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Fig 1. High concentration of V. splendidus AJ01 infection induces coelomocytes lytic coelomocyte death.(A) Sea cucumbers were immersed with different concentrations of AJ01 (107, 108, and 109 CFU/mL) for 0, 3, 6, and 12 h. Sea cucumbers without any treatment served as the controls. Coelomocytes and coelomic fluid supernatants were collected from infection and control groups. 104 coelomocytes were collected from each group, and ATP levels were measured using the CellTiter-Glo kit to determine coelomocyte viability. (B) The collected coelomic fluid supernatants were analyzed for coelomocyte plasma membrane disruption using the CytoTox-Glo cytotoxicity assay kit. (C and D) The morphology of coelomocytes was analyzed using confocal microscopy. Scale bar, 5 μm (C) and FEG-SEM. Dashed lines represent cell membrane boundaries (D), Arrows indicate cells exhibiting necrotic-like features, The dotted lines represent the boundaries of the cell membrane. (E) 108 CFU/mL AJ01 infected coelomocytes for 3, 6 and 12 hours, the forward scatter (FSC) of intact cells was assessed by flow cytometry. (F) The propidium iodide and Annexin V-fluorescein isothiocyanate (FITC)-stained coelomocytes were further analyzed by flow cytometry. All data are plotted as mean ± SEM, asterisks indicate significant differences: *p < 0.05, **p < 0.01, ***p < 0.001.
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Fig 2. AjMLKL is involved in 108 CFU/mL AJ01 induced lytic coelomocyte death independent on its phosphorylation.(A) Expression patterns of AjMLKL protein and phosphorylation levels in coelomocytes after 108 CFU/ml AJ01 challenge as detected using western blotting (Lower) with β-Actin as the loading control. The red arrow represents unphosphorylated AjMLKL, and the blue arrow represents the lower band of AjMLKL. (B) The predicted structure of AjMLKL- STYKc domain adopts a closed conformation with an intact VVIK-αC salt bridge (zoomed panel). (C-D) To confirm AjMLKL involved in AJ01 infection-induced lytic coelomocytes death in sea cucumber coelomocytes, the number of intracellular AJ01 survival (C), coelomocyte viability (D), the number of coelomocytes with lytic morphology, Scale bar, 10 μm (E), the proportion of Annexin V+/PI+ lytic coelomocytes (F) and the coelomocytes plasma membrane disruption (G) were assayed in 108 CFU/mL AJ01-challenged AjMLKL silenced coelomocytes for different times. (H) To address the AjMLKL translocates to the membrane fraction during lytic coelomocytes death, The migration of AjMLKL to the cell membrane was detected at 0, 1.5, 3 and 6 h after AJ01 stimulation through immunocytochemistry. (I) A schematic representation of the phase separation of integral membrane proteins in Trion X-114 solution. (J) AjMLKL and a smaller band of AjMLKL were concentrated in the micelle-rich fraction after phase separation. coelomocytes were treated with AJ01 for the indicated time. The cells were harvested and solubilized in Triton X-114 lysis buffer then separated into aqueous phase (Aq) and detergent phase (Det) as described in the Experimental Procedures and as shown in (I). The samples were analyzed by western blotting using antibodies as indicated. β-actin was shown as loading controls. (K-O) Commercial MLKL-specific inhibitor GW806724X was further used. The 100 μM GW806724X treatment did not reduce coelomocyte viability (K). Treatment of sea cucumber coelomocytes with 100 μM GW806724X for 3 h and then challenged by 108 CFU/mL AJ01. Coelomocyte viability was significantly increased (L), and coelomocytes plasma membrane disruption (M) and the percentage of Annexin V+/PI+ coelomocytes significantly decreased (N). Intracellular AJ01 survival significantly increased in the GW806724X-treated group (O). All data are plotted as mean ± SEM, asterisks indicate significant differences: *p < 0.05, **p < 0.01, ***p < 0.001.
