator that promotes S. iniae infection by impairing the immune defense of host fish.     

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.     

Molecular Characterization and Expression Analysis of ATP-Gated P2X7 Receptor Involved in Japanese Flounder (Paralichthys olivaceus) Innate Immune Response

ATP-gated P2X7 receptor (P2RX7) channel is a key component for purinergic signaling and plays important roles in the innate immune response in mammals. However, the expression, molecular properties and immune significances of P2RX7 in lower vertebrates are still very limited. Here we identified and characterized a novel bony fish P2RX7 homologue cDNA, termed poP2RX7, in Japanese flounder (Paralichthys olivaceus). PoP2RX7 protein shares about 60–88% sequence similarity and 45–78% sequence identity with known vertebrate P2RX7 proteins. Phylogenetic analysis placed poP2RX7 and other P2RX7 proteins within their own cluster apart from other P2RX members. While the functional poP2RX7 channel shares structural features in common with known P2RX7 homologs, electrophysiological studies revealed that BzATP, the more potent agonist for known mammalian and fish P2RX7s, shows similar potency to ATP in poP2RX7 activation. poP2RX7 mRNA constitutively expressed in all examined tissues from unstimulated healthy Japanese flounder with dominant expression in hepatopancreas and the lowest expression in head kidney, trunk kidney, spleen and gill. poP2RX7 mRNA expression, however, was significantly induced in Japanese flounder head kidney primary cells by Poly(I:C) and bacterial endotoxin LPS stimulations. In vivo experiments further revealed that poP2RX7 gene expression was substantially up-regulated by immune challenge with infectious bacteria Edwardsiella tarda and Vibrio anguillarum. Moreover, activation of poP2RX7 results in an increased gene expression of multifunctional cytokines IL-1β and IL-6 in the head kidney primary cells. Collectively, we identified and characterized a novel fish P2RX7 homolog which is engaged in Japanese flounder innate immune response probably through modulation of pro-inflammatory cytokines expression.     

Salt effect of nisin Z isolated from a marine fish on the growth inhibition of Streptococcus iniae, a pathogen of streptococcosis

A bacteriocin-producing Lactococcus lactis subsp. lactis was isolated from the intestine of olive flounder. The bacteriocin was identified as nisin Z. It was active against Gram-positive bacteria. Nisin Z at 3,200 arbitrary units (AU) was more effective in seawater than in PBS; growth of Streptococcus iniae was completely inhibited within 3 h. Nisin Z preparations with 3.5% (w/v) NaCl was the most effective against S. iniae being similar to nisin Z in seawater. Nisin Z is thus a good alternative to antibiotics to prevent streptococcosis caused by S. iniae aquaculture systems.     

Extracellular ATP is a potent signaling molecule in the activation of the Japanese flounder (Paralichthys olivaceus) innate immune responses

Innate immunity is the first line of defense against pathogen infections. Extracellular ATP (eATP) is one of the most studied danger-associated molecular pattern molecules that can activate host innate immune responses through binding with and activating purinergic receptors on the plasma membrane. The detailed actions of eATP on fish innate immunity, however, remain poorly understood. In this study, we investigated bacterial pathogen-induced ATP release in head kidney cells of the Japanese flounder Paralichthys olivaceus. We also examined the actions of eATP on pro-inflammatory cytokine and immune-related gene expression, the activity of induced NO synthase (iNOS), and the production of reactive oxygen species (ROS) and NO in Japanese flounder immune cells. We demonstrate that ATP is dynamically released from Japanese flounder head kidney cells into the extracellular milieu during immune challenge by formalin-inactivated Edwardsiella tarda and Vibrio anguillarum. In addition, we show that eATP administration results in profound up-regulation of pro-inflammatory cytokine gene expression, iNOS activity, and inflammatory mediator production, including ROS and NO, in Japanese flounder immune cells. Altogether, our findings demonstrate that eATP is a potent signaling molecule for the activation of innate immune responses in fish.     

Interactions of Streptococcus iniae with phagocytic cell line

Streptococcus iniae has become one of the most serious aquatic pathogens in the last decade, causing large losses in wild and farmed fish worldwide. There is clear evidence that this pathogen is capable not only of causing serious disease in fish but also of being transferred to and infecting humans. In this study, we investigate the interaction of S. iniae with two murine macrophage cell lines, J774-A1 and RAW 264.7. Cytotoxicity assay demonstrated significant differences between live and UV-light killed IUSA-1 strains. The burst respiratory activity decreased to baseline after 1 and 4 h of exposure for J774-A1 and RAW 264.7, respectively. Immunofluorescent and ultrastructural study of infected cells confirmed the intracellular localization of bacteria at 1 h and 24 h post-infection. Using qRT-PCR arrays, we investigated the changes in the gene expression of immune relevant genes associated with macrophage activation. In this screening, we identified 11 of 84 genes up-regulated, we observed over-expression of pro-inflammatory response as IL-1α, IL-1β, and TNF-α, without a good anti-inflammatory response. Present findings suggest a capacity of S. iniae to modulate a mammalian macrophages cell lines to their survival and replication intracellular, which makes this cell type as a reservoir for continued infection.     

