TRIM27 elicits protective immunity against tuberculosis by activating TFEB-mediated autophagy flux

Abstract

Infectious diseases, such as Mycobacterium tuberculosis (Mtb)-caused tuberculosis (TB), remain a global threat exacerbated by increasing drug resistance. Host-directed therapy (HDT) is a promising strategy for infection treatment through targeting host immunity. However, the limited understanding of the function and regulatory mechanism of host factors involved in immune defense against infections has impeded HDT development. Here, we identify the ubiquitin ligase (E3) TRIM27 (tripartite motif-containing 27) as a host protective factor against Mtb by enhancing host macroautophagy/autophagy flux in an E3 ligase activity-independent manner. Mechanistically, upon Mtb infection, nuclear-localized TRIM27 increases and functions as a transcription activator of TFEB (transcription factor EB). Specifically, TRIM27 binds to the TFEB promoter and the TFEB transcription factor CREB1 (cAMP responsive element binding protein 1), thus enhancing CREB1-TFEB promoter binding affinity and promoting CREB1 transcription activity toward TFEB, eventually inducing autophagy-related gene expression as well as autophagy flux activation to clear the pathogen. Furthermore, TFEB activator 1 can rescue TRIM27 deficiency-caused decreased autophagy-related gene transcription and attenuated autophagy flux, and accordingly suppressed the intracellular survival of Mtb in cell and mouse models. Taken together, our data reveal that TRIM27 is a host defense factor against Mtb, and the TRIM27-CREB1-TFEB axis is a potential HDT-based TB target that can enhance host autophagy flux.Abbreviations: ATG5: autophagy related 5; BMDMs: bone marrow-derived macrophages; CFU: colony-forming unit; ChIP-seq: chromatin immunoprecipitation followed by sequencing; CREB1: cAMP responsive element binding protein 1; CTSB: cathepsin B; E3: ubiquitin ligase; EMSA: electrophoretic mobility shift assay; HC: healthy control; HDT: host-directed therapy; LAMP: lysosomal associated membrane protein; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MCOLN1: mucolipin TPR cation channel 1; Mtb: Mycobacterium tuberculosis; NLS: nuclear localization signal; PBMCs: peripheral blood mononuclear cells; PRKA/PKA: protein kinase cAMP-activated; qRT-PCR: quantitative real-time PCR; RFP: RET finger protein; TB: tuberculosis; TBK1: TANK binding kinase 1; TFEB: transcription factor EB; TRIM: tripartite motif; TSS: transcription start site; ULK1: unc-51 like autophagy activating kinase 1.

Keywords: Autophagy flux; Mycobacterium tuberculosis; cAMP responsive element binding protein 1; transcription factor EB; tripartite motif-containing 27; tuberculosis.

Figure 1.

TRIM27 is a potential host protective factor against mtb infection. (A) expression profiling of the C-IV TRIM family proteins in PBMCs from healthy control (HC) (n = 8) and TB patients (n = 8). (B) scatter map showing the differential expression of C-IV TRIM family genes in PBMCs from HC (n = 8) and TB (n = 8). (C) the expression of TRIM27 in peripheral blood immune cells of HC (n = 3) and TB (n = 3) groups as determined by fluorescence-activated cell sorting (FACS). MFI, mean fluorescence intensity. (D) quantitative real-time PCR (qRT-PCR) analysis of Trim27 mRNA in Trim27^+/+ and trim27^-/- BMDMs. Cells were infected with mtb at a MOI of 5 for 0–12 h, and results are presented relative to those of the control gene Gapdh. (E) survival of mtb in Trim27^+/+ and trim27^-/- BMDMs. Cells were infected as in (D) for 0–24 h. Data are mean ± SEM (n = 5 replicates per group in D and n = 3 replicates per group in E). Statistical significance was determined using two-way ANOVA with Sidak’s multiple comparisons test for (A, C and E) and two-way ANOVA with Tukey’s post-hoc test for (D). Results are representative of three independent experiments.