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Fig 3. The N-terminal domain of AjMLKL directly binds membrane lipids similar to MLKL four-helix bundle domain.(A) Model (Protein Data Bank accession code 4BTF) and schematic of mouse MLKL, showing the structure of mouse MLKL and Its 4HB domain. (B) Model of AjMLKL and schematic, AlphaFold2 to predict the structure of AjMLKL and showing the N-terminal 4HB domain. Bottom, α-helices are shown as white, right-facing arrows. Helicase and peptidase domain annotations are based on NCBI conserved domains. (C) Amino acid sequence alignment for 4HB domains generated using MUSCLE with representative sequences from vertebrates, plants and invertebrates. Helix boundaries are derived from the predicted structure of mouse MLKL. The top row shows the consensus sequence. Conserved amino acid residues are individually coloured. X and dots indicate hypervariable sites. Cladograms (light blue) display known different species relationships. (D) Membranes displaying lipids were incubated with indicated proteins and binding was assessed by blotting for AjMLKL, AjMLKL-4HB and AjMLKL-STYKc. (E) Representative agar plates showing transformed E. coli colonies for AjMLKL, AjMLKL-4HB and AjMLKL-STYKc. (F) bacterial number was counted to test bactericidal activity of AjMLKL (n=3, two-tailed t test) Values are expressed as mean ±S.E.M. (***p<0.001).
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Fig 4. AjMLKL interacts with AjCASP-1 via 4HB and CASc domain.(A) Schematic diagram to detect AjMLKL interactome by precipitation and mass spectrometry. Identification of the protein bound to AjMLKL by mass spectrometry. The protein was identified to be AjCASP-1. (B-D) To define the interaction between AjMLKL and AjCASP-1. AjCASP-1 was found to co-localize with AjMLKL by immunofluorescence (B) (Scale bar, 5 μm.) and Co-IP assays to analyze the interaction between AjMLKL with AjCASP-1 in vivo (C and D). (E-H) Different regions of AjCASP-1 and AjMLKL were generated in vitro, and their interactions were further validated by using pull-down assays.
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Fig 5. AjMLKL is specifically cleaved by the activated form of AjCASP-1(p20/p10).(A) To detect the active product of AjCASP-1, different AjCASP-1 dimers detected in AJ01 challenged coelomocytes after treatment with a cross-linker disuccinimidyl suberate (DSS). (B) A novel 25 kDa active fragment (P20) of AjCASP-1 was detected after AJ01 infection by Western blot. (C and D) AjMLKL was incubated with either different micrograms of activated form of AjCASP-1(p20/p10) for 3 h (C), or with 10 micrograms of activated form of AjCASP-1(p20/p10) for different hours (D). The samples were then coomassie blue staining and immunoblotting as above. The two AjMLKL fragments are indicated by blue and red arrows, respectively. (E) AjMLKL was incubated with different micrograms of inactivated form of AjCASP-1for 3 h. (F and G) Bands from (C) were analyzed by mass spectrometry to identify AjMLKL cleavage sites. Schematic of AjMLKL coverage by Edman sequencing identified peptides and schematic of AjCASP-1 cleavage site in AjMLKL, AjCASP-1 cleavage of AjMLKL at D17-V18 was monitored by Edman sequencing (F). The N-terminal secondary mass spectra of cleaved AjMLKL are shown in (G). (H) Cartoon diagram of mutant AjMLKL structure and the not cleavage by AjCASP-1 (upper panel) and the influence of the D17G substitution on AjMLKL on its cleaving by AjCASP-1(lower panel). (I and J) Effect of mutation of D17G on AjMLKL by pull down assay on its interaction with AjCASP-1. (K) AjMLKL was incubated with AjCASP-1 in the presence of different concentrations of Z-YVAD-FMK, and the cleavage was determined by coomassie blue staining and immunoblotting immunoblotting as above. (L) AjMLKL was incubated with AjCASP-1 in the presence of different caspase inhibitors, and the cleavage was determined by coomassie blue staining and immunoblotting immunoblotting as above.
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Fig 6. Cleaved AjMLKL18-491 breaks phosphatidylinositol phosphate- and cardiolipin-Containing Membranes to induce lytic coelomocytes death with large pore sizes.(A) To search for the molecular mechanism of cleaved AjMLKL membrane association, protein-lipid binding assay was performed with cleaved AjMLKL-4HB domain and cleaved AjMLKL. (B) AjMLKL, AjMLKL-4HB, Cleaved-AjMLKL and Cleaved-AjMLKL-4HB domain binding to PC–PE liposomes containing additional indicated phospholipids (molar proportion of added lipid indicated) was analysed by SDS–PAGE and AjMLKL immunoblot. (C-D) Time course of liposome leakage was monitored as described in the Experimental Procedures. Asterisks indicated the time points when Triton X-100 was added to achieve complete release of Tb3+. Comparison of AjMLKL, AjMLKL-4HB, Cleaved-AjMLKL and Cleaved-AjMLKL-4HB domain in liposome leakage assay. Liposomes containing cardiolipin (CL) (C) or PIP2 (D) were used as indicated. Curves in different colors represent purified recombinant AjMLKL-4HB, AjMLKL, Cleaved-AjMLKL-4HB domain and Cleaved-AjMLKL. (E and F) Uncleaved MLKL or cleaved MLKL overexpressed coelomocytes were treated with AJ01 for 3 h in PBS (CTRL) or PBS containing 30 mM PEG400, PEG1450, PEG6000 or PEG8000. Cells were stained by PI and analyzed under Flow cytometry. (G-I) Intracellular ion concentration were monitored in uncleaved MLKL (G) or cleaved MLKL (H) overexpressed coelomocytes treated with AJ01. Sodium indicator (ANG-2), Calcium indicator (Fluo-4) and Potassium indicator (APG-2) were used together with PI. Trx-His-Tag mRNA overexpressing coelomocytes were treated with AJ01 as a control (I).
