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Streptococcus parauberis , a gram-positive cocci, causes bacterial disease in farmed fish. The recent increase in S. parauberis infection in aquatic farms in South Korea has justified the importance of vaccine development for the prevention of this disease. In this study, we evaluated the effect of subunit vaccines prepared from recombinant M-like protein (SimA) and fibrinogen-binding protein (FBP) candidates with an aluminum hydroxide adjuvant against S. parauberis infection in olive flounder Paralichthys olivaceus. For the in vivo experiment, fish (average length, 7.18 cm; average weight, 3.5 g) were injected intraperitoneally with: phosphate buffer saline (PBS, group 1), PBS/aluminum hydroxide (group 2), FBP/aluminum hydroxide (group 3), SimA/aluminum hydroxide (group 4), and SimA/FBP/aluminum hydroxide (group 5). After 3 weeks, the fish in each group were boosted using PBS (group 1 and 2), FBP (group 3), SimA (group 4), and SimA/FBP (group 5) without adjuvant. We found that the relative percent survival of fish after S. parauberis exposure in group 2, 3, 4, and 5 was 6.25%, 18.75%, 50%, and 12.5%, respectively, whereas the mortality in groups 1 was 80%, respectively. We performed Western blot, ELISA, and quantitative real time RT-PCR (qRT-PCR) after vaccination to investigate the further efficacy of the vaccine. Western blot and ELISA of vaccinated fish serum confirmed the production of specific antibodies against SimA and FBP. Furthermore, results of qRT-PCR showed that recombinant protein SimA induced a remarkably specific-antibody response compared with that in FBP or control and increased the expression of various immune response-related genes including interleukin-8 (IL-8), toll-like receptor 2 (TLR2), tumor necrosis factor-α (TNF-α), CD4-1, and MHC II. Thus, these results indicate that SimA is a potent vaccine candidate for protection against S. parauberis infection. • SimA based vaccine against Streptococcus parauberis in fish prevents infection. • Aluminum hydroxide serves as an effective adjuvant. • SimA/adjuvant-vaccinated fish showed extended survival rate. • In addition expressed immune response-related genes and elevated antibody titer. [ABSTRACT FROM AUTHOR]

The membrane attack complex/perforin (MACPF) superfamily consists of multifunctional proteins that form pores on the membrane surface of microorganisms to induce their death and have various immune-related functions. PFN2 is a perforin-like protein with an MACPF domain, and humans with deficient PFN2 levels have increased susceptibility to bacterial infection, which can lead to fatal consequences for some patients. Therefore, in this study, we confirmed the antimicrobial function of PFN2 in starry flounder (Platichthys stellatus). The molecular properties were confirmed based on the verified amino acid sequence of PsPFN2. In addition, the expression characteristics of tissue-specific and pathogen-specific PsPFN2 mRNA were also confirmed. The recombinant protein was produced using Escherichia coli , and the antimicrobial activity was then confirmed. The coding sequence of PFN2 (PsPFN2) in P. stellatus consists of 710 residues. The MACPF domain was conserved throughout evolution, as shown by multiple sequence alignment and phylogenetic analysis. PsPFN2 mRNA is abundantly distributed in immune-related organs such as the spleen and gills of healthy starry flounder, and significant expression changes were confirmed after artificial infection by bacteria or viruses. We cloned the MACPF domain region of PFN2 to produce a recombinant protein (rPFN2) and confirmed its antibacterial effect against a wide range of bacterial species and the parasite (Miamiensis avidus). • Perforin-2 (PFN2) from starry flounder (Platichthys stellatus) was identified. • PsPFN2 was highly expressed in the spleen, gills and PBLs of healthy starry flounder. • PsPFN2 mRNA expression levels were significantly regulated by S. parauberis and VHSV. • We confirmed the antimicrobial activities of rPsPFN2 against bacteria and M. avidus. [ABSTRACT FROM AUTHOR]

The membrane attack complex/perforin (MACPF) superfamily consists of multifunctional proteins that form pores on the membrane surface of microorganisms to induce their death and have various immune-related functions. PFN2 is a perforin-like protein with an MACPF domain, and humans with deficient PFN2 levels have increased susceptibility to bacterial infection, which can lead to fatal consequences for some patients. Therefore, in this study, we confirmed the antimicrobial function of PFN2 in starry flounder (Platichthys stellatus). The molecular properties were confirmed based on the verified amino acid sequence of PsPFN2. In addition, the expression characteristics of tissue-specific and pathogen-specific PsPFN2 mRNA were also confirmed. The recombinant protein was produced using Escherichia coli , and the antimicrobial activity was then confirmed. The coding sequence of PFN2 (PsPFN2) in P. stellatus consists of 710 residues. The MACPF domain was conserved throughout evolution, as shown by multiple sequence alignment and phylogenetic analysis. PsPFN2 mRNA is abundantly distributed in immune-related organs such as the spleen and gills of healthy starry flounder, and significant expression changes were confirmed after artificial infection by bacteria or viruses. We cloned the MACPF domain region of PFN2 to produce a recombinant protein (rPFN2) and confirmed its antibacterial effect against a wide range of bacterial species and the parasite (Miamiensis avidus). • Perforin-2 (PFN2) from starry flounder (Platichthys stellatus) was identified. • PsPFN2 was highly expressed in the spleen, gills and PBLs of healthy starry flounder. • PsPFN2 mRNA expression levels were significantly regulated by S. parauberis and VHSV. • We confirmed the antimicrobial activities of rPsPFN2 against bacteria and M. avidus. [ABSTRACT FROM AUTHOR]

Thioredoxin-like protein 1 (TXNL1) is a redox-active protein belonging to the thioredoxin family, which mainly controls the redox status of cells. The TXNL1 gene from Amphiprion clarkii (AcTXNL1) was obtained from a pre-established transcriptome database. The AcTXNL1 is encoded with 289 amino acids and is predominantly localized in the cytoplasm and nucleus. The TXN domain of AcTXNL1 comprises a34CGPC37 motif with redox-reactive thiol (SH-) groups. The spatial distribution pattern of AcTXNL1 mRNA was examined in different tissues, and the muscle was identified as the highest expressed tissue. AcTXNL1 mRNA levels in the blood and gills were significantly increased in response to different immunostimulants. In vitro antioxidant capacity of the recombinant AcTXNL1 protein (rACTXNL1) was evaluated using the ABTS free radical-scavenging activity assay, cupric ion reducing antioxidant capacity assay, turbidimetric disulfide reduction assay, and DNA nicking protection assay. The potent antioxidant activity of rAcTXNL1 exhibited a concentration-dependent manner in all assays. Furthermore, in the cellular environment, overexpression of AcTXNL1 increased cell viability under H 2 O 2 stress and reduced nitric oxide (NO) production induced by lipopolysaccharides (LPS). Collectively, the experimental results revealed that AcTXNL1 is an antioxidant and immunologically important gene in A. clarkii. • TXNL1 from A. clarkii was molecularly and functionally characterized. • AcTXNL1 was ubiquitously expressed in all tissues and significantly inducible upon immunostimulants. • Recombinant AcTXNL1 showed potent antioxidant and DNA protection activities. • Overexpression of AcTXNL1 enhanced cell viability under oxidative stress and reduced NO production in macrophages. [ABSTRACT FROM AUTHOR]