Fusion of Streptococcus iniae α-enolase to IMX313 enhanced antibody titer and survival rate in olive flounder (Paralichthys olivaceus).

The polymerization of monomeric antigens can be a strategy to overcome the low immunogenicity of subunit vaccines. IMX313 is a hybrid oligomerization domain of chicken C4bp, and has been demonstrated to have potent activity as adjuvants for the fused antigens in mammals. In the present study, we investigated whether the oligomerization of α-enolase of Streptococcus iniae by fusion with IMX313 affected on antibody induction and on protection against S. iniae infection in olive flounder (Paralichthys olivaceus). The oligomerization of S. iniae enolase by fusion with IMX313 (enolase-IMX313) was verified by non-reducing PAGE, and the antibody titer against enolase in olive flounder immunized with enolase-IMX313 was significantly higher than that in fish immunized with enolase alone. Furthermore, although the survival of olive flounder immunized with enolase alone was low, fish immunized with enolase-IMX313 showed much higher survival (RPS 50%) in accordance with higher serum antibody titer, suggesting that fusion of antigens with IMX313 can be an effective way to enhance protective efficacy of subunit vaccines in olive flounder.

Significance of Glutamate Racemase for the Viability and Cell Wall Integrity of Streptococcus iniae.

Streptococcus iniae is a pathogenic and zoonotic bacterium responsible for human diseases and mortality of many fish species. Recently, this bacterium has demonstrated an increasing trend for antibiotics resistance, which has warranted a search for new approaches to tackle its infection. Glutamate racemase (MurI) is a ubiquitous enzyme of the peptidoglycan synthesis pathway that plays an important role in the cell wall integrity maintenance; however, the significance of this enzyme differs in different species. In this study, we knocked out the MurI gene in S. iniae in order to elucidate the role of glutamate racemase in maintaining cell wall integrity in this bacterial species. We also cloned, expressed, and purified MurI and determined its biochemical characteristics. Biochemical analysis revealed that the MurI gene in S. iniae encodes a functional enzyme with a molecular weight of 30 kDa, temperature optimum at 35°C, and pH optimum at 8.5. Metal ions, such as Cu2+, Mn2+, Co2+ and Zn2+, inhibited the enzyme activity. MurI was found to be essential for the viability and cell wall integrity of S. iniae. The optimal growth of the MurI-deficient S. iniae mutant can be achieved only by adding a high concentration of D-glutamate to the medium. Membrane permeability assay of the mutant showed an increasing extent of the cell wall damage with time upon D-glutamate starvation. Moreover, the mutant lost its virulence when incubated in fish blood. Our results demonstrated that the MurI knockout leads to the generation of S. iniae auxotroph with damaged cell walls.

Purification, Characterization and Inhibition of Alanine Racemase from a Pathogenic Strain of Streptococcus iniae.

Streptococcus iniae is a pathogenic and zoonotic bacteria that impacted high mortality to many fish species as well as capable of causing serious disease to humans. Alanine racemase (Alr, EC 5.1.1.1) is a pyridoxal-5'-phosphate (PLP)-containing homodimeric enzyme that catalyzes the racemization of L-alanine and D-alanine. In this study, we purified alanine racemase from S. iniae that was isolated from an infected Chinese sturgeon (Acipenser sinensis), as well as determined its biochemical characteristics and inhibitors. The alr gene has an open reading frame (ORF) of 1107 bp, encoding a protein of 369 amino acids, which has a molecular mass of 40 kDa. The enzyme has optimal activity at a temperature of 35°C and a pH of 9.5. It belongs to the PLP-dependent enzymes family and is highly specific to L-alanine. S. iniae Alr (SiAlr) could be inhibited by some metal ions, hydroxylamine and dithiothreitol (DTT). The kinetic parameters K m and V max of the enzyme were 33.11 mM, 2426 units/mg for L-alanine, and 14.36 mM, 963.6 units/mg for D-alanine. Finally, the 50% inhibitory concentrations (IC50) values and antibiotic activity of two alanine racemase inhibitors (homogentisic acid and hydroquinone), were determined and found to be effective against both Gram-positive and Gram-negative bacteria employed in this study.Streptococcus iniae is a pathogenic and zoonotic bacteria that impacted high mortality to many fish species as well as capable of causing serious disease to humans. Alanine racemase (Alr, EC 5.1.1.1) is a pyridoxal-5'-phosphate (PLP)-containing homodimeric enzyme that catalyzes the racemization of L-alanine and D-alanine. In this study, we purified alanine racemase from S. iniae that was isolated from an infected Chinese sturgeon (Acipenser sinensis), as well as determined its biochemical characteristics and inhibitors. The alr gene has an open reading frame (ORF) of 1107 bp, encoding a protein of 369 amino acids, which has a molecular mass of 40 kDa. The enzyme has optimal activity at a temperature of 35°C and a pH of 9.5. It belongs to the PLP-dependent enzymes family and is highly specific to L-alanine. S. iniae Alr (SiAlr) could be inhibited by some metal ions, hydroxylamine and dithiothreitol (DTT). The kinetic parameters K m and V max of the enzyme were 33.11 mM, 2426 units/mg for L-alanine, and 14.36 mM, 963.6 units/mg for D-alanine. Finally, the 50% inhibitory concentrations (IC50) values and antibiotic activity of two alanine racemase inhibitors (homogentisic acid and hydroquinone), were determined and found to be effective against both Gram-positive and Gram-negative bacteria employed in this study.

