Fenghua Tan

The expression signatures of neuronal nitric oxide synthase (NOS1) in turbot (Scophthalmus maximus L.) mucosal surfaces against bacterial challenge.

The mucosal surfaces constitute the first immune barrier of host defense and also serve as the dynamic interfaces that simultaneously mediate a diverse array of critical physiological processes. It has been long hypothesized that observed difference of disease resistance among different fish strains and species are strongly correlated to the activities of the immune actors in mucosal surfaces. Particularly, neuronal NOS (nNOS or NOS1) is a constitutively expressed gene that catalyzes the oxidation of l-arginine and water to nitric oxide (NO), which is known as a potent host defence effector in immune system with antimicrobial activity. Moreover, NOS1 was detected to be expressed in fish mucosal surfaces, but its activities in mucosal immune responses were always overlooked. In this regard, we identified the NOS1 of turbot and characterized its expression patterns in mucosal tissues following Vibrio anguillarum and Streptococcus iniae challenge. The results showed that the NOS1 gene had a 4389 bp open reading frame (ORF) that encoded 1462 amino acids. Phylogenetic analysis showed the turbot NOS1 had the strongest relationship to Larimichthys crocea. And the syntenic analysis revealed the similar neighboring genes associated with turbot NOS1, compared with other teleost and mammals. In addition, NOS1 was widely expressed in all examined tissues with the highest expression level in brain, followed by intestine and gill. Finally, the NOS1 showed a general trend of up-regulation in mucosal tissues following both bacterial challenge, with the highest up-regulation in intestine. The significant quick induction of NOS1 in mucosal surfaces against infection indicated its key roles to prevent pathogen attachment and entry in mucosal immunity. More functional studies are needed to conduct in teleost to better understand the roles of NOS1 in maintaining the integrity of the mucosal barriers against infection.

Characterization and expression profiling of NOD-like receptor C3 (NLRC3) in mucosal tissues of turbot (Scophthalmus maximus L.) following bacterial challenge.

Abstract The mucosal surfaces are important for teleost as they are directly and continuously exposed to pathogen‐rich aquatic environments. Scrutinization and recognition of the attached pathogens is the first crucial step of mucosal immunity initiation. Nucleotide oligomerization domain (NOD)‐like receptors (NLRs) are a large group of intracellular pathogen recognition receptors (PRRs) which play key roles in pathogen recognition and subsequent immune signaling pathways activation. In this study, we identified two NLRC3 genes (NLRC3a and NLRC3b), a subfamily of NLRs from turbot, and profiled their expression patterns in mucosal tissues following bacterial challenge. NLRC3a transcript contains an open reading frame (ORF) of 3405 bp that encodes a putative peptide of 1134 amino acids. While NLRC3b has an ORF of 3114 bp encoding 1037 amino acids. A caspase recruitment domain (CARD) at N‐terminus characterized turbot NLRC3a, while NLRC3b seems to be unique to teleost, containing a fish specific NACHT associated (FISNA) domain and an extra B30.2 (PRY/SPRY) domain at C‐terminus. In addition, NLRC3a and NLRC3b were detected in all the examined tissues, with the highest expression levels in kidney and blood, respectively. After bacteria challenge, expression levels of turbot NLRC3 genes were strongly induced in intestine rather than in skin and gill, while NLRC3a had relatively higher expression level than that of NLRC3b. Taken together, NLRC3 genes found in this study were the first NLR members identified in turbot. The different expression signatures of NLRC3a and NLRC3b in mucosal tissues following two bacterial infections indicated they probably have important roles in early response to bacterial infection in the first line of host defense system. HighlightsNLRC3a and NLRC3b were identified in turbot.Turbot NLRC3a is homologous to their counterparts in other vertebrates.NLRC3b appears to be unique to teleost.NLRC3a and NLRC3b were ubiquitously expressed in turbot tissues.Expression of NLRC3a and NLRC3b were significantly induced after bacterial challenge.

