ABSTRACT
The mannan-binding lectin-associated serine protease-1 (MASP-1) gene is a crucial component of the lectin pathway in the complement and coagulation cascade. Although MASP-1 has been found in the immune system of teleosts, its immune functions in response to bacterial infection are unclear. In this study, we identified a MASP-1 homolog (gcMASP-1) in the grass carp (Ctenopharyngodon idella). The full-length 3308-bp gcMASP-1 cDNA includes a 2160-bp open reading frame encoding a protein composed of 719 amino acids with epidermal growth factor-like, complement control protein, and trypsin-like domains. gcMASP-1 shares a high similarity with MASP-1 counterparts in other species, and it is most closely related to Cyprinus carpio MASP-1 and Sinocyclocheilus anshuiensis MASP-1. Transcription of gcMASP-1 was widely distributed in different tissues and induced by Aeromonas hydrophila in vivo and in vitro. Expression of gcMASP-1 was also affected by lipopolysaccharide and flagellin stimulation in vitro. In cells over-expressing gcMASP-1, transcript levels of almost all components, except gcMBL and gcC5, were significantly enhanced, and gcIL1ß, gcTNF-α, gcIFN, gcCD59, gcC5aR1, and gcITGß-2 were significantly upregulated after exposure to A. hydrophila; gcMASP-1 interference downregulated the transcript levels after A. hydrophila challenge. In addition, gcMASP-1 activated NF-κB signaling. These findings indicate the vital role of gcMASP-1 in innate immunity in C. idella.
Subject(s)
Aeromonas hydrophila/immunology , Carps , Fish Diseases/enzymology , Fish Proteins/metabolism , Gram-Negative Bacterial Infections/veterinary , Immunity, Innate/genetics , Mannose-Binding Protein-Associated Serine Proteases/metabolism , Aeromonas hydrophila/physiology , Animals , Cloning, Molecular , DNA, Complementary/genetics , DNA, Complementary/metabolism , Fish Diseases/immunology , Fish Proteins/genetics , Gram-Negative Bacterial Infections/enzymology , Gram-Negative Bacterial Infections/immunology , Mannose-Binding Protein-Associated Serine Proteases/genetics , Phylogeny , RNA, Messenger/genetics , RNA, Messenger/metabolism , Random Allocation , Sequence Analysis, DNA/veterinaryABSTRACT
Rac1, a Rho GTPase, serves critical immunological functions in mammals. Here, a Rac1 homolog (gcRac1) was identified in grass carp (Ctenopharyngodon idella). The full-length 2023-base pair gcRac1 cDNA contained a 579-bp open reading frame encoding a 192-residue protein, including a conserved RHO domain and nuclear localization signal. The gcRac1 protein shares high identity with other Rac1 counterparts and phylogenetically clustered with Danio rerio Rac1. The gcRac1 transcript showed wide tissue distribution and was inducible by Aeromonas hydrophila in vivo and in vitro; its expression also fluctuated with LPS or flagellin stimulation in vitro. With gcRac1 over-expression, gcPAK1, gcIL1-ß, gcTNF-α and gcIFN were basically up-regulated by A. hydrophila and bacterial PAMPs induction, while gcRac1 knockdown decreased these transcripts after A. hydrophila challenge. Over-expression of gcRac1 reduced, while its suppression facilitated, bacterial invasion. Moreover, gcRac1 could activate NF-κB signaling. These findings implicate the vital role of gcRac1 in grass carp innate immunity.
Subject(s)
Carps/genetics , Carps/immunology , Fish Proteins/genetics , Fish Proteins/immunology , rac1 GTP-Binding Protein/genetics , rac1 GTP-Binding Protein/immunology , Amino Acid Sequence , Animals , Base Sequence , Carps/metabolism , Cloning, Molecular , Fish Proteins/metabolism , Gene Expression Profiling , Molecular Sequence Data , Phylogeny , Polymerase Chain Reaction , Sequence Analysis, DNA , Transcriptome/genetics , rac1 GTP-Binding Protein/metabolismABSTRACT
Toll-like receptors (TLRs) play a critical role in the innate immune system. Although TLR18 is an important member of this family of receptors in fish, the role of the tlr18 gene in responses to pathogen infection is still unclear. In this study, we identified and characterized the grass carp tlr18 gene (gctlr18) to further clarify the function of TLR18 in teleost fish. Gctlr18 spans over 3600 bp and encodes a polypeptide of 852 amino acids. Analysis of the deduced amino acid sequence showed that gctlr18 encodes structures typical of the TLR family, including a signal peptide, seven leucine-rich repeats (LRRs), a transmembrane region, and a (Toll-interleukin-1 receptor) TIR domain. Quantitative RT-PCR analysis showed that gctlr18 was constitutively expressed in all investigated tissues, with abundant expression in spleen, gill, heart, intestine, kidney and fin and low expression in skin, liver and brain. Following grass carp reovirus-challenge and Aeromonas hydrophila inoculation, gctlr18 transcripts were upregulated significantly in immune-relevant tissues. Stimulation of Ctenopharyngodon idella kidney (CIK) cells with purified flagellin from Salmo typhimurium, lipopolysaccharide and polyinosinic-polycytidylic acid stimulation in vitro resulted in significantly increased gctlr18 expression, reaching a peak followed by restoration of normal levels. Overexpression of gctlr18 reduced A. hydrophila invasion by 83.4%. In CIK cells, gctlr18 induced the expression of proinflammatory cytokines, including il-8, inf-1 and tnf-α. Our results indicate that gctlr18 plays a key role in innate immune responses in teleost fish.