A Streptococcus iniae DNA vaccine delivered by a live attenuated Edwardsiella tarda via natural infection induces cross‐genus protection

Edwardsiella tarda and Streptococcus iniae are important fish pathogens. We have reported previously a live E. tarda vaccine based on the attenuated strain TX5RM and a S. iniae DNA vaccine based on the antigen Sia10. In this study, we examined the possibility of constructing a cross‐genus vaccine by taking advantage of the residual infectivity of TX5RM and using it as a carrier host for the natural delivery of a S. iniae DNA vaccine. For this purpose, the recombinant TX5RM, TX5RMS10, was created, which harbours and retains stably the DNA vaccine plasmid pCS10 that expresses Sia10. When flounder were vaccinated with TX5RMS10 via oral and immersion routes, TX5RMS10 was detected in multiple tissues within 12–14 days postvaccination (p.v.). At 7 and 14 days p.v., expression of the DNA vaccine was detected in spleen, kidney and liver. Following E. tarda and S. iniae challenge at one and 2 months p.v., the vaccinated fish exhibited relative per cent survival rates of 69–83%. Immunological analysis indicated that TX5RMS10‐vaccinated fish produced specific serum antibodies and exhibited enhanced expression of a wide range of immune genes.     

Rapid MALDI biotyper-based identification and cluster analysis of <Emphasis Type="Italic">Streptococcus iniae</Emphasis>

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.     

High cell density fed-batch fermentation for the production of recombinant <Emphasis Type="Italic">E. coli</Emphasis> K-12 ghost vaccine against streptococcal disease

The bacterial ghost system is a novel vaccine delivery method which provides versatile carrier functions for foreign antigens with excellent natural intrinsic adjuvant properties. In this study, ghost bacteria of E. coli K-12/pHCE-InaN-GAPDH-ghost 27 SDM were created for mass production of a Streptococcus iniae ghost vaccine. The optimal fed-batch process for high cell density culture of E. coli K-12/pHCE-InaN-GAPDH-ghost 27 SDM was developed using the nutrient feeding strategy with Riesenberg defined medium. Fermentation was conducted in four phases as follow: (1) initial batch phase, (2) fed-batch phase for high cell density culture, (3) thermal induction phase for the formation of ghost by the expression of lysis gene E, and (4) high temperature holding phase to increase ghost formation efficiency. The maximum ghost bacteria vaccine (GBV) was obtained from the fed-batch fermentation of 34.9 g dry cell weight (dcw)/L. The expression of antigen glyceraldehyde 3-phosphate dehydrogenase (GAPDH) on the ghost cell with a high temperature holding phase was confirmed with outer-membrane protein fractionation using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Results indicate no damage to the expressed antigen on the ghost cell surfaces even after the temperature was increased to 47°C for high efficiency ghost cell formation. Efficacy of the GBV was evaluated by the challenge test in which vaccinated Olive flounder were infected with live S. iniae. The E. coli K-12 host strain, E. coli K-12/pHCE vector control, and formalin-killed cell (FKC) -treated vaccine groups showed 100, 100, and 65% cumulative mortality, respectively. The GBV-treated groups showed 50% cumulative mortality with increased survival ratios. Hence, the immunoprotective efficacy of GBV against S. iniae was better than that of the FKC vaccine. Therefore, the GBV is proposed as an effective vaccine in aquaculture for the prevention of streptococcal disease.     

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.     

Evolution of the Capsular Operon of Streptococcus iniae in Response to Vaccination

Streptococcus iniae causes severe septicemia and meningitis in farmed fish and is also occasionally zoonotic. Vaccination against S. iniae is problematic, with frequent breakdown of protection in vaccinated fish. The major protective antigens in S. iniae are the polysaccharides of the capsule, which are essential for virulence. Capsular biosynthesis is driven and regulated by a 21-kb operon comprising up to 20 genes. In a long-term study, we have sequenced the capsular operon of strains that have been used in autogenous vaccines across Australia and compared it with the capsular operon sequences of strains subsequently isolated from infected vaccinated fish. Intriguingly, strains isolated from vaccinated fish that subsequently become infected have coding mutations that are confined to a limited number of genes in the cps operon, with the remainder of the genes in the operon remaining stable. Mutations in strains in diseased vaccinated fish occur in key genes in the capsular operon that are associated with polysaccharide configuration (cpsG) and with regulation of biosynthesis (cpsD and cpsE). This, along with high ratios of nonsynonymous to synonymous mutations within the cps genes, suggests that immune response directed predominantly against capsular polysaccharide may be driving evolution in a very specific set of genes in the operon. From these data, it may be possible to design a simple polyvalent vaccine with a greater operational life span than the current monovalent killed bacterins.