Identification and mucosal expression analysis of cathepsin B in channel catfish (Ictalurus punctatus) following bacterial challenge

The mucosal surfaces of fish (skin, gill and intestine) constitute the primary line of defense against pathogen invasion. Although the importance of fish mucosal surfaces as the first barriers against pathogens cannot be overstated, the knowledge of teleost mucosal immunity are still limited. Cathepsin B, a lysosomal cysteine protease, is involved in multiple levels of physiological and biological processes, and playing crucial roles for host immune defense against pathogen infection. In this regard, we identified the cathepsin B (ctsba) of channel catfish and investigated the expression patterns of the ctsba in mucosal tissues following Edwardsiella ictaluri and Flavobacterium columnare challenge. Here, catfish ctsba gene was widely expressed in all examined tissues with the lowest expression level in muscle, and the highest expression level in trunk kidney, followed by spleen, gill, head kidney, intestine, liver and skin. In addition, the phylogenetic analysis showed the catfish ctsba had the strongest relationship to zebrafish. Moreover, the ctsba showed a general trend of up-regulated in mucosal tissues following both Gram-negative bacterial challenge. Taken together, the increased expression of ctsba in mucosal surfaces indicated the protective function of ctsba against bacterial infection, and the requirement for effective clearance of invading bacteria. Further studies are needed, indeed, to expand functional characterization and examine whether ctsba may play additional physiological and biological roles in catfish mucosal tissues.

The involvement of cathepsin F gene (CTSF) in turbot (Scophthalmus maximus L.) mucosal immunity

Abstract Cathepsin F (CTSF) is a recently described papain‐like cysteine protease and unique among cathepsins due to an elongated N‐terminal pro‐region, which contains a cystatin domain. CTSF likely plays a regulatory role in processing the invariant chain which is associated with the major histocompatibility complex (MHC) class II. In this regard, we identified the CTSF gene of turbot as well as its protein structure, phylogenetic relationships, and expression patterns in mucosal tissues following Vibrio anguillarum and Streptococcus iniae challenge. We also determined the expression patterns of CTSF in mucosal tissues after vaccinated with the formalin‐inactivated V. vulnificus whole‐cell vaccine. Briefly, turbot CTSF gene showed the closest relationship with that of Paralichthys olivaceus in phylogenetic analysis. And CTSF was ubiquitously expressed in all tested tissues with the highest expression level in gill. In addition, CTSF gene showed different expression patterns following different bacterial challenge. The significant quick regulation of CTSF in mucosal surfaces against infection indicated its roles in mucosal immunity. Functional studies should further characterize avail utilization of CTSF function to increase the disease resistance of turbot in maintaining the integrity of the mucosal barriers against infection and to facilitate selection of the disease resistant family/strain in turbot. HighlightsCTSF is homologous to their counterparts in other vertebrates.CTSF was ubiquitously expressed in turbot tissues.CTSF gene showed different expression patterns following different bacterial challenge and vaccination.

Identification, characterization and expression analysis of TLR5 in the mucosal tissues of turbot ( Scophthalmus maximus L.) following bacterial challenge

Abstract TLRs (Toll‐like receptors) are very important pathogen pattern recognition receptors, which control the host immune responses against pathogens through recognition of molecular patterns specific to microorganisms. In this regard, investigation of the turbot TLRs could help to understand the immune responses for pathogen recognition. Here, transcripts of two TLR5 (TLR5a and TLR5b) were captured, and their protein structures were also predicted. Meanwhile, we characterized their expression patterns with emphasis on mucosal barriers following different bacterial infection. The phylogenetic analysis revealed the turbot TLR5 genes showed the closest relationship to Paralichthys olivaceus. These two TLR5 genes were ubiquitously expressed in healthy tissues although expression levels varied among the tested tissues. In addition, the two copies of turbot TLR5 showed different expression patterns after bacterial infections. After Vibrio anguillarum infection, TLR5a was generally up‐regulated in intestine and skin while down‐regulated in gill, while TLR5b showed a general down‐regulation in mucosal tissues. After Streptococcus iniae infection, the TLR5a was down‐regulated at 2 h while generally up‐regulated after 4 h in mucosal tissues. Interestingly, the TLR5b was up‐regulated in intestine while down‐regulated in skin and gill after Streptococcus iniae infection. These findings suggested a possible irreplaceable role of TLR5 in the immune responses to the infections of a broad range of pathogens that include Gram‐negative and Gram‐positive bacteria. Future studies should apply the bacteriological and immune‐histochemical techniques to study the main sites on the mucosal tissue for bacteria entry and identify the ligand specificity of the turbot TLRs after challenge. HighlightsTLR5 genes are homologous to their counterparts in other vertebrates.TLR5 genes were ubiquitously expressed in turbot tissues.TLR5 genes showed different expression patterns following different bacterial challenge.

