Jianmin Ye

Identification and characterization of C1 inhibitor in Nile tilapia (Oreochromis niloticus) in response to pathogenic bacteria

Abstract C1 inhibitor (C1INH) is a multi‐functional serine protease inhibitor in plasmatic cascades, not only inactivating various proteases, but also regulating both complement and contact system activation. In this study, we described the identification and characterization of a C1INH ortholog from Nile tilapia (Oreochromis niloticus) at molecular, protein and cellular levels. The full‐length cDNA of Oreochromis niloticus C1INH (OnC1INH) consisted of 1791 bp of nucleotide sequence encoding polypeptides of 596 amino acids. The deduced protein possessed a serpin domain at the C‐terminal domain, and two Ig‐like domains in the N‐terminal domain with significant homology to teleost. Expression analysis revealed that the OnC1INH was extremely highly expressed in the liver; however, much weakly exhibited in other tissues including spleen, kidney, blood and heart. After the in vivo challenges of the lipopolysaccharide (LPS) and Streptococcus agalactiae, the expression of OnC1INH was significantly up‐regulated in liver and spleen at the late phase, which was confirmed at the protein level with immunohistochemical analysis. The up‐regulation of OnC1INH expression was also demonstrated in head kidney monocytes/macrophages in vitro stimulated with LPS, Aeromonas hydrophila and Streptococcus agalactiae, which was positively correlated with the protein expression pattern in the culture media. Taken together, the results of this study indicated that OnC1INH might be involved in the immune response of Nile tilapia against to bacterial challenge. HighlightsC1INH homolog was identified from Nile tilapia (OnC1INH).OnC1INH was up‐regulated by Streptococcus agalactiae challenge.The up‐regulation was demonstrated in monocytes/macrophages stimulated with S. agalactiae.

Identification and characterization of a mannose-binding lectin from Nile tilapia (Oreochromis niloticus)

Abstract Mannose‐binding lectin (MBL) is a pattern recognition protein that plays an important role in innate immunity capable of activating the lectin pathway of the complement system. In this study, a MBL homologue (OnMBL) was identified from Nile tilapia (Oreochromis niloticus) and characterized at expression and agglutination functional levels. The open reading frame of OnMBL is 687 bp of nucleotide sequence encoding polypeptides of 228 amino acids. The deduced amino acid sequence is highly homology to teleost and similar to mammalian MBL, containing a canonical collagen‐like region, a carbohydrate recognition domain and a neck region. Expression analysis revealed that the OnMBL was highly expressed in the liver, and also exhibited in other tissues including hind kidney, intestines, head kidney and spleen. In addition, the OnMBL expression was significantly up‐regulated in spleen and head kidney following challenges with a Gram‐positive bacterial pathogen (Streptococcus agalactiae) and a Gram‐negative bacterial pathogen (Aeromonas hydrophila). Recombinant OnMBL ((r)OnMBL) protein was able to agglutinate both S. agalactiae and A. Hydrophila in vitro. Taken together, the results of this study indicated that OnMBL, possessing apparent agglutination ability to bacterial pathogens, might be involved in host defense against bacterial infection in Nile tilapia. HighlightsThe MBL gene was identified in Nile tilapia (OnMBL).OnMBL was significantly up‐regulated following challenges with Streptococc galactiae and Aeromonas hydrophila.rOnMBL possessed agglutination ability to both bacteria (Streptococc galactiae and Aeromonas hydrophila).

