Cloning,Expression And Functional Analysis Of TLR5/TLR22 And Associated Downstream Signaling Molecules In Qihe Crucian Carp (Carassius Auratus)

Specific immunityis not perfected in fish, which isthe lower vertebrate, and the non-specific immunity plays a critical role in immune response against invasion of pathogens.As the first and best characterized pattern recognition receptors(PRRs), Toll-like receptors(TLRs) play an important role in the innate immune system. TLRs can recognize diverse highly conserved pathogen-associated molecular patterns(PAMPs)specificallyand trigger the signaling pathways that activate immune cells in response to pathogen infection.The Qihe crucian carp(Carassius auratus), a triploid freshwater fish with natural gynogenesis, is an endemic fish andhas been widely cultivated throughout the north of Henan Province, China. However,in recent years, the high density and intensive farming has resulted in the weaker immunity and outbreak of fish diseases, which causes extensive losses in aquaculture. Therefore, in order to understand the immune responsesand developing preventive and therapeutic measures against pathogens, we should clarify the mechanism of immune response based on the PRRsin Qihe crucian carp after pathogen infection. Aeromonas hydrophila, as the most devastating pathogenic bacteria for freshwater fish, is responsible for the bacterial septicemia which restricts the development of Qihe crucian carp aquaculture seriously. To date, research of the immune response of Qihe crucian carp against A. hydrophilainfection mainly focuses on the determination of changes about biochemical index and expression analysis of immune-related genes. However, the mechanism of immune response based on the PRRsin Qihe crucian carp is still poorly understood.In the present study, we firstly cloned partial sequences of 13 TLRs in C. auratus and investigated the expression profiles of the TLRs in spleen and head kidney to screen which TLRs play essential roles in immune response against A.hydrophila infection. Secondly, the complete c DNA and genomic DNA sequences of TLR5, TLR22, My D88, and TRAF6 in Qihe crucian carp were cloned and characterized. Protein structures, homology alignment, and phylogenetic analysis were performed using the bioinformatics. Thirdly, tissue distribution, expression during embryonic development, and the response to PolyI:C, flagllin, and A. hydrophila infection were investigated using real-time quantitative PCR(q RT-PCR). Furthermore, the responses of three cytokines which were downstream genes of TLR signaling pathways were also analyzed. Fourthly, recombinant TLR5, TLR22, MyD88, and TRAF6 proteins were expressed in E. coli, and the polyclonal antibodies were produced using mice. Lastly, recombinant plasmids were constructed and transfected into HEK293 T cell. Subcellular localization of fusion proteins and the effects of gene over-expression on activity of NF-κB were determined.And the main results were described as follows:(1) Fragments of 13 members of TLR family(TLR1, 2, 3, 4, 5, 7, 8, 9, 18, 19, 20, 21, and 22) in C. auratuswere cloned and sequenced. And the expression profiles of these TLRs in spleen and head kidney at different time points(3, 6, 12, 24, and 48 h) after A. hydrophila infection were investigated. The results showed that the expressions of TLR5 and TLR22 were significantly up-regulated after infection, and the expression levels were the highest in spleen with 52.56-fold and 28.14-fold increase, respectively. These findings suggested that TLR5 and TLR22 may play important roles in the immune responses of C. auratusagainst A. hydrophila infection, and these two TLRs were the most likely receptors to identify the A. hydrophila.This is the first report to investigate the expression patterns of a complete set of TLRs in freshwater fish during gram-negative bacteria infection.(2) Complete c DNA and genomic DNA sequences of TLR5, TLR22, MyD88, and TRAF6 in Qihe crucian carp were cloned and characterized. The full-length c DNA of TLR5 was 2972 bp, with a 140 bp 5’-UTR, a 183 bp 3’-UTR, and a 2649 bp ORF encoding an 882 amino acid polypeptide with a Mw of 101.5 kD and a p I of 5.33. Protein structure analysis indicated that TLR5 possesses a signal peptide, 13 LRR motifs, a LRR-CT motif, a transmembrane region, and a TIR domain. There is an 223 bp intron locating in the 5’-UTR of TLR5 genomic DNA as similar as in zebrafish TLR5 s.The full-length cDNA of TLR22 was 3613 bp, with a 228 bp 5’-UTR, a 547 bp 3’-UTR, and a 2838 bp ORF encoding an 945 amino acid polypeptide with a Mw of 108.4 k D and a p I of 8.65. Protein structure analysis indicated that TLR22 possesses a signal peptide, 15 LRR motifs, a LRR-CT motif, a transmembrane