Expression profiling and microbial ligand binding analysis of high-mobility group box-1 (HMGB1) in turbot ( Scophthalmus maximus L.)

ABSTRACT High‐mobility group box 1 (HMGB1), a highly conserved DNA‐binding protein, was involved in nucleosome formation and transcriptional regulation, and could also act as an extracellular cytokine to trigger inflammation and immune responses. In this study, we identified a HMGB1 gene in turbot (Scophthalmus maximus L.). The full‐length SaHMGB1 cDNA includes an open reading frame of 615 bp which encoded a 204 amino acid polypeptide with an estimated molecular mass of 23.19 kDa. SaHMGB1 was closely related to several fish HMGB1 and shared 74.4% overall identity with human. In addition, phylogenetic analyses revealed SaHMGB1 showed the closest relationship to Larimichthys crocea. Furthermore, QPCR analysis showed that SaHMGB1 was expressed in all examined tissues with abundant expression levels in brain, gill, intestine, and head kidney, and showed different expression patterns following different bacterial challenge. The significant quick regulation of SaHMGB1 in mucosal surfaces against infection suggest that HMGB1 might play critical roles in mucosal immunity against bacterial challenge. Finally, the in vitro binding assay showed that SaHMGB1 had strong binding ability to LPS, LTA, and PGN. Functional studies should further characterize HMGB1 function to understand the importance of the integrity of the mucosal barriers against infection, and to facilitate selection of the disease resistant family/strain in turbot. HIGHLIGHTSSaHMGB1 gene is homologous to their counterparts in other vertebrates.SaHMGB1 gene was ubiquitously expressed in turbot tissues.SaHMGB1 gene showed different expression patterns following different bacterial challenge.SaHMGB1 had strong binding ability to LPS, LTA, and PGN.

l-rhamnose-binding lectins (RBLs) in turbot (Scophthalmus maximus L.): Characterization and expression profiling in mucosal tissues.

&NA; Rhamnose‐binding lectin (RBL) were mostly identified from egg cortex and ovary cells from vertebrates and invertebrates, with the specific binding activities to l‐rhamnose or d‐galactose. Previously, we found that a RBL gene was dramatically down‐regulated (−11.90 fold at 1 h, −48.95 fold at 4 h, −905.94 fold at 12 h) in the intestine of turbot following Vibrio anguillarum challenge using RNA‐seq expression analysis. In this regard, we sought here to identify RBLs in turbot, as well as the analysis of genomic structure, phylogenetic relationships, basal tissue distribution and the expression patterns following different bacteria challenge in mucosal tissues. In this study, two RBLs were captured in turbot with two conserved type 5 CRD5s, which were belong to type IIIc RBL. In phylogenetic tree analysis, turbot RBLs were clustered with tilapia, European sea bass and snakehead. In addition, in comparison of genomic architecture of turbot RBLs with the available published RBL genes revealed a high degree of conservation in the exon/intron organization among the teleost species. Moreover, both RBLs were significantly up‐regulated in mucosal tissues following V. anguillarum and Streptococcus iniae challenge, indicated their critical roles in turbot mucosal immunity. Further studies are needed to expand functional characterization of detailed mechanisms of RBLs in fish innate immunity. HighlightsRBL genes are homologous to their counterparts in other vertebrates.RBL genes were ubiquitously expressed in turbot tissues.RBL genes showed different expression patterns following different bacterial challenge.

