Shao Y et al. (2022), Xenophagy of invasive bacteria is differentiall...

ECB-ART-50525

J Biol Chem 2022 Mar 01;2983:101667. doi: 10.1016/j.jbc.2022.101667.

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Xenophagy of invasive bacteria is differentially activated and modulated via a TLR-TRAF6-Beclin1 axis in echinoderms.

Shao Y , Wang Z , Chen K , Li D , Lv Z , Zhang C , Zhang W , Li C .

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In marine environments, organisms are confronted with numerous microbial challenges, although the differential regulation of xenophagy in response to different pathogenic bacterial species remains relatively unknown. Here, we addressed this issue using Apostichopus japonicus as a model. We identified 39 conserved autophagy-related genes by genome-wide screening, which provided a molecular basis for autophagy regulation in sea cucumbers. Furthermore, xenophagy of two Gram-negative bacteria, Vibrio splendidus and Escherichia coli, but not a Gram-positive bacteria, Micrococcus luteus, was observed in different autophagy assays. Surprisingly, a significantly higher autophagy capacity was found in the E. coli-challenged group than in the V. splendidus-challenged group. To confirm these findings, two different lipopolysaccharides, LPSV. splendidus and LPSE. coli, were isolated; we found that these LPS species differentially activated coelomocyte xenophagy. To explore the molecular mechanism mediating differential levels of xenophagy, we used an siRNA knockdown assay and confirmed that LPSV. splendidus-mediated xenophagy was dependent on an AjTLR3-mediated pathway, whereas LPSE. coli-mediated xenophagy was dependent on AjToll. Moreover, the activation of different AjTLRs resulted in AjTRAF6 ubiquitination and subsequent activation of K63-linked ubiquitination of AjBeclin1. Inversely, the LPSV. splendidus-induced AjTLR3 pathway simultaneously activated the expression of AjA20, which reduced the extent of K63-linked ubiquitination of AjBeclin1 and impaired the induction of autophagy; however, this finding was no t evident with LPSE. coli. Our present results provide the first evidence showing that xenophagy could be differentially induced by different bacterial species to yield differential autophagy levels in echinoderms.

