Characterization, expression and function analysis of pfTLR5S and pfTLR5M in yellow catfish (Pelteobagrus fulvidraco) responding to bacterial challenge.

Toll-Like Receptors (TLRs) are important pattern recognition receptors, playing critical roles in the early innate immune response to defensing against pathogen invasion. In this study, we found both soluble form TLR5 (pfTLR5S) and membrane form TLR5 (pfTLR5M) in yellow catfish Pelteobagrus fulvidraco. The open reading frames (ORFs) of pfTLR5M and pfTLR5S genes were 2655 bp and 1947 bp in length, encoding 884 and 648 amino acids, respectively. pfTLR5M was composed of thirteen LRR domains, one TIR domain and one transmembrane domain. However, pfTLR5S have only fifteen LRR domains, without any TIR domain and transmembrane domain. Both pfTLR5M and pfTLR5S genes had the highest expression in liver, especially for pfTLR5S, which showed a noticeable high expression in liver. We also compared the relative mRNA expression levels of pfTLR5M and pfTLR5S in digestive and immune-related tissues after challenge of three different bacteria. In addition, we also found that pfTLR5S can interact with pfTLR5M, and inhibit the expression of pfTLR5M protein, while induced the expression of downstream proinflammatory factors, such as TNFα and IL8. These results revealed that both pfTLR5M and pfTLR5S play important and different roles in defensing against the invasion of flagellated bacteria, and they may function by binding to each other.

Immunoglobulin (Ig) heavy chain gene locus and immune responses upon parasitic, bacterial and fungal infection in loach, Misgurnus anguillicaudatus.

Teleost fish are the most primitive bony vertebrates that contain immunoglobulin (Ig). Although teleost Ig is known to be important during tetrapod evolution and comparative immunology, little is known about the genomic organization of the immunoglobulin heavy-chain (IgH) locus. Here, three Ig isotype classes, IgM, IgD and IgT, were firstly identified in dojo loach (Misgurnus anguillicaudatus), and the IgH locus covering τ, µ and δ genes was also illustrated. Variable (V) gene segments lie upstream of two tandem diversity (D), joining (J) and constant (C) clusters and the genomic organization of the IgH locus presented as Vn-Dn-Jn-Cτ-Dn-Jn-Cµ-Cδ, similar to some other teleost fish. However, unlike some other teleost fish, ten VH, ten D and nine J genes were observed in this locus, which suggest teleost Igs might be conserved and diverse. Thus, it would be interesting to determine how Igs divide among themselves in immune response to different antigens. To address this hypothesis, we have developed three models by bath infection with parasitic, bacterial and fungal pathogens, respectively. We found that IgM, IgD and IgT were highly upregulated in the head kidney and spleen after infection with Ichthyophthirius multifiliis (Ich), suggesting that the three Igs might participate in the systemic immune responses to Ich. Moreover, the high expression of IgT in mucosal tissue, such as skin or gills, appeared after being infected with three different pathogens infection, respectively, in which the expression of IgT increased more rapidly in response to Ich infection. Interestingly, the expression of IgD showed a higher increase in spleen and head kidney being challenged with fungi, suggesting that IgD might play an important role in antifungal infection.

T cell receptor (TCR) α and ß genes of loach (Misgurnus anguillicaudatus): Molecular cloning and expression analysis in response to bacterial, parasitic and fungal challenges.

In vertebrates, the T cell receptor (TCR) plays a crucial role in immune system. To date, the roles of fish TCRs in response to pathogen infection are still poorly understood. In the present study, we firstly cloned and identified the TCRα and TCRß from dojo loach (Misgurnus anguillicaudatus) by RACE approaches. The full-length cDNAs of Ma-TCRα and Ma-TCRß include an open reading frame (ORF) of 723 and 879 bp encoding a polypeptide of 241 and 293 amino acids, respectively. Structural analysis indicated that Ma-TCRα and Ma-TCRß had a signal peptide, IgV domain, IgC domain, a connecting peptide (CPS), a transmembrane region (TM) and a cytoplasmic (CYT), which are similar to their counterparts described in other teleost. Phylogenetic analysis supported that Ma-TCR Cα and Ma-TCR Cß were closely related to the Cα and Cß region of Cyprinidae family, respectively. Transcriptional expression analysis indicated that Ma-TCRα and Ma-TCRß mRNAs were ubiquitously expressed in a wide array of tissues and most abundantly found in skin, brain, kidney, gill and spleen. The expression patterns of Ma-TCRα and Ma-TCRß after bacteria (F. columnare G4), parasite (Ichthyophthirius multifiliis) and fungus (Saprolegnia) infection were detected by qRT-PCR. Additionally, the morphological changes of gill and skin following the three infection models were investigated. The results clearly indicated that Ma-TCRα and Ma-TCRß was significant up-regulated not only in spleen and kidney, but also in skin and gill. In summary, our present findings suggested that Ma-TCRα and Ma-TCRß might play significantly roles in the modulation of immune response and protect loach from different pathogens infection.

IgM and IgD heavy chains of yellow catfish (Pelteobagrus fulvidraco): Molecular cloning, characterization and expression analysis in response to bacterial infection.