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Fig 7. Cleaved AjMLKL directly kills AJ01.(A and B) AJ01 were untreated or treated with recombinant AjMLKL-4HB domain, AjMLKL, Cleaved-AjMLKL-4HB domain or Cleaved-AjMLKL18-491 (200 nM or indicated concentrations) for 30 min before samples were collected and bacterial growth was assessed by monitoring turbidity by optical density (representative experiments, left) (A). The time to reach OD600 of 0.05 above background, which is a quantitative measure of the lag in detectable growth because of fewer viable bacteria, was defined as Tthreshold (right). The right graph shows the mean ± s.d. of three technical replicates (B). (C and D) AJ01 viability assays, Bacterial viability after 20 min incubation with indicated, proteins (200 nM) or isopropanol. Syto-9 enters live and dead bacteria, PI only enters dead bacteria (representative images, left; percent live cells, right). Data shown are representative of results of three independent experiments. Statistical differences are relative to untreated samples; * P < 0.05, **P < 0.01 (two-tailed t-test). Scale bars, 5 μm.
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Fig 8. Activation of AjCASP-1 is dependent on the assembly of AjNLRC4/AjCASP-1 inflammasome.(A) Coelomocyte AjNLRC4 and AjCASP-1 were found to be colocalized in cytoplasm by immunocytochemistry analysis, and the signal intensity was enhanced after AJ01 infection. (Scale bar, 5 μm.). (B and C) The coelomocytes were harvested after infection with 108 CFU/ml AJ01 at different time points (0 and 3 h). The interaction between AjNLRC4 and AjCASP-1 was further determined by Co-IP and Western blotting. (D) Different domain recombinant proteins of AjNLRC4 and AjCASP-1 were generated in vitro. (E-K) The interaction between AjNLRC4 and AjCASP-1 were further validated by using pull-down assays. The product was isolated by SDS-PAGE and detected by Coomassie blue staining. Dotted boxes of different colors represent recombinant proteins of different domains of AjNLRC4 and AjCASP-1 (L) To further validate AjNLRC4’s role in activating AjCASP-1, coelomocyte CASP-1 activity was detected with specific Ac-YVAD-pNA fluorescent substrates after the silencing of AjCASP-1 or AjNLRC4 and challenge with 108 CFU/ml AJ01 infection for 0, 3, 6, and 12 h. (M) To further elucidate AjCASP-1 involved in AJ01-mediated lytic coelomocytes death, commercial CASP-1 specific inhibitor Z-YVAD-FMK did not affect coelomocyte viability at 25 μM. (N) CASP-1 activity was assayed by treatment with 25 μM Z-YVAD-FMK or DMSO (control) for 3 h and then challenged with 108 CFU/ml AJ01 for 3 h. (O) Coelomocyte CASP-1 activity was assayed under AjNLRC4 overexpression conditions. Consistently, the overexpression of AjNLRC4 promoted the production of P20, the addition of Z-YVAD-FMK inhibited the AjNLRC4 on AjCASP-1 self-cleavage. All data are plotted as mean ± SEM, asterisks indicate significant differences: *p < 0.05, **p < 0.01, ***p < 0.001.
