Chengbin Gao

Transcriptomic profiling revealed the signatures of intestinal barrier alteration and pathogen entry in turbot (Scophthalmus maximus) following Vibrio anguillarum challenge

The mucosal immune system serves as the frontline barriers of host defense against pathogen infection, especially for the fishes, which are living in the pathogen rich aquatic environment. The intestine constitutes the largest surface body area in constantly contact with the external pathogens, and plays a vital role in the immune defense against inflammation and pathogen infection. Previous studies have revealed that fish intestine might serves as the portal of entry for Vibrio anguillarum. To characterize the immune actors and their associated immune activities in turbot intestine barrier during bacterial infection, here we examined the gene expression profiles of turbot intestine at three time points following experimental infection with V.xa0anguillarum utilizing RNA-seq technology. A total of 122 million reads were assembled into 183,101 contigs with an average length of 1151 bp and the N50 size of 2302 bp. Analysis of differential gene expression between control and infected samples at 1xa0h, 4xa0h, and 12xa0h revealed 2079 significantly expressed genes. Enrichment and pathway analysis of the differentially expressed genes showed the centrality of the pathogen attachment and recognition, antioxidant/apoptosis, mucus barrier modification and immune activation/inflammation in the pathogen entry and host immune responses. The present study reported the novel gene expression patterns in turbot mucosal immunity, which were overlooked in previous studies. Our results can help to understand the mechanisms of turbot host defense, and may also provide foundation to identify the biomarkers for future selection of disease-resistant broodstock and evaluation of disease prevention and treatment options.

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.

Identification and expression analysis of TLR2 in mucosal tissues of turbot (Scophthalmus maximus L.) following bacterial challenge.

The pathogen recognition receptors (PRRs), which can recognize the conserved pathogen-associated molecular patterns (PAMPs) of the bacteria, play key roles in the mucosal surfaces for pathogen recognition and activation of immune signaling pathways. However, our understanding of the PRRs and their activities in mucosal surfaces in the critical early time points during pathogen infection is still limited. Towards to this end, here, we sought to identify the Toll-like receptor 2 (TLR2) in turbot as well as its expression profiles in mucosal barriers following bacterial infection in the early time points. The full-length TLR2 transcript consists of open reading frame (ORF) of 2451 bp encoding the putative peptide of 816 amino acids. The phylogenetic analysis revealed the turbot TLR2 showed the closest relationship to Paralichthys olivaceus. The TLR2 mRNA expression could be detected in all examined tissues, with the most abundant expression level in liver, and the lowest expression level in skin. In addition, TLR2 showed different expression patterns following Vibrio anguillarum and Streptococcus iniae infection, but was up-regulated following both challenge, especially post S.xa0iniae challenge. Characterization of TLR2 will probably contribute to understanding of a number of infectious diseases and broaden the knowledge of interactions between host and pathogen, which will eventually help in the development of novel intervention strategies for farming turbot.

Identification and expression analysis of toll-like receptor genes (TLR8 and TLR9) in mucosal tissues of turbot (Scophthalmus maximus L.) following bacterial challenge

Mucosal immune system is one of the most important components in the innate immunity and constitutes the front line of host defense against infection, especially for teleost, which are living in the pathogen-rich aquatic environment. The pathogen recognition receptors (PRRs), which can recognize the conserved pathogen-associated molecular patterns (PAMPs) of bacteria, are considered as one of the most important component for pathogen recognition and immune signaling pathways activation in mucosal immunity. In this regard, we sought to identify TLR8 and TLR9 in turbot (Scophthalmus maximus), as well as their mucosal expression patterns following different bacterial infection in mucosal tissues for the first time. The full-length TLR8 transcript consists of an open reading frame (ORF) of 3108 bp encoding the putative peptide of 1035xa0amino acids. While the TLR9 was 6730 bp long, containing a 3168 bp ORF that encodes 1055xa0amino acids. The phylogenetic analysis revealed both TLR8 and TLR9 showed the closest relationship to large yellow croaker. Moreover, both TLR8 and TLR9 could be detected in all examined healthy turbot tissues, with the lowest expression level in liver and a relatively moderate expression pattern in healthy mucosal tissues. Distinct expression patterns of TLR8 and TLR9 were comparatively observed in the mucosal tissues (intestine, gill and skin) following Vibrio anguillarum and Streptococcus iniae infection, suggesting their different roles for mucosal immunity. Further functional studies are needed to better characterize TLR8 and TLR9 and their family members, to better understand the ligand specificity and to identify their roles in different mucosal tissues in protecting fish from the pathogenically hostile environment.

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.

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.

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.

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.