Expression profiling and functional characterization of galectin-3 of turbot (Scophthalmus maximus L.) in host mucosal immunity

Galectins, a family of evolutionary conserved β-galactoside-binding proteins, have been characterized in a wide range of species. Galectin-3 is the only member in the chimera type, which is a monomeric lectin with one CRD domain. A growing body of evidence have indicated vital roles of galectin-3 in innate immune responses against infection. Here, one galectin-3 gene was captured in turbot (SmLgals3) with a 1203 bp open reading frame (ORF). In comparison to other species, SmLgals3 showed the highest similarity and identity to large yellow croaker and medaka, respectively. The genomic structure analysis showed that SmLgals3 had 5 exons similar to other vertebrate species. The syntenic analysis revealed that galectin-3 had the same neighboring genes across all the selected species, which suggested the synteny encompassing galectin-3 region during vertebrate evolution. Subsequently, SmLgals3 was widely expressed in all the examined tissues, with the highest expression level in brain and the lowest expression level in skin. In addition, SmLgals3 was significantly down-regulated in intestine following both Gram-negative bacteria Vibrio anguillarum, and Gram-positive bacteria Streptococcus iniae immersion challenge. Finally, the rSmLgals3 showed strong binding ability to all the examined microbial ligands. Taken together, our results suggested SmLgals3 played vital roles in fish innate immune responses against infection. However, the knowledge of SmLgals3 are still limited in teleost species, further studies should be carried out to better characterize its detailed roles in teleost mucosal immunity.

Expression profiling and microbial ligand binding analysis of galectin-4 in turbot (Scophthalmus maximus L.)

Galectins are a family of galactoside-binding proteins with an affinity for β-galactosides, involved in mediating fundamental processes including development, inflammation, cell migration and apoptosis. Galectin-4 is a member of tendem-repeat galectins, plays vital roles in intestinal epithelial barrier. Here, one galectin-4 gene was captured in turbot (SmLgals4) contains a 1197 bp open reading frame (ORF). In comparison to other species, SmLgals4 showed the highest similarity and identity both to large yellow croaker. The genomic structure analysis showed that SmLgals4 had conserved exons in the CRD domains compared to other vertebrate species. The syntenic analysis revealed that galectin-4 had the same neighboring genes across all the selected species, which suggested the synteny encompassing galectin-4 region during vertebrate evolution. Subsequently, SmLgals4 was widely expressed in all the examined tissues, with the highest expression level in intestine and the lowest expression level in skin. In addition, SmLgals4 was significantly down-regulated in intestine following both Gram-negative bacteria Vibrio anguillarum, and Gram-positive bacteria Streptococcus iniae immersion challenge. Finally, the rSmLgals4 showed strong binding ability to all the examined microbial ligands. Taken together, our results suggested SmLgals4 plays vital roles in fish intestinal immune responses against infection, but the detailed roles of galectin-4 in teleost are still lacking, further studies are needed to be carried out to characterize whether galectin-4 plays similar roles in teleost intestinal immunity.

Characterization, expression profiling and functional characterization of cathepsin Z (CTSZ) in turbot (Scophthalmus maximus L.)

Cathepsin Z (CTSZ) is a lysosomal cysteine protease of the papain superfamily. It participates in the host immune defense via phagocytosis, signal transduction, cell-cell communication, proliferation, and migration of immune cells such as monocytes, macrophages, and dendritic cells. In this study, we reported the identification of SmCTSZ, a CTSZ homolog from turbot (Scophthalmus maximus L.). SmCTSZ was 317 residues in length and contains a Pept-C1 domain. In multiple species comparison, SmCTSZ shared 65-93% overall sequence identities with the CTSZ counterparts from human, rat, and several fish species. In the phylogenetic analysis, SmCTSZ showed the closest relationship to Cynoglossus semilaevis. The syntenic analysis revealed the similar neighboring genes of CTSZ across all the selected species, which suggested the synteny encompassing CTSZ region during vertebrate evolution. Subsequently, SmCTSZ was constitutively expressed in various tissues, with the lowest and highest levels in brain and intestine respectively. In addition, SmCTSZ was significantly up-regulated in intestine following both Gram-negative bacteria Vibrio anguillarum, and Gram-positive bacteria Streptococcus iniae immersion challenge. Finally, the rSmCTSZ showed strong binding ability to all the examined microbial ligands, and the agglutination effect to different bacteria. Taken together, these results indicated SmCTSZ could play important roles in mucosal immune response in the event of bacterial infection in teleost. However, the knowledge of CTSZ are still limited in teleost species, further studies should be carried out to better characterize its detailed roles in teleost mucosal immunity.