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Fig 9. AjNLRC4 modulates AJ01-induced lytic coelomocyte death depending on AjCASP-1.(A-G) Sea cucumbers were transfected with specific AjNLRC4 siRNA or AjCASP-1 siRNA for 24 h. The nontargeting siRNA (NC) was transfected as the control group. Then, 108 CFU/mL AJ01 at final concentration was added to the experimental and NC groups for another 3, 6, and 12 h. The collected coelomocytes were used to determine coelomocyte viability (A), and coelomocytes plasma membrane disruption (B). (C and D) Coelomocytes from the control group (untreated sea cucumber), NC group, and experimental group (3 h) were used for annexin V and PI double staining (C) and tunnel staining assays (D), detected by fluorescence microscopy. (E and F) The proportion of Annexin V+/PI+ necrotic coelomocytes were texted by Flow Cytometry. (G) The intracellular AJ01 survival was determined by plate counts. (H-K) To further elucidate the regulation of CASP-1 activity by AjCASP-1, commercial CASP-1 specific inhibitor Z-YVAD-FMK did not affect coelomocyte viability at 25 μM. Sea cucumbers were injected with Z-YVAD-FMK inhibitor for 3 h, followed by 108 CFU/mL Aj01 infection for another 3 h to determine coelomocyte viability (H) and coelomocytes plasma membrane disruption (I). The proportion of Annexin V+/PI+ lytic coelomocytes death (J) and intracellular AJ01 survival (K) were also assayed in the AJ01 + Z-YVAD-FMK and AJ01 + DMSO groups. (L-O) To confirm whether AjCASP-1-mediate lytic coelomocytes death is associated with AjNLRC4, AjNLRC4 mRNA was overexpressed by transfection of AjNLRC4 mRNA, and then Z-YVAD-FMK was injected into the overexpression group for 3 h, followed by 108 CFU/mL AJ01 infection for another 3 h. The transfection of Trx-His tag mRNA served as the control group for AjNLRC4 overexpression group. DMSO served as the control for Z-YVAD-FMK treatment. Coelomocyte viability (L), coelomocytes plasma membrane disruption (L), and the number of intracellular AJ01 survival (N) were assayed in different groups. The percentage of annexin V+/PI+ coelomocytes were assayed by flow cytometry (M). FEG-SEM was used in observing morphological changes in coelomocytes (Q), (scale bar, 10 μm). All data are plotted as mean ± SEM, asterisks indicate significant differences: *p < 0.05, **p < 0.01, ***p < 0.001.
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Fig 10. AjNLRC4/AjCASP-1 induces cleaving of AjMLKL and mediates lytic coelomocytes death by regulating the migration of cleaved AjMLKL to the cell membrane.(A-H) To confirm whether AjMLKL-mediated lytic coelomocytes death was associated with AjNLRC4 or AjCASP-1, the mRNA of AjNLRC4 or AjCASP-1, and then GW806724X was injected into the overexpression group for 3 h, followed by 108CFU/mL AJ01 for another 3 h. Coelomocyte viability (A and B) and coelomocytes plasma membrane disruption (C and D) were assayed. The percentage of Annexin V+/PI+ coelomocytes (E and F) and intracellular AJ01 survival were determined under the same condition (G and H). (I) Interference with AjNLRC4 or AjCASP-1 and treatment with CASP-1 inhibitor Z-YVAD-FMK inhibited MLKL cleavage. (J) Interference by AjNLRC4 or AjCASP-1 or GW806724X treatment significantly inhibited the migration of AjMLKL and its cleavage products from the cytoplasm to the cell membrane, (scale bar, 5 μm). (K) Interference with AjNLRC4 or AjCASP-1 and treatment with the MLKL inhibitor GW806724X both blocked translocation of cleaved MLKL to the membrane fraction. The cells were harvested and then separated into the aqueous phase and detergent phase, performed as described in (Fig 2D). The samples were analyzed by western blotting using antibodies as indicated. All data are plotted as mean ± SEM, asterisks indicate significant differences: *p < 0.05, **p < 0.01, ***p < 0.001.
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Fig 11. A schematic diagram of atypical NLRC4-CASP-1-MLKL axis-mediated lytic coelomocytes death that is not dependent on MLKL phosphorylation.AJ01 was identified by AjNLRC4 to promote AjNLRC4 dimer formation and internalized into cytoplasm. The interlined AjNLRC4 without CARD domain interacted with the C-terminal of AjCASP-1 through its Ig domain, which also lacked the CARD domain. The interaction further promoted AjCASP-1 activation and generated the active p20/p10 form, which subsequently specifically cleaves AjMLKL at Asp17 in the 14-LESD-17 tetrapeptide. Then, the cleaved AjMLKL is transported to the membrane on the one hand by binding to lipids, forming non-selective ion channels and inducing lytic coelomocytes. On the other hand, it also directly kills AJ01.
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