Baofeng Su

The mucosal expression signatures of g-type lysozyme in turbot (Scophthalmus maximus) following bacterial challenge.

The mucosal surfaces constitute the first line of host defense against infection, and also serve as the dynamic interfaces that simultaneously mediate a diverse array of critical physiological processes, while in constantly contact with a wide range of pathogens. The lysozymes are considered as key components for innate immune response to pathogen infection with their strong antibacterial activities. But their activities in mucosal immune responses were always overlooked, especially for g-type lysozymes, whose expression patterns in mucosal tissues following bacterial challenge are still limited. Towards to this end, here, we characterized the g-type lysozymes, Lyg1 and Lyg2 in turbot, and determined their expression patterns in mucosal barriers following different bacterial infection. The phylogenetic analysis revealed the turbot g-type lysozyme genes showed the closest relationship to Cynoglossus semilaevis. The two lysozyme genes showed different expression patterns following challenge. Lyg2 was significantly up-regulated in mucosal tissues following Vibrio anguillarum and Streptococcus iniae challenge, while Lyg1 showed a general trend of down-regulation. The significant mucosal expression signatures of g-type lysozyme genes indicated their key roles to prevent pathogen attachment and entry in the first line of host defense system. Further functional studies should be carried out to better characterize the availability of utilization of g-type lysozyme to increase the disease resistance in the mucosal surfaces and facilitate the disease resistant breeding selection.

Characterization and expression analysis of a peptidoglycan recognition protein gene, SmPGRP2 in mucosal tissues of turbot (Scophthalmus maximus L.) following bacterial challenge

Peptidoglycan recognition receptor proteins (PGRPs), a group of pattern recognition receptors (PRRs), can recognize peptidoglycan (PGN) of the bacteria cell wall and play an important role in host immune defense against pathogen infection. They are highly structurally conserved through evolution, but with different function in innate immunity between invertebrates and vertebrates. In teleost fish, several PGRPs have been characterized recently. They have both amidase activity and bactericidal activity and are involved in indirectly killing bacteria and regulating multiple signaling pathways. However, the knowledge of PGRPs in mucosal immunity of teleost fish is still limited. In this study, we identified a PGRPs gene (SmPGRP2) of turbot and investigated its expression patterns in mucosal tissues after challenge with Gram-positive bacteria Streptococcus iniae and Gram-negative bacteria Vibrio anguillarum. Phylogenetic analysis showed the strongest relationship of turbot PGRP to halibut, which was consistent with their phylogenetic relationships. In addition, SmPGRP2 was ubiquitously expressed in turbot tissues, and constitutive expression levels were higher in classical immune tissues (including liver, spleen, and head-kidney) than mucosal tissues (intestine, gill and skin). After bacterial challenge, the expression of SmPGRP2 was induced and showed a general trend of up-regulation in mucosal tissues, except in intestine following V. anguillarum infection. These different expression patterns varied depending on both pathogen and tissue type, suggesting its distinct roles in the host immune response to bacterial pathogen.

Mucosal expression signatures of two Cathepsin L in channel catfish (Ictalurus punctatus) following bacterial challenge.

The mucosal surfaces of fish are the first line of host defense against various pathogens. The mucosal immune responses are the most critical events to prevent pathogen attachment and invasion. Cathepsins are a group of peptidases that involved in different levels of immune responses, but the knowledge of the roles of Cathepsin in mucosal immune responses against bacterial infection are still lacking. Therefore, in the present study we characterized the Cathepsin L gene family in channel catfish, and profiled their expression levels after challenging with two different Gram-negative bacterial pathogens. Here, two Cathepsin L genes were identified from channel catfish and were designated CTSL1a and CTSL.1. Comparing to other fish species, the catfish CTSL genes are highly conserved in their structural features. Phylogenetic analysis was conducted to confirm the identification of CTSL genes. Expression analysis revealed that the CTSL genes were ubiquitously expressed in all tested tissues. Following infection, the CTSL genes were significantly induced at most timepoints in mucosal tissues. But the expression patterns varied depending on both pathogen and tissue types, suggesting that CTSL genes may exert disparate functions or exhibit distinct tissue-selective roles in mucosal immune responses. Our findings here, clearly revealed the key roles of CTSL in catfish mucosal immunity; however, further studies are needed to expand functional characterization and examine whether CTSL may also play additional physiological roles in catfish mucosal tissues.