Figure 2.

TRIM27 nuclear localization increases upon mtb infection. (A) confocal microscopy analysis of the nuclear localization of TRIM27. Left, Representative confocal images of the colocalization of TRIM27 and the nucleus in HC PBMCs. Cells were uninfected or infected with Alexa Fluor^TM 488-labeled mtb (green) at a MOI of 5 for 8 h, and were then fixed and stained with the antibody against TRIM27 (red). Nuclei were stained with DAPI (blue). Scale bars: 10 µm. Right, average fluorescence intensity of TRIM27 in the cytoplasm and nucleus from HC PBMCs. About 50 cells were counted and analyzed for each biological replicate. (B) immunoblotting of total TRIM27 and TUBA1A (loading control) in Trim27^+/+ and trim27^-/- BMDMs. Cells were infected with mtb strains at a MOI of 5 for 0 − 8 h. (C) immunoblotting of TRIM27, TUBA1A (loading control for the cytoplasm) and PARP (loading control for the nucleus) in the cytoplasm and the nucleus from Trim27^+/+ BMDMs (left). Cells were infected as in (B). Middle, the relative abundance of TRIM27 in the cytoplasm and nucleus from Trim27^+/+ BMDMs. Right, the ratio of nuclear:cytoplasmic TRIM27. Densitometry of triplicate samples was performed for quantification. The relative abundance of TRIM27 in the cytoplasm and nucleus was calculated as the ratios of TRIM27:TUBA1A and the ratios of TRIM27:PARP, respectively. (D) genomic distribution of potential TRIM27-binding regions in HC PBMCs. Cells were infected as in (B). (E) biotype distribution of potential TRIM27-binding regions in HC PBMCs. Cells were infected as in (B). (F) the total 16 candidate protein-coding genes (fold-enrichment >10 and FDR < 0.05) whose promoter region is regulated by TRIM27 in mtb-infected PBMCs. Data are mean ± SEM (n = 5 replicates per group in A and n = 3 replicates per group in C). Statistical significance was determined using two-way ANOVA with Tukey’s post-hoc test for (A), two-way ANOVA with Sidak’s multiple comparisons test for (C, middle) and unpaired two-sided Student’s t-test for (C, right). Results are representative of three independent experiments.

Figure 3.

Nuclear TRIM27 promotes TFEB gene transcription. (A) qRT-PCR analysis of the potential genes targeted by TRIM27. The mRNA levels of potential TRIM27-targeted genes were determined in HC PBMCs infected with mtb at a MOI of 5 for 0 or 8 h. The copy numbers of mRNA were calculated based on the GAPDH standard curve. TEX30: testis expressed 30; NCR1: natural cytotoxicity triggering receptor 1; ACOT11: acyl-CoA thioesterase 11; ECSCR: endothelial cell surface expressed chemotaxis and apoptosis regulator; ZNF561: zinc finger protein 561; SLC25A16: solute carrier family 25 member 16; DBNL: drebrin like; EIF4G3: eukaryotic translation initiation factor 4 gamma 3; DPF2: double PHD fingers 2. (B) qRT-PCR analysis of tfeb mRNA in Trim27^+/+ and trim27^-/-BMDMs. Cells were infected with mtb at a MOI of 5 for 0–12 h, and results are presented relative to those of the control gene Gapdh. (C) qRT-PCR analysis of TRIM27 mRNA (left) and TFEB mRNA (right) in PBMCs from HC (n = 5) and TB (n = 5) individuals. Results are presented relative to those of the control gene GAPDH. (D) ChIP-qPCR assay for TRIM27 enrichment in the promoters of potential target gene TFEB. ChIP assay was conducted using anti-TRIM27 rabbit antibody followed by qRT-PCR in PBMCs uninfected or infected with mtb for 8 h. Enrichment was calculated relative to normal rabbit IgG control. (E) luciferase reporter assay of the transcriptional regulation activity of TRIM27 on its potential target gene TFEB in HEK293T cells. TFEB promoter-pGL2-basic vector was co-transfected into HEK293T cells with 1 μg of empty vector or vectors encoding full-length TRIM27. Resultant luciferase activities were expressed as relative luciferase activities normalized to the pRL-TK activity. Data are mean ± SEM (n = 3 replicates per group in A and E, n = 4 replicates per group in B and D). Statistical significance was determined using two-way ANOVA with Sidak’s multiple comparisons test for (A), two-way ANOVA with Tukey’s post-hoc test for (B and D) and unpaired two-sided Student’s t-test for (C and E). Results are representative of three independent experiments.