Expression and functional characterization of transferrin in Nile tilapia (Oreochromis niloticus) in response to bacterial infection

ABSTRACT Transferrin (TF), an iron‐binding glycoprotein, plays an important role in host defense against pathogenic infection, which inhibits the growth and proliferation of pathogens, deprives iron from invading pathogens, and activates anti‐microbial responses in macrophages. In this study, a TF homologue (OnTF) was identified from Nile tilapia (Oreochromis niloticus) and characterized at expression pattern against bacterial infection and capability binding bacterial pathogens. The open reading frame of OnTF is 2118 bp of nucleotide sequence encoding polypeptides of 705 amino acids. The deduced protein is highly homology to the other species, containing two conserved iron binding lobes: N‐lobe and C‐lobe. Expression analysis revealed that the OnTF was extremely highly expressed in liver tissue; however, much weakly exhibited in other examined tissues including spleen and head kidney. The OnTF expression was significantly up‐regulated in the liver, spleen and head kidney following infection of a Gram‐positive bacterial pathogen (Streptococcus agalactiae) and a Gram‐negative bacterial pathogen (Aeromonas hydrophila). The up‐regulation of OnTF expression was also demonstrated in hepatocytes and macrophages in vitro stimulated with S. agalactiae and A. hydrophila. In addition, recombinant OnTF ((r)OnTF) protein possessed capability to bind both S. agalactiae and A. hydrophila in vitro. Taken together, the present study indicated that OnTF might be involved in host defense against bacterial infection in Nile tilapia. HIGHLIGHTSOnTF was significantly up‐regulated following challenges with Streptococcus galactiae and Aeromonas hydrophila in vivo.Bacterial stimulation induced the OnTF expression in hepatocytes and macrophages in vitro.Recombinant OnTF possessed binding capability with both bacteria (Streptococcus galactiae and Aeromonas hydrophila), LTA and LPS.

C1r and C1s from Nile tilapia (Oreochromis niloticus): Molecular characterization, transcriptional profiling upon bacterial and IFN-γ inductions and potential role in response to bacterial infection

ABSTRACT The complement components C1r and C1s play a vital role in immunity with the activation of C1 complex in the classical complement pathway against pathogen infection. In this study, Nile tilapia (Oreochromis niloticus) C1r and C1s orthologs (OnC1r and OnC1s) were identified and characterized. The cDNA of OnC1r and OnC1s ORFs consisted of 1902 bp and 2100 bp of nucleotide sequence encoding polypeptides of 633 and 699 amino acids, respectively. The deduced OnC1r and OnC1s proteins both possessed CUB, EGF, CCP and SP domains, which were significantly homology to teleost. Spatial mRNA expression analysis revealed that the OnC1r and OnC1s were highly expressed in liver. After the in vivo challenges of Streptococcus agalactiae (S. agalactiae) and lipopolysaccharide (LPS), the mRNA expressions of OnC1r and OnC1s were significantly up‐regulated in liver and spleen, which were consistent with immunohistochemical detection at the protein level. The up‐regulation of OnC1r and OnC1s expressions were also demonstrated in head kidney monocytes/macrophages in vitro stimulated with LPS, S. agalactiae, and recombinant OnIFN‐&ggr;. Taken together, the results of this study indicated that OnC1r and OnC1s were likely to get involved in the immune response of Nile tilapia against bacterial infection. HighlightsThe C1r and C1s gene were identified in Nile tilapia (OnC1r and OnC1s).OnC1r and OnC1s possessed similar structure by bioinformatic analysis.OnC1r and OnC1s were up‐regulated following challenges with LPS and Streptococcus agalactiae.OnC1r and OnC1s were induced by recombinant OnIFN‐&ggr;.

Spleen tyrosine kinase from Nile tilapia (Oreochromis niloticus): Molecular characterization, expression pattern upon bacterial infection and the potential role in BCR signaling and inflammatory response

