Chen K et al. (2021), A unique NLRC4 receptor from echinoderms mediat...

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PLoS Pathog 2021 Dec 13;1712:e1010145. doi: 10.1371/journal.ppat.1010145.

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A unique NLRC4 receptor from echinoderms mediates Vibrio phagocytosis via rearrangement of the cytoskeleton and polymerization of F-actin.

Chen K , Zhang S , Shao Y , Guo M , Zhang W , Li C .

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Many members of the nucleotide-binding and oligomerization domain (NACHT)- and leucine-rich-repeat-containing protein (NLR) family play crucial roles in pathogen recognition and innate immune response regulation. In our previous work, a unique and Vibrio splendidus-inducible NLRC4 receptor comprising Ig and NACHT domains was identified from the sea cucumber Apostichopus japonicus, and this receptor lacked the CARD and LRR domains that are typical of common cytoplasmic NLRs. To better understand the functional role of AjNLRC4, we confirmed that AjNLRC4 was a bona fide membrane PRR with two transmembrane structures. AjNLRC4 was able to directly bind microbes and polysaccharides via its extracellular Ig domain and agglutinate a variety of microbes in a Ca2+-dependent manner. Knockdown of AjNLRC4 by RNA interference and blockade of AjNLRC4 by antibodies in coelomocytes both could significantly inhibit the phagocytic activity and elimination of V. splendidus. Conversely, overexpression of AjNLRC4 enhanced the phagocytic activity of V. splendidus, and this effect could be specifically blocked by treatment with the actin-mediated endocytosis inhibitor cytochalasin D but not other endocytosis inhibitors. Moreover, AjNLRC4-mediated phagocytic activity was dependent on the interaction between the intracellular domain of AjNLRC4 and the β-actin protein and further regulated the Arp2/3 complex to mediate the rearrangement of the cytoskeleton and the polymerization of F-actin. V. splendidus was found to be colocalized with lysosomes in coelomocytes, and the bacterial quantities were increased after injection of chloroquine, a lysosome inhibitor. Collectively, these results suggested that AjNLRC4 served as a novel membrane PRR in mediating coelomocyte phagocytosis and further clearing intracellular Vibrio through the AjNLRC4-β-actin-Arp2/3 complex-lysosome pathway.

