Dai F et al. (2022), Vibrio splendidus flagellin C binds tropomoduli...

ECB-ART-50296

J Biol Chem 2022 Jul 01;2987:102091. doi: 10.1016/j.jbc.2022.102091.

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Vibrio splendidus flagellin C binds tropomodulin to induce p38 MAPK-mediated p53-dependent coelomocyte apoptosis in Echinodermata.

Dai F , Guo M , Shao Y , Li C .

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As a typical pathogen-associated molecular pattern, bacterial flagellin can bind Toll-like receptor 5 and the intracellular NAIP5 receptor component of the NLRC4 inflammasome to induce immune responses in mammals. However, these flagellin receptors are generally poorly understood in lower animal species. In this study, we found that the isolated flagellum of Vibrio splendidus AJ01 destroyed the integrity of the tissue structure of coelomocytes and promoted apoptosis in the sea cucumber Apostichopus japonicus. To further investigate the molecular mechanism, the novel intracellular LRR domain-containing protein tropomodulin (AjTmod) was identified as a protein that interacts with flagellin C (FliC) with a dissociation constant (Kd) of 0.0086 ± 0.33 μM by microscale thermophoresis assay. We show that knockdown of AjTmod also depressed FliC-induced apoptosis of coelomocytes. Further functional analysis with different inhibitor treatments revealed that the interaction between AjTmod and FliC could specifically activate p38 MAPK, but not JNK or ERK MAP kinases. We demonstrate that the transcription factor p38 is then translocated into the nucleus, where it mediates the expression of p53 to induce coelomocyte apoptosis. Our findings provide the first evidence that intracellular AjTmod serves as a novel receptor of FliC and mediates p53-dependent coelomocyte apoptosis by activating the p38 MAPK signaling pathway in Echinodermata.