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	Figure 1. Complete autophagy machinery of A. japonicus according to in silico analysis. Sea cucumbers contain the main processes of autophagy, namely autophagy initiation, phagophore elongation, autophagosome formation, and lysosome fusion, and the presence of all processes guarantees the integrity of the autophagy machinery and the accomplishment of xenophagy. The domains of 13 key proteins were detected using the SMART program and compared with those of Homo sapiens, Mus musculus, Danio rerio, and Strongylocentrotus purpuratus.
	Figure 2. Characterization of A. japonicus coelomocyte xenophagy after challenge with V. splendidus, E. coli, and M. luteus for 24 h.A, TEM detection after V. splendidus and E. coli injection following with 10 nM Baf-A1 treatment, the red arrows indicated the double-membraned autophagosomes surrounding bacteria; B, LC3 fluorescence intensity detection without CQ or Baf-A1 injection; C, LC3 fluorescence intensity detection with CQ injection; D, LC3-positive autophagosomes colocalized with lysosomes with CQ injection. E, LC3-positive autophagosomes colocalized with lysosomes with Baf-A1 injection. After challenge, the cells were fixed and stained with anti-LC3 or combined with anti-LAMP antibodies at the indicated time point of 24 h. After nuclear staining with DAPI, green and red signals that represent autophagosomes and lysosomes, respectively, were visualized under a confocal microscope and statistically analyzed; scale bar = 5 μm. The relative LC3 positivity in 1000 cells from each indicated sample was determined. The data are presented as the means ± SDs (n = 3) relative to the control (0 h), are shown in bar graphs (lower panel in D and E). Asterisks indicate significant differences compared with the control group: ∗p < 0.05 and ∗∗p < 0.01 (t test).
	Figure 3. Characterization of A. japonicus coelomocyte xenophagy after challenge with M. luteus, V. splendidus, and E. coli by Western blotting analysis. Sea cucumbers were injected with or without 10 nM Baf-A1 following M. luteus (A), V. splendidus (B), and E. coli (C) challenge for 12 and 24 h, respectively. The protein band density was calculated using ImageJ software. The data, which are presented as the means ± SDs (n = 3) relative to the 0 h (control), are shown in bar graphs (right panel in D and E). Asterisks indicate significant differences compared with the control group: ∗p < 0.05 and ∗∗p < 0.01 (t test).
	Figure 4. Characterization of A. japonicus primary coelomocyte xenophagy after LPSV. splendidus, LPSE. coliand PGNM. luteusexposure. Primary cultured coelomocyte were exposed to LPSV. splendidus, LPSE. coli, and PGNM. luteus at a final concentration of 10 μg ml−1, and the coelomocytes were collected at 0 h (control), 6, and 12 h following with CQ or Baf-A1 treatments for subsequent autophagy assays. A, TEM detection after LPSV. splendidus and LPSE. coli exposure following with 2 nM Baf-A1 treatment, the red arrows indicated autophagosomes; B, Western blotting analysis after LPSE. coli and LPSV. splendidus exposure following with 2 nM Baf-A1 treatment; C, autophagosome (LC3) and lysosome (LAMP) colocalization detection with CQ treatment. D, autophagosome (LC3) and lysosome (LAMP) colocalization detection with Baf-A1 treatment. The protein band density was calculated using ImageJ software. The data, which are presented as the means ± SDs (n = 3) relative to the 0 h, are shown in bar graphs (right panel in B). For LC3-positive autophagosomes colocalized with lysosomes, the cells were fixed and stained with anti-LC3 in combination with anti-LAMP antibodies at the indicated time points (0 and 12 h). After nuclear staining with DAPI, green and red signals that represent autophagosomes and lysosomes, respectively, were visualized under a confocal microscope and statistically analyzed; scale bar = 5 μm. The relative LC3 positivity in 1000 cells from each indicated sample was determined. The data are presented as the means ± SDs (n = 3) relative to the control, are shown in bar graphs (lower panel in C and D). Asterisks indicate significant differences compared with the control group: ∗p < 0.05 and ∗∗p < 0.01 (t test).
	Figure 5. The AjLC3-I-to-AjLC3-II conversion and protein level of Ajp62 in coelomocyte were detected after 0, 6, and 12 h of exposure to LPSV. splendidusand LPSE. colifollowing the mRNA silencing of AjTLR3, AjToll, AjSR-B, AjNLRC-4, and AjULK.A, Western blotting analysis of LPSV. splendidus-challenged group following mRNA silencing of AjTLR3, AjToll, AjSR-B, AjNLRC-4, and AjULK; B, Western blotting analysis of LPSE. coli-challenged group following mRNA silencing of AjTLR3, AjToll, AjSR-B, AjNLRC-4, and AjULK. The NC group was transfected with nontargeted double-stranded siRNA and served as the control. The protein band density was calculated using ImageJ software. The data, which are presented as the means ± SDs (n = 3) relative to the 0 h (control) are shown in bar graphs (lower panel in A and B). The asterisks indicate significant differences compared with the control group: ∗p < 0.05 and ∗∗p < 0.01 (t test).
	Figure 6. LPSV. splendidusand LPSE. colimediate the K63-linked ubiquitination of AjTRAF6 based on AjTLR3 and AjToll cascades, which interacts with AjBeclin1 to regulate xenophagy in A. japonicus coelomocyte. Sea cucumber primary coelomocyte were stimulated with LPSV. splendidus (A) or LPSE. coli (B) for the indicated times (0 and 12 h) following AjTLR3 (C), AjToll (D), and AjTRAF6 (E and F) silencing for 24 h. G, LC3 fluorescence intensity detection after interference of AjTRAF6 followed by LPSV. splendidus or LPSE. coli exposure for 12 h. H, LC3 fluorescence intensity detection after interference of AjULK followed by LPSV. splendidus or LPSE. coli exposure for 12 h. Western blotting analysis of immunoprecipitated (IP) AjTRAF6 samples was performed to determine the presence of K63-linked ubiquitin (Ub K63). The membranes in (A) and (B) were stripped and analyzed for interaction with AjBeclin-1. Whole-cell lysates were analyzed by western blotting as indicated. The protein band density was calculated using ImageJ software. The heavy chain of the TRAF6 antibody was detected by the secondary antibody and was labeled Ig band. The data, which are presented as the means ± SDs (n = 3) relative to the 0 h (control) in Fig. S2. For LC3 positive signal, the cells were fixed and stained with anti-LC3 at the indicated time points (0 and 12 h). After nuclear staining with DAPI, green signal represents autophagosomes was visualized under a confocal microscope and statistically analyzed; scale bar = 5 μm. The relative LC3 positivity in 1000 cells from each indicated sample was determined. The data are presented as the means ± SDs (n = 3) relative to the control, are shown in bar graphs (lower panel in G and H). The asterisks indicate significant differences compared with the control group: ∗p < 0.05 and ∗∗p < 0.01 (t test).
	Figure 7. LPSV. splendidusand LPSE. colidifferentially induce A. japonicus coelomocyte xenophagy by AjA20. Sea cucumber primary coelomocyte were stimulated with LPSV. splendidus (A) or LPSE. coli (B) for the indicated times (0 and 12 h) following AjTLR3 (C), AjToll (D), and AjTRAF6 (E and F) silencing for 24 h. Western blotting analysis of immunoprecipitated (IP) AjBeclin1 samples was performed to determine the presence of K63-linked ubiquitin (Ub K63). Whole-cell lysates were analyzed by western blotting as indicated. The protein band density was calculated using ImageJ software. The heavy chain of the Beclin1 antibody was detected by the secondary antibody and was labeled Ig band. The data are presented as the means ± SDs (n = 3) relative to the 0 h (control) in Fig. S3. The V indicate significant differences compared with the control group: ∗p < 0.05 and ∗∗p < 0.01 (t test).
	Figure 8. Differential TLR-mediated xenophagy in A. japonicus after V. splendidus and E. coli infection. The exposure of coelomocyte to LPSV. splendidus and LPSE. coli were mediated by AjTLR3 and AjToll, respectively. Activated AjTLR3 and AjToll both promoted AjTRAF6 ubiquitination, which further increased the K63-linked ubiquitination of AjBeclin1 and triggered the formation of autophagosomes. Inconsistently, the engagement of AjTLR3 also triggered a signaling pathway that led to the expression of AjA20. The increased abundance of AjA20 might limit AjTLR3-induced autophagy through the deubiquitination of AjBeclin1. The precise mechanism of A20 and deubiquitination should be verified in future studies. The yellow solid line indicates E. coli-induced autophagy based on the AjToll signaling pathway; the green solid and imaginary lines indicate V. splendidus-induced autophagy based on the AjTLR3 signaling pathway; the red solid line indicates the common xenophagy processes in both V. splendidus- and E. coli-induced autophagy.

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