Abstract Spleen tyrosine kinase (SYK), a member of non‐receptor tyrosine kinase family, plays an important role in immune responses against pathogen infection, which is capable of activating B cells signaling pathway and regulating inflammatory response. In this study, Nile tilapia (Oreochromis niloticus) ortholog (OnSYK) was identified and characterized at expression pattern against bacterial infection, function in B cells activation pathway and inflammatory response. The cDNA of OnSYK ORF contained 1851 bp of nucleotide sequence encoding polypeptides of 616 amino acids. The deduced OnSYK protein was highly homologous to other species SYK, containing two SH2 domains and a TyrKc domain. Spatial mRNA expression analysis revealed that OnSYK had wide tissue distribution and was highly expressed in the liver. After challenge of Streptococcus agalactiae (S. agalactiae) in vivo, mRNA expression of OnSYK was significantly up‐regulated in the head kidney, spleen and liver. The up‐regulation of OnSYK transcript was also displayed in the head kidney and spleen leukocytes stimulation with S. agalactiae and LPS in vitro, which was confirmed at protein level in the head kidney leukocytes by FACS analysis. In addition, after induction with mouse anti‐OnIgM monoclonal antibody in vitro, the expressions of OnSYK and its downstream molecules (OnLYN, OnBLNK and OnAP‐1) were significantly up‐regulated in the head kidney leukocytes, and pharmacological inhibition of SYK activity with inhibitor (P505‐15) significantly attenuated the expressions of OnLYN, OnBLNK and OnAP‐1. Moreover, upon LPS challenge, the expressions of OnSYK, OnTNF‐&agr;, OnIL‐6 and OnAP‐1 were also up‐regulated in the head kidney monocytes/macrophages. After treatment with SYK inhibitor (BAY 61–3606), the expressions of OnTNF‐&agr;, OnIL‐6 and OnAP‐1 were inhibited in the LPS‐challenged head kidney monocytes/macrophages. Taken together, the results of this study indicated that OnSYK, playing potential roles in BCR signaling and inflammatory response, was likely to get involved in host defense against bacterial infection in Nile tilapia. HighlightsThe SYK gene was identified in Nile tilapia (OnSYK).OnSYK expression was significantly up‐regulated upon challenge with S. agalactiae.OnSYK was involved in BCR signaling pathway.OnSYK played an important role in regulation of inflammatory response.

Identification and characterization of a B-type mannose-binding lectin from Nile tilapia (Oreochromis niloticus) in response to bacterial infection

Lectins are a group of carbohydrate-binding proteins, which play an important role in innate immune system against pathogen infection. In this study, a B-type mannose-binding lectin (OnBML) was identified from Nile tilapia (Oreochromis niloticus), and characterized at expression patterns against bacterial infection and capability to promote phagocytosis by macrophages. The open reading frame of OnBML is 354 bp of nucleotide sequence encoding polypeptides of 117 amino acids. The deduced protein is highly homologous to other teleost BMLs, containing two repeats of the conserved mannose-binding motif QXDXNXVXY. Expression of OnBML was widely exhibited in all examined tissues, with the most abundance in spleen and following gill, peripheral blood, and head kidney. The OnBML expressions were significantly up-regulated following two major bacterial infections including a Gram-positive bacterium (Streptococcus agalactiae) and a Gram-negative bacterium (Aeromonas hydrophila) in vivo and in vitro. Recombinant OnBML protein possessed capacities of mannose-binding and calcium-dependent agglutination to S. agalactiae and A. hydrophila, and promoted the phagocytosis by macrophages. Taken together, the present study indicated that OnBML is likely to get involved in host defense against bacterial infection in Nile tilapia.

High-efficiency inorganic nitrogen removal by newly isolated Pannonibacter phragmitetus B1

An aerobic heterotrophic nitrogen removal bacterium strain, B1, was isolated from aquaculture water and identified as Pannonibacter phragmitetus (99% similarity) by 16S rRNA sequencing analysis. When ammonium, nitrite or nitrate was the sole nitrogen source, with an initial nitrogen concentration of 14 mg/L, the nitrogen removal efficiencies were 98.66%, 99.96% and 98.73%, respectively, and the corresponding maximum removal rates reached as high as 1.16, 0.77 and 0.81 mg/L/h, respectively. In the presence of NH4+-N, the removal efficiency of 56 mg/L NO2--N within 27 h increased by 83.50%, and the corresponding removal rate reached as high as 1.72 mg/L/h. Additionally, different carbon sources (dl-malic acid, sucrose, sodium citrate, and glucose) could be utilized in nitrogen removal. Sequence amplification indicates that the denitrification genes nirK, norB and narG are present in strain B1. All results demonstrate that strain B1 has high promise for future applications of removing inorganic nitrogen from wastewater.