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	Figure 1. Morphological observations of the Vibrio splendidus AJ01 flagellum.A, transmission electron micrograph of AJ01. The scale bars represent 2 μm. B, fluorescence staining of AJ01. DAPI was used to stain the nucleus, and green fluorescence was used to stain the flagellum. The images were taken under 40× magnification. The scale bars represent 10 μm.
	Figure 2. Immune function of the AJ01 flagellum in Apostichopus japonicus.A, tissue structure changes in sea cucumbers at 24 h after 100 μg flagellum treatment. The first column presents healthy sea cucumbers without any treatment that served as the blank control. The second column presents sea cucumbers treated with 100 μg flagellum for 24 h. The third column presents sea cucumbers treated with 100 μg BSA for 24 h. The symptoms of skin ulceration of sea cucumbers are indicated with red rectangles. Red arrows indicate obvious tissue damage. The scale bars represent 200 μm. B, flow cytometry analysis of the changes in coelomocyte apoptosis at 24 h after AJ01 (107 CFU/ml) and flagellum treatment (100 μg). C, statistical analysis of the change in coelomocyte apoptosis from (B) (n = 3). ∗∗p < 0.01, ∗∗∗p < 0.001. BSA, bovine serum albumin.
	Figure 3. FliC specifically binds AjTmod.A, differential bands were obtained by HisFliC pull-down and identified as AjTmod by mass spectrometry. M: protein marker; Lane 1, purified rFliC; Lane 2, sea cucumber coelomocyte lysates; Lane 3, rFliC and binding protein eluted by Ni-NTA elution buffer. B, pull-down assays of the interaction between purified GSTFliC and HisTmod. SDS–PAGE and Western blotting assays to detect GSTFliC and HisTmod interactions are shown. Upper Panel M: Protein marker; Lane 1, purified rGST; Lane 2, purified GSTFliC; Lane 3, purified HisTmod; Lane 4, rGST pulled HisTmod sample after six washes; Lane 5, rGST pulled HisTmod elution sample; Lane 6, GSTFliC pulled HisTmod sample after six washes; Lane 7, GSTFliC pulled HisTmod elution sample. Lower Panel M: Protein marker; Lane 1, purified HisTmod; Lane 2, purified rGST; Lane 3, purified GSTFliC; Lane 4, HisTmod-pulled rGST sample after six washes; Lane 5, HisTmod-pulled rGST elution sample; Lane 6, HisTmod-pulled GSTFliC sample after six washes; Lane 7, HisTmod-pulled GSTFliC elution sample. The first panel in each row presents the SDS–PAGE results. The second panel in each row presents Western blotting analysis of the GST tag signal performed using a GST-labeled mouse monoclonal antibody. The third panel in each row presents Western blotting analysis of the His tag signal performed using a His-labeled mouse monoclonal antibody. C, immunofluorescence of AjTmod and FliC colocalization. Upper panels present sea cucumber primary coelomocytes infected with AJ01 (107 CFU/ml), and lower panels present primary coelomocytes infected with flagellum (20 μg) using FliC-labeled mouse polyclonal antibody and AjTmod-labeled rabbit polyclonal antibody by laser confocal technology. Green and red fluorescence represent the expression of FliC and AjTmod, respectively. The second panel in each row presents the nuclei stained by DAPI. The fourth panel in each row presents the image of the front three panels with digital overlays to visualize the colocalization. The images were taken under 4× lenses. The scale bars represent 5 μm. D, binding curves and dissociation constant (Kd) of AjTmod with GSTFliC or GST using microscale thermophoresis (MST). The results are expressed as the mean ± SD derived from three independent repeats (n = 3). E, confirmation of the FliC–AjTmod interaction by far-western assay. Upper panel: Lane 1, 5 μg of HisTomd; Lane 2, 10 μg of HisTomd; Lane 3, 20 μg of HisTomd; Lane 4, 20 μg of rHis tag. Lower panel: Lane 1, 5 μg GSTFliC; Lane 2, 10 μg GSTFliC; Lane 3, 20 μg GSTFliC; Lane 4, 20 μg rGST tag. All samples were run on an SDS–PAGE gel and transferred onto a PVDF membrane. The proteins were renatured on the membrane by adding a series of concentrations of guanidine-HCl. rGSTFliC/rHisTmod was added to recognize rHisTmod/rGSTFliC. Attached proteins were detected by GST- and His-labeled antibodies. F, recognition of GSTFliC by HisTmod. GSTFliC (10 μg, 1 μg, 0.1 μg, and 0.01 μg per well) was coated in 96-well plates. After the wells were blocked with 5% BSA, 600 ng of HisTmod was added to the well to bind for 3 h at 28 °C. The bound proteins were detected using conventional ELISA with a His-labeled antibody. G, recognition of HisTmod by GSTFliC. HisTmod (10 μg, 1 μg, 0.1 μg, and 0.01 μg per well) was coated in 96-well plates. After the wells were blocked with 5% BSA, 600 ng of GSTFliC was added to the wells to bind for 3 h at 28 °C. The bound proteins were detected using conventional ELISA with a GST-labeled antibody. The results were expressed as the mean ± SD derived from three independent repeats. BSA, bovine serum albumin; FliC, flagellin C; rFliC, recombinant FliC.
	Figure 4. AjTmod interacts with FliC through its LRR domain.A, the corresponding region of Flic and different recombinant plasmids of AjTmod. B, immunoprecipitation assay of AjTmod and FliC. HeLa cells were cotransfected with the indicated EGFP-FliC and different recombinant plasmids of AjTmod and cultured for 24 h. The cells were then lysed in lysis buffer and immunoprecipitated with protein A + G for 4 h. GFP- and Flag-labeled antibodies were used for immunoblotting analysis. FliC, flagellin C.