region, and a TIR domain. Genomic DNA of TLR22 was consisted of a single exon. The full-length cDNA of My D88 was 2463 bp, with a 191 bp 5’-UTR, a 1417 bp 3’-UTR, and an 855 bp ORF encoding a 284 amino acid polypeptide with a Mw of 33.1 k D and a p I of 5.53. Protein structure analysis indicated that My D88 possesses a DEATH domain and a TIR domain. Genomic DNA of My D88 spanned five exons and was separated by four introns. The full-length cDNA of TRAF6 was 2555 bp, with a 52 bp 5’-UTR, an 871 bp 3’-UTR, and a 1632 bp ORF encoding a 543 amino acid polypeptide with a Mw of 61.7 k D and a pI of 5.81. Protein structure analysis indicated that TRAF6 contains five domains including a RING domain, two Zf-TRAF domains, a CCR domain, and a MATH domain. Genomic DNA of TRAF6 spanned six exons and was separated by five introns. Homology alignment based on full-length amino acid sequences indicated that these four proteins were highly conserved with other fish ortholog, and the close evolutionary relationship may possibly suggest their vital functions in fish.(3) The constitutive expression of TLR5,TLR22, My D88, and TRAF6 in all embryonic development stagessuggested the importance of TLR5/22 signaling pathways in protecting the early development stages of fish from various pathogenic invasions. High expression level was detected in fertilized eggs followed by decreased level and the minimum level was found at gastrula stage(TLR5), tail-bud stage(TLR22), and hatching stage(My D88 and TRAF6), respectively. That implied the presence and consumption of maternal genes mRNA. Then, expression level was increased significantly after hatching. These results suggested that, for protecting the embryons from pathogenic infection, we should pay more attention on care of embryons before hatching because of the poor immunity.(4) TLR5,TLR22, My D88, and TRAF6 of Qihe crucian carp were constitutively expressed in all examined eleven tissues but the mRNA expression level varied considerably amid the tissues. The hightest expression of TLR5 was detected in the kidney followed by the liver, head kidney, heart, and muscle, and the lowest expression was in the gill. Expression of TLR22 was higher in the head kidney, kidney, muscle, liver, and intestine, and was lower in heart and brain. The hightest expression of My D88 was detected in the spleen followed by the liver, blood, muscle, head kidney, kidney, and gill, and the lowest expression was in the skin. Expression of TRAF6 was higher in muscle and blood followed by spleen, kidney, head kidney, skin, and intestine, and was lower in the gill.(5) The expression of TLR5/TLR22 and associated downstream signaling molecules were regulated significantly by the Poly I:C, flagellin, and A. hydrophila infection, suggested that TLR5/22 signaling pathways were involved in the immune response of Qihe crucian carp against pathogenic invasions. The highest expression of TLR5 and TLR22 was detected at 3-12 h post infection but My D88 at 24 h post infection. These results indicated that there is a time difference in the expression response of different components in the signal pathway. Additionally, the expression of proinflammatory cytokine including IL-1β, IL-8, and TNFα was up-regulated after stimulation, especially IL-1β, suggested they play an important role in fish immunity.(4) Recombinant TLR5, TLR22, MyD88, and TRAF6 proteins were expressed in E. coli, and purified by cutting and extracting proteins from SDS-PAGE gel. Mice anti-serum was obtained against purified proteins and had marked antigen-specific reactivity. The polycloned antibodies can be used to research the location and expression profile of proteins in Qihe crucian carp tissues.(5) To investigated the subcellular localization of TLR5-TIR, TLR22-TIR, My D88, and TRAF6 proteins, recombinant plasmids with GFP marked were constructed and transfected into HEK293 T cell. TLR5/22-TIR distributed in the cytoplasm location near the cell membrane, and these results suggested that TLR5/22 may locate in the plasma membrane of HEK293 T cell. Additionally, My D88 and TRAF6 fusion proteins located in the cytoplasm.(6) Dual-luciferase reporter assay was used to determine the effects of gene over-expression on activity of NF-κB. The results indicated that over-expression of the TLR5/22-TIR and full length of My D88 can up-regulate the activity of NF-κB significantly and suggested that function of TLR5, TLR22, and My D88 was performed normally in HEK293 T cell. However, over-expression of TRAF6 whole protein had no significant effect on activity of NF-κB. Therefore, we speculated that TRAF6 in Qihe crucian carp cannot play its function in HEK293 T cell because protein struture was different from ortholog in mammals.