Recombinant phosphoglucomutase and CAMP factor as potential subunit vaccine antigens induced high protection against Streptococcus iniae infection in flounder (Paralichthys olivaceus).

AIMS: The aim of this study was to screen vaccine candidates from virulence factors of Streptococcus iniae in flounder model. METHODS AND RESULTS: The immunogenicity of recombinant phosphoglucomutase (rPGM) and rCAMP factor was confirmed by Western blot. The percentage of surface membrane immunoglobulin-positive (sIg+ ) lymphocytes in peripheral blood leucocytes, the specific and total serum IgM and the activity of acid phosphatase (ACP) and peroxidase (POD) in flounder were determined with flow cytometry, ELISA and commercial enzyme activity kits, respectively, after intraperitoneal immunization with rPGM and rCAMP factor. The results showed that rPGM and rCAMP factor could induce significant rise in sIg+ lymphocytes, specific serum IgM and activities of ACP and POD. Additionally, the relative percent survival rate of the vaccinated flounder was 64 and 54% in challenge experiment using S. iniae, respectively. These results indicated that rPGM and rCAMP factor could evoke humoural and innate immune response in flounder and provide high-efficiency immunoprotection against S. iniae infection. CONCLUSIONS: Phosphoglucomutase (PGM) and CAMP factor were promising vaccine candidates against S. iniae in flounder. SIGNIFICANCE AND IMPACT OF THE STUDY: Phosphoglucomutase and CAMP factor have the potential to be vaccine candidates, which provide important information for us to develop the effective subunit vaccines, especially the multivaccine, against S .iniae in aquaculture.

Orthologues of Streptococcus pyogenes nuclease A (SpnA) and Streptococcal 5'-nucleotidase A (S5nA) found in Streptococcus iniae.

Streptococcus pyogenes nuclease A (SpnA) and streptococcal 5' nucleosidase A (S5nA) are two recently described virulence factors from the human pathogen S. pyogenes. In vitro studies have shown that SpnA is a nuclease that cleaves ssDNA and dsDNA, including the DNA backbone of neutrophil extracellular traps. S5nA was shown to hydrolyse AMP and ADP, but not ATP, to generate the immunomodulatory molecule adenosine. S5nA also generates the macrophage-toxic deoxyadenosine from dAMP. However, detailed in vivo studies of the two enzymes have been hampered by difficulties with using current animal models for this exclusive human pathogen. Here we report the identification of two novel enzymes from the fish pathogen Streptococcus iniae that show similarities to SpnA and S5nA in amino acid sequence, protein domain structure and biochemical properties. We propose that SpnAi and S5nAi are orthologues of the S. pyogenes enzymes, providing a rationale to analyse the in vivo function of the two enzymes using a S. iniae-zebrafish infection model.

Comprehensive characterization of three glutathione S-transferase family proteins from black rockfish (Sebastes schlegelii).

Glutathione S-transferases (GSTs, EC 2.5.1.18) are categorized as phase II enzymes, which form an important multifunctional family associated with a wide variety of catalytic activities. GSTω, GSTρ, and GSTθ are cytosolic GSTs which have been extensively studied in a variety of organisms; however, few studies have focused on teleosts. Those paralogs from black rockfish (Sebastes schlegelii; RfGSTω, RfGSTρ, and RfGSTθ, respectively) were molecularly, biochemically, and functionally characterized to determine their antioxidant extent and protective aptitudes upon pathogenic stress. RfGSTω, RfGSTρ, and RfGSTθ, contained open reading frames of 717bp, 678bp, and 720bp respectively, which encoded respective proteins of 239, 226, and 240 amino acids in length. In silico analysis revealed that all RfGSTs possessed characteristic N-terminal domains bearing glutathione (GSH)-binding sites, and C-terminal domains containing substrate-binding sites. Recombinant RfGSTω (rRfGSTω) catalyzed the conjugation of GSH to dehydroascorbate (DHA), while rRfGSTθ and rRfGSTρ catalyzed to the model GST substrate 1-Chloro-2,4-dinitrobenzene (CDNB). Kinetic analysis revealed variation in Km and Vmax values for each rRfGST, indicating their different conjugation rates. The optimum conditions (pH and temperature) and inhibition assays of each protein demonstrated different optimal ranges showing their wide range of activity as an assembly. RfGSTω and RfGSTθ paralogs demonstrated their antioxidant potential towards H2O2 and heavy metals (Cd, Zn, and Cu) in vitro, while RfGSTρ had an antioxidant potential only towards heavy metals (Zn and Cu). Though all the paralogs were ubiquitously expressed in different magnitudes, RfGSTω was highly expressed in blood, whereas RfGSTρ and RfGSTθ were highly expressed in liver. The mRNA expression of RfGSTω and RfGSTθ, upon Streptococcus iniae and poly I:C stimulation, revealed a significantly up-regulated expression, whereas RfGSTρ mRNA expression was significantly down-regulated. Collectively, our findings suggest that RfGSTω, RfGSTρ, and RfGSTθ paralogs are potent in detoxifying xenobiotic toxics, capable of protecting cells from oxidative stress generated by both H2O2 and heavy metals, and finally, yet importantly, stimulated under pathogenic stress signals.