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	Fig 1. AjNLRC4 is located in the membrane of sea cucumber coelomocytes.(A) The domain architecture of sea cucumber AjNLRC4 predicted by SMART (http://www.smart.embl-heidelberg.de/). (B) Analysis of AjNLRC4 subcellular distribution in sea cucumber coelomocytes. Coelomocytes were subjected to immunofluorescence staining with the cell membrane probe Dil. AjNLRC4 is localized to the cell membrane and precisely colocalized with Dil signals. Scale bar = 5 μm. (C) Coelomocyte binding of rAjNLRC4-EX and rAjNLRC4-IN. rAjNLRC4-EX and rAjNLRC4-IN were injected into sea cucumbers, and coelomocytes were isolated for immunohistochemistry assays to detect binding abilities with an anti-His tag antibody (green). rAjNLRC4-EX binds to the coelomocyte surface, and rAjNLRC4-IN cannot bind to the coelomocyte surface. Scale bar = 5 μm.
	Fig 2. The Ig domain of AjNLRC4 performs an immune recognition function.(A) The bacterial binding activities of rAjNLRC4-EX and rAjNLRC4-IN were analyzed by using western blotting. Different bacteria were incubated with rAjNLRC4-EX and rAjNLRC4-IN, washed with PBS four times and then analyzed by western blotting with an anti-His antibody. TBS instead of rAjNLRC4-EX and rAjNLRC4-IN was used as the negative control. (B-E) The binding activities of rAjNLRC4-EX to different polysaccharides were analyzed with ELISA. Four polysaccharides were used for ELISA analysis (n = 5), including LPS from E. coli or V. splendidus, PGN and MAN. (F) rAjNLRC4-EX agglutinates different microbes in the presence of Ca2+. FITC-labeled microbes (green) were mixed with an equal volume of rAjNLRC4-EX (1 mg/ml) in the presence or absence of 10 mM CaCl2 and incubated at room temperature for approximately 1 h. BSA instead of rAjNLRC4-EX was used as the negative control. After incubation, agglutination reactions were observed under a fluorescence microscope.
	Fig 3. V. splendidus infection can induce AjNLRC4 dimerization and internalization into the cytoplasm of coelomocytes.(A) Purified rAjNLRC4-EX was analyzed using native PAGE and stained with Coomassie blue. (B) A dimer of AjNLRC4 was detected in coelomocytes in vivo using western blotting after treatment with a crosslinker (BS3). Sea cucumbers were injected with V. splendidus for 30 min, and then, coelomocytes were collected and treated with BS3. These coelomocytes were homogenized, and the extracted proteins were separated by SDS–PAGE. Western blotting was then performed using anti-AjNLRC4 antibodies. (C) AjNLRC4 expression in the coelomocytes of sea cucumbers was detected at 0 (untreated), 6, and 12 h postinfection with V. splendidus. Scale bar = 5 μm. (D) Sea cucumbers were challenged with V. splendidus, and then, the membrane and cytoplasm proteins were extracted from the coelomocytes. AjNLRC4 expression in the membrane and cytoplasm of coelomocytes was analyzed using western blotting at 0, 6, and 12 h postinfection with V. splendidus. The lower panels show the statistical analysis of three independent experiments. ***p < 0.001.
	Fig 4. AjNLRC4 acts as a receptor of V. splendidus and mediates its endocytosis.(A) Immunocytochemistry was used to detect the colocalization of AjNLRC4 and Dil-labeled V. splendidus in coelomocytes. The coelomocytes were collected at different time points (0.5, 3, and 6 h) post-V. splendidus injection. Scale bar = 5 μm. The graphs are representative of three independent assays, and the proportions were calculated from those three assays, p < 0.05. (B-C) The efficiency of AjNLRC4-RNAi in coelomocytes was determined using qPCR and western blotting analysis. The graphs are representative of three independent assays, and the proportions were calculated from those three assays, p < 0.05, p < 0.01. (D) Flow cytometry was performed after knockdown of AjNLRC4 to investigate the phagocytic activity of coelomocytes against V. splendidus. The graphs are representative of three independent assays, and the phagocytic activity was calculated from those three independent assays, p < 0.01.
	Fig 5. V. splendidus enters coelomocytes of sea cucumber through multiple endocytic pathways.(A) The effect of chlorpromazine (CPZ) on the cell viability of sea cucumbers. Sea cucumber coelomocytes were treated with increasing concentrations of CPZ for 3 h, and cell viability was calculated. (B) Flow cytometry was performed after CPZ inhibitor treatment to investigate the phagocytic activity of V. splendidus. Sea cucumbers were treated with 10 μM CPZ for 3 h and then infected with FITC-labeled V. splendidus for 3 h. Flow cytometry was used to investigate the phagocytic activity of V. splendidus. The graphs are representative of three independent assays, and the proportions were calculated from those three assays, p < 0.01. (C) The effect of cytochalasin D on the cell viability of sea cucumbers. (D) Flow cytometry was performed after cytochalasin D inhibitor treatment to investigate the phagocytic activity of V. splendidus. The graphs are representative of three independent assays, and the proportions were calculated from those three assays, p < 0.01. (E) The effect of Mitmab on the cell viability of sea cucumbers. (F) Flow cytometry was performed after mitmab inhibitor treatment to investigate the phagocytic activity of V. splendidus. The graphs are representative of three independent assays, and the proportions were calculated from those three assays, p < 0.01. (G) The effect of IPA-3 on the cell viability of sea cucumbers. (H) Flow cytometry was performed after IPA-3 inhibitor treatment to investigate the phagocytic activity of V. splendidus. The graphs are representative of three independent assays, and the proportions were calculated from those three assays, p < 0.01. (I) The effect of nystatin on the cell viability of sea cucumbers. (J) Flow cytometry was performed after nystatin inhibitor treatment to test the phagocytic activity of V. splendidus. The graphs are representative of three independent assays, and the proportions were calculated from those three assays.
	Fig 6. Internalized AjNLRC4 promotes V. splendidus phagocytosis via the actin-mediated endocytic pathway.(A-B) The efficiency of AjNLRC4 overexpression in coelomocytes, as determined using qPCR and western blotting analysis. The graphs are representative of three independent assays, and the proportions were calculated from those three assays, p < 0.05. (C) Flow cytometry was performed to investigate the phagocytic activity of V. splendidus after the overexpression of AjNLRC4. The graphs are representative of three independent assays, and the proportions were calculated from those three assays, *p < 0.01. (D-E) After the overexpression of AjNLRC4, the inhibitors were added, and then, flow cytometry was performed to detect the phagocytic activity.
