Orange-spotted Grouper(Epinephelus Coioides) Toll-like Receptor22:Molecular Characterization, Expression Pattern And Pertinent Signaling Pathways

Toll-like receptors (TLRs) are an important gene family for host innate immunologic surveillance. TLRs belong to the type I transmembrane proteins, containing an extracellular leucine-rich repeats (LRR) domain and a cytoplasmic Toll/interleukin (IL)-1receptor (TIR) domain. After recognizing PAMPs, TLRs mainly recruite two important adaptors, MyD88and TRIF, to mediate two individual TLR signaling cascades, the MyD88-dependent pathway and the TRIF-dependent pathway, resulting in the activation of NF-κB (nuclear factor kappa B) and MAPK(mitogen-activated protein kinase) as well as the release of inflammatory cytokines and the type I interferons. Toll-like receptor22(TLR22) is only found in aquatic animals. After challenge, TLR22recruits MyD88and TRIF adaptors for activating the innate immune system to defense the invasion of microorganisms. In present study, we have obtained the full cDNA sequence of Toll-like receptor22(TLR22) from orange-spotted grouper (Epinephelus coioides), challenged the grouper with pathogen in vivo and incubated the leucocytes with mitogen in vitro to analyze its expression profiles. The main results are as follows:1. We have cloned the grouper TLR22gene with an open reading frame (ORF) of2,880bp, encoding a putative peptide of960amino acids which has three highly typical domains with the feature of TLR family members. The hydrophobicity analysis of the propeptide revealed that grouper TLR22contained a signal peptide (37amino acids), sixteen LRR domains, one C-terminal LRR domain (LRRCT) at the extracellular region and a TIR domain in the cytoplasmic region. Multiple alignments demonstrated that grouper TLR22had a highly homologous with the Percoidea TLR22(more than50%identities), and a higher similarity to the TIR domain of teleosts (more than80%identities). Phylogenetic tree exhibited that the grouper TLR22grouped with teleost TLR22s, and closely located to marine fishes of the Percoidea.2. Quantitative expression analysis of grouper TLR22was examined in almost all tissues of healthy fish. The highest expression of TLR22mRNA was detected in grouper trunk kidney and head kidney, with low expression in PBLs, spleen and heart and lower expression in gill, eyes, intestine, skin and liver, indicating that grouper TLR22would have a potential role in participating in host defense.3. Real-time Quantitative PCR was used to explore the expression levels of grouper TLR22gene at different time in spleen when infected with Vibrio alginolyticus. Post infection, the mRNA expression of TLR22enhanced obviously in the grouper spleen and the peak expression occurred at24h. Then gene expression maintained at comparatively high levels at48h even to72h.4. LPS and Poly I:C were used to mimic bacterial and viral infection respectively, the gene expression change of TLR22was detected in grouper head-kidney leucocytes and spleen leucocytes. Stimulation of Poly I:C for3h, the grouper TLR22expression levels were further raised significantly and prolonged for a longer time. After LPS induction, the gene expression increased at1.5h, and still had a high mRNA expression levels at6h.5. LPS and Poly I:C were used to mimic bacterial and viral infection respectively, the gene expressions change of TLR22signaling pathway were detected in grouper head-kidney leucocytes. Four important molecules for signal transduction, MyD88, TRIF, TNF-a and IRF3, were chosen to research the signaling pathway for anti-pathogen responses. Post Poly I:C stimulation, the mRNA expressions of MyD88and TNF-a enhanced obviously at1.5h. It was also occurred the peak expressions after6-h LPS stimulation. Upon LPS challenging for1.5h, TRIF and IRF3mRNA expressions were induced and showed an augmentative tendency in head kidney leukocytes.