Expression and functional characterization of collection-K1 from Nile tilapia (Oreochromis niloticus) in host innate immune defense

HIGHLIGHTSThe CL‐K1 gene was identified in Nile tilapia (OnCL‐K1).OnCL‐K1 expression was significantly up‐regulated following challenges with Streptococcus galactiae and Aeromonas hydrophila.Recombinant OnCL‐K1 possessed agglutination and opsonization ability to both bacteria (Streptococcus galactiae and Aeromonas hydrophila).Recombinant OnCL‐K1 was able to participate in the regulation of inflammatory and migration reaction. ABSTRACT Collectin‐K1 (CL‐K1), a multifunctional Ca2+‐dependent lectin, is able to bind carbohydrates on pathogens and inhibit infection by direct neutralization, agglutination, opsonization and killing, which plays an important role in innate immunity. In this study, a CL‐K1 homolog (OnCL‐K1) was identified from Nile tilapia (Oreochromis niloticus) and characterized at expression and agglutination functional levels. The open reading frame of OnCL‐K1 is 720 bp of nucleotide sequence encoding a polypeptide of 239 amino acids. The deduced amino acid sequence has two characteristic structures, containing a collagen‐like region and a carbohydrate recognition domain. Expression analysis revealed that the OnCL‐K1 was highly expressed in the liver, and widely exhibited in other tissues including kidney, intestine and spleen. In addition, the OnCL‐K1 expression was significantly up‐regulated in spleen and anterior kidney following challenges with a Gram‐positive bacterial pathogen (Streptococcus agalactiae) and a Gram‐negative bacterial pathogen (Aeromonas hydrophila). The up‐regulation of OnCL‐K1 expression was also demonstrated in hepatocytes and monocytes/macrophages in vitro stimulation with S. agalactiae and A. hydrophila. Recombinant OnCL‐K1 protein was able to agglutinate both S. agalactiae and A. hydrophila in vitro, and participate in the regulation of inflammatory, migration reaction and promote the phagocytosis by monocytes/macrophages. Taken together, the results of this study indicated that OnCL‐K1, possessing apparent agglutination, opsonization and killing ability to bacterial pathogens and participating in the regulation mechanisms of the non‐specific cellular immune, might be involved in host defense of innate immunity against bacterial infection in Nile tilapia.

Affinity maturation occurs in channel catfish (Ictalurus punctaus) following immunization with a T-cell dependent antigen

Affinity maturation of the antibody response, a process of antibody affinity increasing over response, is one of the key features of the mammalian immune system. However, the process is incompletely understood in teleost, including channel catfish (Ictalurus punctaus). In this study, IgM affinity maturation in channel catfish was investigated by estimating the kinetics of antibody affinity using ELISA and ELISPOT assays. Fish were immunized with a T-cell dependent antigen (TNP-KLH), and individual serum IgM antibody titers and affinities, and IgM+ antibody-secreting cells (ASCs) in peripheral blood were analyzed over a period of 14 weeks. A detectable serum anti-TNP response developed by 2-weeks post-immunization, and the maximal antibody production was observed by 6-weeks post-immunization. The average affinity of anti-TNP serum antibody increased consistently and reached the maximum by 10-weeks post-immunization. The increase of antibody affinity beyond the point of optimal antibody titer revealed that the affinity maturation of IgM antibody response occurred in channel catfish. Dissection of dynamics of individual affinity subpopulations indicated that a significant proportion of low affinity subpopulations appeared at early response, and high affinity subpopulations appeared predominantly at later, resulting in a 100-fold increase in affinity over response. Additional, TNP+ IgM+ ASCs was detected by 2-weeks post-immunization and achieved the maximal number by 6-weeks post-immunization. Using an inhibition ELISPOT assay, the findings of a consistent increase in the average affinity of secreted IgM antibody by peripheral blood ASCs, as the immune response progressed, confirmed the occurrence of the affinity maturation. Taken together, the results of this study indicated that affinity maturation occurred in channel catfish following immunization with a TD antigen TNP-KLH.

