Figure 1.
The structural organization of TLRs from C. gigas, M. galloprovincialis (MgTLR-j, KC357777 and MgTLR-k, KC357778) and C. farreri (CfTLR, ABC73693).
Each of the four CgTLRs belongs to sccTLRs and contains a TIR domain, a TM domain, multiple extracellular LRRs and an N-terminal signal peptide.
Figure 2.
The MDS analysis of evolutionary relationships among chordata, mollusca and arthropoda TLRs.
The sequences used for MDS analysis are listed in table S2. Different colors are used to distinguish between phylum species. The distance between any two points on the plot refers to their molecular distance or divergence. Mollusk and arthropod TLRs are much less divergent than that within vertebrata TLR family. The TLRs of ancient non-vertebrate chordates like S. purpuratus and B. lanceolatum is closer to CgTLRs than to arthropoda TLRs.
Figure 3.
The NF-κB activity activated by CgTLRs.
(A) The four CgTLRs can activate NF-κB-responsive reporter in a dose-dependent manner by transfection of plasmids increasing from 100 to 500 ng per well. HEK293 cells were transiently transfected with empty vector pcDNA or CgTLR expression vectors with 20 ng NF-κB-responsive reporter vector and 20 ng pRL-TK internal control vector per well in 24-well plate, respectively. Luciferase activities were tested at 24 h post transfection. (B) No PAMP can enhance higher induction of CgTLR-triggered activation of NF-κB. Transfected cells were then treated with 13 PAMPs, respectively. The groups without PAMP treatment were taken as blank. Luciferase activities were tested at 12 h post treatment. Each column and error bar represents the mean±S.D. (n = 5).
Figure 4.
Subcellular localization of CgTLRs in HEK293 cells.
Empty plasmid pEGFP-N1was used as control. Red, green and blue colors show localization of Rab7-Red, the GFP tag or GFP-tagged CgTLRs and nucleus, respectively. Rab7-Red was a marker for late endosome. The four CgTLRs have similar intracellular localizations, majoring on late endosomes and minoring on plasma membranes.
Figure 5.
The NF-κB activity triggered by CgTLR-CgMyD88 linked pathway.
(A) CgMyD88 induced NF-κB activation is CgTLR1-dependent. HEK293 cells were transiently transfected with combination of 100 ng CgTLR1 and 100 ng CgMyD88 expression vectors with 20 ng NF-κB-responsive reporter vector and 20 ng pRL-TK vector per well in 24 well plates. The triangle increasing symbol indicated that dose of CgTLR1 vector is transfected from 100 ng to 500 ng per well. (B) CgMyD88-TIR blocked the CgTLR1-triggered activation of NF-κB. HEK293 cells were transiently transfected with combination of 100 ng CgTLR1 and 100 ng deletion mutant CgMyD88-TIR expression vectors with 20 ng NF-κB-responsive reporter vector and 20 ng pRL-TK vector per well in 24 well plates. The triangle increasing symbol indicated that dose of CgMyD88-TIR vector is transfected from 100 to 500 ng per well. (C) CgMyD88-TIR inhibited CgTLR2-, CgTLR3-, CgTLR4-dependent NF-κB activation. HEK293 cells were transiently transfected with 100 ng of each CgTLR2, CgTLR3 and CgTLR4 expression vectors with 200 ng CgMyD88-TIR expression vector. Luciferase activities were tested at 24 h post transfection. Each column and error bar represents the mean±S.D. (n = 5).
Figure 6.
The mRNA expression patterns of CgTLRs and CgMyD88 in response to pathogenic ligands in hemocytes of C. gigas (n = 3).
The mRNA expression was determined using real-time PCR in primary cultured hemocyte cells challenged with HKVP, HKLM and eight PAMPs post 3, 6, 12, and 24 h. All of the expression levels were normalized with geometric averaging of three reference genes EF1α, rpl, and GAPDH. The color code indicates the fold change of TLR mRNA after logarithm transformation, and the dendrograms were construed based on algorithm of hierarchical clustering.
Figure 7.
Blocking peptide targeted BB-loop of CgMyd88 inhibited TLR signaling.
(A) Sequences of conserved motif of BB-loop from human and oyster. (B) 3D structure shows the location of a BB-loop on a TIR domain. (C) Inhibitory effect of blocking peptide on NF-κB activation. HEK293 cells were transiently co-transfected with 100ng CgTLR, 100 ng CgMyd88, 20 ng NF-κB-responsive reporter vector and 20 ng pRL-TK vector per well in 24 well plates under treatment of either CP or BP in a concentration of 10, 20 and 50 µM, respectively. Each column and error bar represents the mean±S.D. (n = 5). A start indicates p<0.05. CP: control peptide, BP: blocking peptide.
Figure 8.
Blocking peptide (BP) inhibits degranulation caused by infection.
(A) The granulocytes population in hemocytes under bacterial infection in presence of BP or CP. The hemocyte cells were plotted in a side scatter (SSC, Y-axis) versus forward scatter (FSC, X-axis) dot plot, and the different populations were analyzed and quantified using CellQuest. R1: granulocytes, R2: intermediate cells and hylinocytes. (B) The percentage of granulocytes population in hemocytes under treatments. Each column and error bar represents the mean±S.D. (n = 7).
Figure 9.
The mRNA expression levels of CgTNF1 (A) and CgTNF2 (B) in TLR signaling trigged by V. parahaemolyticus infection and controlled by blocking peptide.
The mRNA expression was investigated by real-time PCR in primary cultured hemocyte cells infected with V. parahaemolyticus under treatment of either CP or BP. Each column and error bar represents the mean±S.D. (n = 7). Different letters indicate significant differences in expression between each treatment (p < 0.05). Con: control group, infe: infected group.