	Figure 5. AjTmod mediates AJ01 flagellum-induced coelomocyte apoptosis.A, qRT–PCR analysis of AjTmod mRNA expression profiles in coelomocytes post siTmod transfection (n = 3). B–G, flow cytometry analysis of the changes in coelomocyte apoptosis after siNC transfection (20 μM, 12 h), siTmod transfection (20 μM, 12 h), flagellum treatment (100 μg, 12 h), rTmod recovery after siTmod (100 μg, 12 h), flagellum treatment+siTmod, and rTmod recovery+flagellum treatment. H, apoptosis rate of coelomocytes detected under different conditions from (B–G). Data are the means of three independent experiments and are presented as the means ± SD. siTmod, siRNA targeting AjTmod. ∗p < 0.05, ∗∗p < 0.01.
	Figure 6. FliC/AjTmod-mediated coelomocyte apoptosis is dependent on the p38-MAPK pathway.A, Western blot analysis of the specificity of p38, JNK, ERK, p53, and Tmod antibodies. B, protein abundances and phosphorylation levels of p38, JNK, ERK, and p53 at 3 h, 12 h, and 24 h after AJ01 infection (107 CFU/ml) and flagellum stimulation (100 μg). p38 protein and phosphorylation expression in coelomocytes was determined using p38-labeled mouse polyclonal antibody and phospho-p38 (Thr180/Tyr182) polyclonal antibody. JNK protein expression and phosphorylation in coelomocytes was determined using a JNK-labeled rabbit monoclonal antibody and phospho-JNK (Thr183/Tyr185) monoclonal antibody. ERK protein expression and phosphorylation in coelomocytes was determined using ERK1/2-labeled rabbit monoclonal antibody and phospho-ERK1/2 (Thr202/Tyr204) monoclonal antibody. p53 protein expression and phosphorylation in coelomocytes was determined using p53-labeled mouse monoclonal antibody and phospho-p53 (Ser15) polyclonal antibody, and β-Tubulin-labeled rabbit antibody served as the control. C, band density was quantified using ImageJ, and phosphorylation and protein levels of p38, JNK, ERK, and p53 were quantified and normalized to β-Tubulin. Data (means ± SD) are representative of at least three experiments. Asterisks indicate significant differences (∗p< 0.05, ∗∗p< 0.01, ∗∗∗p< 0.001). D–F, screening of the MAPK pathways in response to flagella activation. The coelomocytes were treated with VX-702 (p38 inhibitor, 20 nM), SP600125 (JNK inhibitor, 90 nM), and FR180204 (ERK inhibitor, 0.2 μM), and then 2 μl of flagellum (2.5 μg/μl) was added to each well with inhibitor-treated coelomocytes. The first column of the upper panels presents coelomocytes treated with the same volume of PBS for 12 h as the control. The second column of the upper panels presents coelomocytes treated with inhibitors for 12 h. The third column of the upper panels presents coelomocytes treated with 5 μg flagellum for 12 h after treatment with inhibitors. The antibodies described above were used to detect the protein expression and phosphorylated of p38, JNK, ERK, and p53. Band density was quantified using ImageJ in the lower panels as described above. G–L, the apoptosis rate of cells treated with different inhibitors was detected as previously described. Data are the means of three independent experiments and presented as the means ± SD. FliC, flagellin C.
	Figure 7. FliC/AjTmod-p38-MAPK-induced coelomocyte apoptosis requires p53 activation.A, Western blotting analysis of p38 and p53 protein expression and phosphorylation profiles in coelomocytes post siNC transfection (20 μM; 3 h, 12 h, and 24 h), siTmod transfection (20 μM; 3 h, 12 h, and 24 h) and flagellum treatment after 12 h of siTmod transfection (100 μg; 3 h, 12 h, and 24 h). B, band density of (A) was quantified using ImageJ. C, Western blotting analysis of p38 and p53 protein and phosphorylation expression profiles in coelomocytes post siNC transfection (20 μM; 12 h), siTmod transfection (20 μM; 12 h), siTmod+His tag recovery (100 μg; 12 h), siTmod+His tag recovery+flagellum treatment (100 μg; 12 h), siTmod+HisTmod recovery (100 μg; 12 h), and siTmod+HisTmod recovery+flagellum treatment (100 μg; 12 h). D, band density of (C) was quantified using ImageJ. E, nuclear protein level of p38 after 3 h, 12 h, and 24 h in the 100 μg flagellum treatment. After flagellum treatment, the collected coelomocytes were used to extract nuclear proteins, and the protein level of p38 was detected by Western blotting analysis. F, nuclear protein level of p38 at 3 h, 12 h, and 24 h in the 100 μg flagellum treatment at 12 h after siTmod transfection. Under the above conditions, the collected coelomocytes were used to extract nuclear proteins, and the protein level of p38 was detected by Western blotting analysis. β-Tubulin antibody was used as a cytosolic marker. Histone-H3 was used as a nuclear marker. G, the optimal concentration of the p53 inhibitor pifithrin-μ was detected by the MTT Cell Proliferation and Cytotoxicity Assay Kit. Sea cucumber coelomocytes were cultured in 96-well plates at more than 5000 cells per well, and different concentrations of pifithrin-μ were added. After the addition of 10 μM MTT (28 °C for 4 h) and 100 μM formazan solution (37 °C for 4 h), the absorbance value was measured at 570 nm. H and I, coelomocyte apoptosis detection after flagellum treatment (Fla, 5 μg, 12 h), pifithrin-μ treatment (Pif, 1 μM, 12 h), and flagellum treatment after pifithrin-μ treatment (Pif+Fla). The X and Y axes represent PI and Annexin V, respectively. The cells in the red box indicate a portion of all examined cells. Data are the means of three independent experiments and are presented as the means ± SD. FliC, flagellin C; siTmod, siRNA targeting AjTmod. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.
	Figure 8. Schematic of the role of FliC in promoting coelonocyte apoptosis by binding AjTmod. Following AJ01 infection, Tmod bound to the internalized AJ01 flagellum component FliC based on recognition through the LRR domain and then promoted p38 phosphorylation. Phosphorylated p38 entered the nucleus to activate p53 expression, which directly modulated coelomocyte apoptosis. FliC, flagellin C.

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