	Fig 7. The actin-mediated endocytic pathway is activated in a NACHT domain- and actin interaction-dependent manner.(A) Identification of AjNLRC4-NACHT interacting proteins from host cells. Coelomocyte homogenates (20 mL) were incubated with 20 mg rGST-AjNLRC4-NACHT or rGST tag with rotation at 4°C for 3 h. The mixture was then passed through a GST-resin column. The bound proteins were eluted and analyzed by SDS–PAGE. The bands of interest were excised and analyzed by MS/MS. (B-C) Interactions between His-tagged β-actin and GST-tagged AjNLRC4-NACHT were detected using pull-down assays. (D) Microscale thermophoresis (MST) assays of the interaction between GST-AjNLRC4-IN and His-β-Actin. The recombinant proteins were contained in NT standard capillaries. The solid curve is the fit of the data points to the standard Kd-fit function. The experiment was repeated at least three times. (E) Immunocytochemistry was used to detect the colocalization of AjNLRC4, F-actin and FITC-labeled V. splendidus in coelomocytes. Scale bar = 5 μm.
	Fig 8. AjNLRC4 mediates the endocytosis of V. splendidus by regulating the Arpc2/3 complex to mediate actin polymerization and cytoskeletal rearrangement.(A) G-actin and F-actin were extracted from the coelomocytes of healthy sea cucumbers. (B-C) The change in the ratio F-actin/G-actin after treatment with AjNLRC4-RNAi or the cytoskeletal nucleation inhibitor CK666 was detected by western blot. (D) Quantification of the number of V. splendidus cells internalized into coelomocytes. After V. splendidus was coincubated with coelomocytes, the unattached bacterial cells were washed away. The numbers of cells associated with and/or internalized into coelomocytes were counted on solid plates after the detachment of the coelomocytes with trypsin-like enzyme and the lysis of the coelomocytes with Triton X-100. The V. splendidus cells internalized into the coelomocytes were counted after killing the external V. splendidus with gentamicin, detaching the coelomocytes with trypsin-like enzyme and lysing the coelomocytes with Triton X-100. The horizontal bars represent the medians. The p values were calculated by the t test for paired samples, and p <0.05 indicated significant differences. (E) AjArpc2/3/4/5 mRNA expression levels were measured after treatment with AjNLRC4-RNAi. The graphs are representative of three independent assays, and the proportions were calculated from those three assays, p < 0.05, *p < 0.01. (F) AjArpc4/5 mRNA expression levels were measured after CK666 treatment. The graphs are representative of three independent assays, and the proportions were calculated from those three assays, p < 0.05, *p < 0.01. (G), Analysis of AjArpc4/5 expression in V. splendidus-infected sea cucumbers. The vertical bars represent the mean ± S.D. (N = 3). (H-I) The efficiency of AjArpc4-RNAi in coelomocytes was determined using qPCR and western blotting analysis. The graphs are representative of three independent assays, and the proportions were calculated from those three assays, p < 0.05. (J) western blotting revealed the change in the ratio F-actin/G-actin after treatment with AjArpc4-RNAi. The graphs are representative of three independent assays, and the proportions were calculated from those three assays, p < 0.01. (K-M) Immunocytochemistry was performed after knockdown of AjNLRC4 or AjArpc4 or treatment with CK666 (actin polymerization inhibitor) to investigate the depolymerization of actin cytoskeleton microfilaments. (N), Sea cucumbers were injected with CK666 inhibitor for 24 hours and then injected with V. splendidus. After 3 h, coelomocytes were collected from the sea cucumbers, and immunocytochemistry was performed to analyze the interactions related to the internalization of AjNLRC4 using AjNLRC4 antisera as the primary antibody. The secondary antibody was labeled with Cy3 (red). The cell nuclei were stained with DAPI (blue), and then, the cells were observed under a laser scanning confocal microscope. Scale bar = 5 μm. (O-P), Flow cytometry was performed to investigate the phagocytic activity of V. splendidus after AjArpc4-RNAi or CK666 inhibitor treatment. The graphs are representative of three independent assays, and the proportions were calculated from those three assays, p < 0.01.
	Fig 9. Endocytosed V. splendidus is further cleared via lysosome degradation.(A) Analysis of the V. splendidus clearance ability of AjNLRC4. Each dot represents a group. The horizontal bars represent the medians. (B) Colocalization of ingested V. splendidus and lysosomes. Twenty-four hours after the injection of 40 μM CLQ, FITC-labeled V. splendidus was injected into sea cucumbers. After another 3 h, coelomocytes were collected, incubated with LysoBrite Red to label the lysosomes, stained with DAPI and then observed under a laser-scanning confocal microscope. Scale bar = 5 μm. (C) Effect of chloroquine (CLQ) on the cell viability of sea cucumbers. Sea cucumbers were treated with increasing concentrations of CLQ for 24 h, and the cell viability was calculated. The graphs are representative of three independent assays, and the proportions were calculated from those three assays, p < 0.01. (D) The relative number of intracellular V. splendidus after CLQ injection was determined by internalization assay. The values are presented as the mean ± SD (n = 3). The asterisks indicate significant differences: p < 0.05.
	Fig 10. Schematic of the role of AjNLRC4 in promoting V. splendidus endocytosis as a transmembrane receptor.V. splendidus binds to the extracellular domains of AjNLRC4 and activates receptor-mediated endocytosis. AjNLRC4 oligomerizes, and the intracellular domain of AjNLRC4 interacts with Ajβ-Actin, forming a protein complex that facilitates V. splendidus internalization through the actin-mediated endocytosis pathway by Arp2/3 complex-mediated cytoskeleton polymerization and rearrangement. Finally, V. splendidus is degraded in coelomocyte phagolysosomes, which effectively restricts V. splendidus infection in sea cucumbers.

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