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.

Expression profile analysis of two cathepsin S in channel catfish (Ictalurus punctatus) mucosal tissues following bacterial challenge

Cathepsin S belongs to the papain family of cysteine protease, and is considered to play key roles in immune responses after bacterial challenge. However, despite the recognized importance of Cathepsin S in immunity, no studies have systematically characterized Cathepsin S in catfish. In this regard, here, we characterized the Cathepsin S gene family in channel catfish, and investigated their expression patterns following two different Gram-negative bacterial challenge. In the present study, two Cathepsin S genes (ctss and ctssa) were captured in channel catfish. In comparison to other species, the catfish Cathepsin S genes are highly conserved in their structural features. Phylogenetic analysis indicated the strongest phylogenetic relationship with zebrafish, which is consistent with their evolutional relationships. Tissue distribution analysis revealed that Cathepsin S genes were ubiquitously expressed in catfish tissues. Following bacterial infection, the Cathepsin S genes were significantly up-regulated at most time-points in mucosal surfaces, with an acute response post Edwardsiella ictaluri infection. Obviously, the expression profiles were quite distinct between two Cathepsin S genes, across the tissues and between pathogens, suggesting that Cathepsin S genes may exert disparate roles in mucosal immune responses. Our findings here, provide early insight into the immune functions of Cathepsin S in catfish; however, further studies are needed to determine the mechanisms of Cathepsin S for antigen presentation during inflammatory processes and innate host defense.

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.

Expression profiling analysis of immune-related genes in channel catfish (Ictalurus punctatus) skin mucus following Flavobacterium columnare challenge

Fish are covered by a watery gel-mucus, mainly secreted by the goblet cells, serving as the physical and biochemical barrier between the external environment and the interior milieu, playing more important roles in fish that without scale. Despite the important roles of mucus in fish immunity, the knowledge of detailed molecular events happened during infection process is still limited. While most studies were focused on characterizing the protein and enzyme activities in the mucus following challenge, no studies have examined the gene expression profiles in fish mucus. In this regard, herein we carried out the first gene profiling analysis in catfish mucus using real-time PCR. Ten important immune-related genes were selected according to our previous studies. Their expression levels were examined in the early timepoints (namely, 1 h, 2 h, 4 h, 8 h, and 24 h) following Flavobacterium columnare challenge. Notably, expression levels of most of the selected genes were rapidly altered by the challenge. Seven genes were down-regulated, while only three genes were up-regulated. In addition, the gene expression patterns in mucus were very different from the mucosal surfaces (skin, gill and intestine) and the classical immune organs (liver, spleen and kidney). The unique expression patterns obtained here may be resulted from the great advantage of the large amount of attached bacteria in the mucus than the internal tissues, and resulted from the bacteria virulent actors to suppress the host immune response. Taken together, our results can expand our knowledge of fish mucosal immunity, and the un-lethal mucus sampling can provide early insight for developing the strategies for selection of disease resistant families and strains in catfish as well as other fish species.

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.

Characterization and expression analysis of chitinase genes (CHIT1, CHIT2 and CHIT3) in turbot (Scophthalmus maximus L.) following bacterial challenge

ABSTRACT Chitinases are hydrolytic enzymes which have been employed to breakdown chitin coats of pathogenic microorganisms, thereby weaken the defense system of several pathogens and insects. In this regard, we identified the chitinase genes of turbot and characterized their expression patterns in mucosal tissues following Vibrio anguillarum and Streptococcus iniae challenge. In present study, transcripts of three chitinase genes (CHIT1, CHIT2 and CHIT3) were captured, as well as their protein structures and expression patterns following different bacterial infection were also characterized. The chitinases were widely expressed in all tested tissues with the highest expression levels of CHIT1 and CHIT2 in intestine, and CHIT3 in skin. Finally, these three genes showed different expression patterns following bacterial challenge. The significant quick induction of chitinases in mucosal surfaces against infection indicated their key roles to prevent pathogen attachment and entry in mucosal immunity. Functional studies should further characterize the chitinases and avail utilization of their function to increase the disease resistance in maintaining the integrity of the mucosal barriers against infection and facilitating the disease resistant family/strain selection in turbot. HIGHLIGHTSThree chitinase genes were identified in turbot.Chitinase genes were ubiquitously expressed in turbot tissues.Chitinase genes were significantly regulated in the early timepoints after bacterial challenge.