Figure 4.

Nuclear TRIM27 promotes TFEB-mediated activation of autophagy. (A−G) qRT-PCR analysis of Map1lc3 mRNA (A), Sqstm1 mRNA (B), ctsb mRNA (C), Atg5 mRNA (D), Mcoln1 mRNA (E), Lamp1 mRNA (F) and Lamp2 mRNA (G) in Trim27^+/+ and trim27^-/- BMDMs. Cells were infected with mtb at a MOI of 5 for 0 or 8 h, and results are presented relative to those of the control gene Gapdh. (H) immunoblotting of MAP1LC3, TRIM27 and ACTB (loading control) in Trim27^+/+ and trim27^-/- BMDMs. Cells were infected with mtb at a MOI of 5 for 0 − 8 h in the presence of DMSO or bafilomycin A1 (baf A1), and were then lysed and analyzed by immunoblotting (left). Right, the ratios of MAP1LC3-II:ACTB densitometry of quintuplicate samples was performed for quantification. (I) confocal microscopy analysis of autophagy in Trim27^+/+ and trim27^-/- BMDMs. Left, Representative confocal images of fixed Trim27^+/+ and trim27^-/- BMDMs. Cells were infected with mcherry-GFP-MAP1LC3-lentivirus for 24 h, and were then infected with Alexa Fluor^TM 405-labeled mtb (blue) for 8 h. Uninfected cells were prepared as the negative control. Scale bars: 10 μm. Middle and right, the numbers of total MAP1LC3 puncta (middle) and the ratio of yellow:total MAP1LC3 puncta (right) from 50 cells for each biological replicate. Data are mean ± SEM (n = 3 replicates per group in A−G and I, n = 5 replicates per group in H). Statistical significance was determined using two-way ANOVA with Tukey’s post-hoc test for (A−I). Results are representative of three independent experiments.

Figure 5.

TRIM27 promotes autophagy activation depending on its nuclear localization. (A) schematic diagram representing the distribution region of nuclear localization signals (NLS) in TRIM27. The NLS were predicted with the cNLS mapper website. (B) confocal microscopy analysis of the nuclear localization of TRIM27 or TRIM27ΔNLS. Left, Representative confocal images of the colocalization of TRIM27 or TRIM27ΔNLS with the nucleus. RAW264.7 cells were transfected with vector encoding MYC-tagged TRIM27 or MYC-tagged TRIM27ΔNLS. After 24 h, transfected RAW264.7 cells were uninfected or infected with Alexa Fluor^TM 488-labeled mtb (green) at a MOI of 5 for 8 h, and were then fixed and stained with the antibody against MYC-tag (red). Nuclei were stained with DAPI (blue). Scale bars: 5 µm. Right, average fluorescence intensity of TRIM27 or TRIM27ΔNLS in the cytoplasm and nucleus. About 50 cells were counted and analyzed for each biological replicate. (C) immunoblotting of TRIM27, TUBA1A (loading control for the cytoplasm) and PARP (loading control for the nucleus) in the cytoplasm and nucleus in TRIM27- or TRIM27ΔNLS-complementing trim27^-/- BMDMs. Left, cells were transfected with MYC-TRIM27 or MYC-TRIM27ΔNLS for 24 h, and were then uninfected or infected with mtb for 8 h, followed by immunoblotting analysis. *: nonspecific bands. Right, the relative abundance of TRIM27 or TRIM27ΔNLS in the cytoplasm and nucleus. Densitometry of quintuplicate samples was performed for quantification. The relative abundance of TRIM27 or TRIM27ΔNLS in the cytoplasm and nucleus was calculated as the ratios of TRIM27:TUBA1A or TRIM27ΔNLS:TUBA1A and the ratios of TRIM27:PARP or TRIM27ΔNLS:PARP, respectively. (D) immunoblotting of MAP1LC3, TRIM27 and ACTB (loading control) in TRIM27- or TRIM27ΔNLS-complementing trim27^-/- BMDMs. Cells were infected as in (C), followed by immunoblotting analysis (left). *: nonspecific bands. Right, the ratios of MAP1LC3-II:ACTB. Densitometry of triplicate samples was performed for quantification. Data are mean ± SEM (n = 5 replicates per group in B −D). Statistical significance was determined using two-way ANOVA with Tukey’s post-hoc test for (B −D). Results are representative of three independent experiments.