Three different immunoglobulin (Ig) isotypes, namely IgM, IgD, and IgT/IgZ have been described in most teleost, among which IgM and IgT are considered crucial in systematic and mucosal immunity, respectively. However, some teleost have no IgT/IgZ and it is unclear how other Ig isotypes interact to perform immune-protective roles in both systematic and mucosal sites. In this study, the complete cDNA sequences of IgM and IgD heavy chains were cloned and analyzed from yellow catfish (Pelteobagrus fulvidraco). The full-length cDNA of Pf-IgM and Pf-IgD heavy chains contained an open reading frame (ORF) of 1710 and 2991 bp encoding a predicted protein of 570 and 997 amino acids, respectively. Tissue-specific expression analysis indicated that both IgM and IgD were highly expressed in kidney and spleen, and higher expression levels were found at zygote and 13th day post hatching during early development. Multiple sequence alignment and phylogenetic analysis showed IgM and IgD of yellow catfish are closely related to other fish of Siluriformes. Moreover, we also constructed the infection model of yellow catfish with bacteria (Flavobacterium columnare G4) for the first time to study the function of Pf-IgM and Pf-IgD heavy chain genes in immune response. Quantitative real-time PCR (qRT-PCR) showed that significantly up-regulated expression of Pf-IgM was not only detected in liver and spleen, but also in mucosal tissues including skin and intestine, while Pf-IgD was just significantly increased in liver and spleen, which might suggest the main immune-protecting roles of IgM in mucosal tissues of yellow catfish.

Molecular characterization and expression analysis of T cell receptor (TCR) γ and δ genes in dojo loach (Misgurnus anguillicaudatus) in response to bacterial, parasitic and fungal challenge.

In mammalian, T-cell receptors (TCRs) play a key role in recognizing the presented antigen from external to protect organisms against environmental pathogens. To understand the potential roles of TCRγ and TCRδ in dojo loach (Misgurnus anguillicaudatus), Ma-TCRγ and Ma-TCRδ cDNAs were cloned and their gene expression profiles were investigated after bacterial, parasitic and fungal challenge. The open reading frame (ORF) of Ma-TCRγ and Ma-TCRδ cDNAs contained 948 and 867 bp, encoding 316 and 288 amino acid residues, respectively. Structurally, Ma-TCRγ and Ma-TCRδ were consisted of a signal peptide, a variable region, a constant region (IgC), a connecting peptide (CPS), a transmembrane region (TM) and a cytoplasmic domain (CYT), which were similar to those of other vertebrates. Multiple sequence alignment and phylogenetic analysis showed Ma-TCRγ and Ma-TCRδ were closely related to fish of Cyprinidae family. Ma-TCRγ and Ma-TCRδ were widely expressed in all tested organs/tissues, as the highest expressions of Ma-TCRγ and Ma-TCRδ were detected in kidney and gill, respectively. In addition, three infection models of dojo loach with bacteria (F. columnare G4), parasite (Ichthyophthirius multifiliis) and fungus (Saprolegnia sp.) were constructed. The morphological changes of gills and skin after challenged with F. columnare G4 and Ichthyophthirius multifiliis were investigated. Compared to F. columnare G4 infection, mRNA expression of both TCRγ and TCRδ showed higher sensitivity in classical immune organs (kidney and spleen) and mucosal tissues (skin and gill) after challenge with Ichthyophthirius multifiliis and Saprolegnia sp. Our results first indicated that TCRγ and TCRδ of dojo loach might function differently in response to challenge with different pathogens.

Molecular characterization and expression analysis of interleukin 15 (IL15) and interleukin-15 receptor subunit alpha (IL15Rα) in dojo loach (Misgurnus anguillicaudatus): Their salient roles during bacterial, parasitic and fungal infection.

Interleukin 15 (IL15) is a pleiotropic cytokine that participates in innate and adaptive immunity along with its receptor α-chain (IL15Rα). In order to investigate the potential roles of IL15 and IL15Rα in dojo loach (Misgurnus anguillicaudatus), we firstly cloned the cDNA sequence of Ma-IL15 and Ma-IL15Rα, which contain 1096bp and 1236bp and code proteins of 193 amino acids and 210 amino acids, respectively. A short signal peptide and Pfam IL15 domain were found in Ma-IL15, while a highly conserved sushi domain existed in Ma-IL15Rα. Ontogeny analysis indicated that significantly increased expression of Ma-IL15 and Ma- IL15Rα mRNA were detected in larvae from 1d to 7d post hatching, while relative high expression levels were detected in both systematic and mucosal immune-related tissues of adult dojo loach. Then three dojo loach infection models with F. columnare G4, I. multifiliis and Saprolegnia parasitica were constructed, which resulted in increased skin goblet cells and serious lesions in gill. Ma-IL15 and Ma-IL15Rα showed different expression patterns in different tissues during three infection models. Ma-IL15Rα mRNA was found to be more significantly elevated than Ma-IL15 after infection with F. columnare G4 in all examined tissues including kidney, spleen, gill and skin. I. multifiliis infection induced higher expression of Ma-IL15 in mucosal tissues including skin and gill, while it mainly increased Ma-IL15Rα expression in kidney. Moreover, our study firstly evaluated the influence of fungal infection on IL15 and IL15Rα expression in teleost, and it is interesting to find that both Ma-IL15 and Ma-IL15Rα expression showed consistent up-regulation after Saprolegnia parasitica infection compared to two other infection models. Therefore, our results suggest that Ma-IL15 and Ma-IL15Rα possess important defensive roles in systematic and mucosal tissues of dojo loach during bacterial, fungal and parasitic infection.