Oxidative burst activity in haemocytes of the freshwater prawn Macrobrachium rosenbergii

ABSTRACT Oxidative burst, release of reactive oxygen species/reactive nitrogen species (ROS/RNS) contributed to microorganisms killing, is a vital immune response of crustacean haemocyte. Three morphologic haemocyte types (hyaline cells, HC; semigranular cells, SGC; granular cells, GC) have been defined in crustaceans, and found to play different roles in immune defense. However, oxidative burst activities of different haemocyte subpopulations in crustaceans are currently not documented. In the present study, we investigated the oxidative burst activities of the three haemocyte types in the freshwater prawn Macrobrachium rosenbergii using the common ROS fluorescent probe dichlorofluorescin‐diacetate (DCFH‐DA). Nitric oxide (NO) donor sodium nitroprusside (SNP) improved the DCF fluorescence in haemocytes, while NO scavenger C‐PTIO (2‐(4‐carboxyphenyl)‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl‐3‐oxide) and NO‐synthase inhibitor NG‐monomethyl‐l‐arginine (L‐NMMA) reduced the fluorescence, suggesting that DCF fluorescence intensity could also be modified by intracellular NO level and activity of NO‐synthase pathway. ROS/RNS was also produced in the untreated haemocytes. GC contained most non‐induced ROS/RNS production, while oxidative activity of HC was rather weak. No significant impact of PMA could be observed on ROS/RNS level in all the three cell types. Both zymosan A (ZA) and lipopolysaccharide (LPS) significantly triggered the production of ROS/RNS in SGC and GC, whereas they had no effect on those of HC, suggesting that SGC and GC were the primary cell types involved in pathogens killing by ROS/RNS pathway. Cytochalasin B (Cyt B) inhibited the ZA‐induced ROS/RNS production, but could not change the ROS/RNS level stimulated by LPS. For unstimulated haemocytes, ROS/RNS productions decreased 29.6%, 44.1% and 48.6% in SGC, and decreased 44.5%, 28.4% and 57.3% in GC, in the presence of L‐NMMA, Fccp and DPI respectively, whereas apocynin could not modulate DCF fluorescence in both SGC and GC, suggesting that mitochondrial oxidative pathway was relatively more dominant in SGC, and NO‐synthase (NOS) pathway appeared more active in GC. For LPS‐stimulated haemocytes, oxidative activities decreased 22.9%, 42.9%, 29.6% and 60.0% in SGC, and reduced 40.6%, 25.2%, 26.7% and 70.6% in GC with the presence of L‐NMMA, apocynin, Fccp and DPI respectively, suggesting that NADPH‐oxidase (NOX) pathway in both SGC and GC was activated by LPS, and it became the predominant oxidative pathway in stimulated SGC, while NOS pathway was the relative main source for ROS/RNS production in stimulated GC. HIGHLIGHTSGranular cell (GC) and semigranular cell (SGC) were the main cell types for basal and LPS‐induced ROS/RNS production.Cytochalasin B inhibited ZA‐stimulated oxidative activity, but had no effect on LPS‐stimulated oxidative activity.Without activation, NO‐synthase pathway was more dominant in GC, while mitochondria pathway was more active in SGC.NADPH‐oxidase pathway was activated in GC and SGC by LPS, and became the most dominant approach in SGC.