Figure 6.

Nuclear TRIM27 functions as a transcription activator to enhance TFEB gene transcription during mtb infection. (A) electrophoretic mobility shift assay (EMSA) analysis of interactions between his-tagged TRIM27 and nine promoter fragments of TFEB. The promoter fragments of TFEB (10 nM) were amplified and incubated with 10 μg of purified his-TRIM27. (B) EMSA analysis of interactions of his-tagged TRIM27 and its truncations with TFEB promoter fragment (−400 bp to the TSS) as in (A). (C) ChIP-qPCR analysis for the binding site of TRIM27 in the promoter of TFEB gene. ChIP assay was conducted using anti-TRIM27 rabbit antibody followed by qRT-PCR with specific primers for TFEB promoter fragments in PBMCs. Enrichment was calculated relative to the normal rabbit IgG control. (D) luciferase reporter assay of the transcriptional regulation activity of TRIM27 or its truncations on the potential target gene TFEB or TFEB Δ-200bp~TSS in HEK293T cells. TFEB promoter- or TFEB Δ-200bp~TSS-pGL2-basic vector was co-transfected into HEK293T cells with 1 μg of empty vector or vectors encoding full-length TRIM27 or its truncations. Resultant luciferase activities were expressed as relative luciferase activities normalized to the pRL-TK activity. (E and F) qRT-PCR analysis of Trim27 mRNA (E) and tfeb mRNA (F) in Trim27^+/+ and trim27^-/- cells stably expressing nontargeting control shRNA (shNC) or Creb1-targeting shRNA (shCreb1). Cells were infected with mtb at a MOI of 5 for 0 or 8 h, and results are presented relative to those of the control gene Gapdh. (G) immunoprecipitation (IP) of CREB1 by MYC-tagged TRIM27 or its truncations in RAW264.7 cells. Cells were transfected with empty vector or vector encoding MYC-TRIM27 for 16 h and were then infected with mtb at a MOI of 5 for additional 8 h. Cells were lysed and immunoprecipitated with the antibody against MYC. (H) luciferase reporter assay of the transcriptional regulation activity of TRIM27 and its truncations on the potential target gene TFEB in HEK293T cells as in (D). (I) DNA-binding affinity of CREB1 in the presence of MYC-tagged TRIM27 or its truncations. (J) immunoblotting of p-CREB1, total CREB1 and GAPDH (loading control) in Trim27^+/+ and trim27^-/-BMDMs. Cells were infected with mtb strains at a MOI of 5 for 0 − 8 h. (K) immunoblotting of TRIM27-mediated phosphorylation of CREB1 in vitro in the absence or presence of TRIM27. (L) IP of PRKA by CREB1 in Trim27^+/+ and trim27^-/- BMDMs. Cells were infected as in (J), and were then lysed and immunoprecipitated with the antibody against CREB1. Data are mean ± SEM (n = 3 replicates per group in C−F, H and I). Statistical significance was determined using two-way ANOVA with Sidak’s multiple comparisons test for (C), two-way ANOVA with Tukey’s post-hoc test for (D−F) and one-way ANOVA with Tukey’s post-hoc test for (H and I). Results are representative of three independent experiments.

Figure 7.

CREB1 is essential for TRIM27 to enhance TFEB-mediated autophagy flux. (A−G) qRT-PCR analysis of Map1lc3 mRNA (A), Sqstm1 mRNA (B), ctsb mRNA (C), Atg5 mRNA (D), Mcoln1 mRNA (E), Lamp1 mRNA (F) and Lamp2 mRNA (G) in Trim27^+/+ and trim27^-/- cells stably expressing shNC or shCreb1. Cells were infected with mtb at a MOI of 5 for 0 or 8 h, and results are presented relative to those of the control gene Gapdh. (H) confocal microscopy analysis of autophagy in Trim27^+/+ and trim27^-/- BMDMs with or without compound 3i treatment. Upper, Representative confocal image of fixed Trim27^+/+ and trim27^-/- BMDMs. Cells were infected with mCherry-GFP-MAP1LC3-lentivirus for 24 h, and were then infected with Alexa Fluor^TM 405-labeled mtb (blue) at a MOI of 5 for 8 h with or without compound 3i treatment. Uninfected cells were prepared as the control. Scale bars: 10 μm. Bottom, the numbers of total MAP1LC3 puncta (left) and the ratio of yellow:total MAP1LC3 puncta (right) from 50 cells for each biological replicate. (I) survival of mtb in Trim27^+/+ and trim27^-/- cells stably expressing shNC or shCreb1. Data are mean ± SEM (n = 3 replicates per group in A−I). Statistical significance was determined using two-way ANOVA with Tukey’s post-hoc test for (A−I). Results are representative of three independent experiments.

Figure 8.

TRIM27 promotes TFEB-mediated autophagy flux to enhance host anti-mtb immunity in vivo. (A and B) qRT-PCR analysis of Trim27 mRNA (A) and Tfeb mRNA (B) in the lungs of Trim27^fl/fl and trim27^Lyz2 mice infected with mtb at the indicated time points. (C and D) the MFI of MAP1LC3 (C) and ATG5 (D) in lung macrophages. The Trim27^fl/fl and trim27^Lyz2 mice were infected with mtb and treated with oil or TFEB activator 1 for 7 days, and the lungs were harvested for analyzation by FACS. (E and F) bacterial loads of mtb in the lungs (E) and spleens (F) of Trim27^fl/fl and trim27^Lyz2 mice. Mice were infected and treated as in (C). (G) H&E and acid-fast staining of lung sections of Trim27^fl/fl and trim27^Lyz2 mice. Mice were infected and treated as in (C). (H and I) quantitation of inflammatory areas (H) and average mtb counts (I) in lung sections of Trim27^fl/fl and trim27^Lyz2 mice. Mice were infected and treated as in (C). Data are mean ± SEM (n = 3 replicates per group in A−D, n = 5 replicates per group in E, F and H, n = 10 replicates per group in I). Statistical significance was determined using two-way ANOVA with Tukey’s post-hoc test for (A−F, H and I). Results are representative of one experiment with two independent biological replicates.

Figure 9.

The schematic model showing that nuclear TRIM27 enhances TFEB transcription to promote autophagy flux and Mtb clearance. Briefly, during Mtb infection, nuclear TRIM27 increases and interacts with TFEB promoter and CREB1, followed by enhancing CREB1-TFEB promoter binding affinity and promoting phosphorylation of CREB1 at Ser133 catalyzed by PRKA, thus promoting TFEB gene transcription and the ensuing autophagy-related gene expression and autophagy activation to accelerate the clearance of pathogenic bacteria.