Figures and data
NLSs are required for the nuclear translocation of MmpE.
(A) Expression and nuclear localization of MmpE. (upper) qRT-PCR analysis of mmpE mRNA in HEK293T cells over 48 h post-transfection. (bottom) Western blot analysis of nuclear fractions showing time-dependent nuclear accumulation of MmpE-EGFP. Histone H3 and β-actin served as nuclear and cytoplasmic markers, respectively.
(B) Domain architecture of MmpE. The schematic representation of MmpE shows its annotated functional domains, including a Tat signal peptide (1-54 amino acids, with a twin-arginine translocation motif) and two nuclear localization signals (NLS1: 20-22 amino acids; NLS2: 460-462 amino acids). The structure of the MmpE protein was predicted using AlphaFold 2.2.0, where the position of NLS1 is highlighted in red and NLS2 is highlighted in green.
(C) Subcellular localization of EGFP-tagged wild-type and NLS-deleted MmpE. (left) Schematic representation of EGFP-tagged constructs, including wild-type MmpE and its mutants (MmpEΔNLS1, MmpEΔNLS2, and MmpEΔNLS1-2). (right) Confocal microscopy images of HEK293T cells transfected with the indicated constructs for 36 hpt. EGFP fluorescence (green) and nuclear staining with DAPI (blue) were visualized using an FLUOVIEW software (v5.0). Scale bar, 10 µm. Images were acquired with a ×100 oil immersion objective (NA = 1.4).
(D) Nuclear EGFP intensity of wild-type and mutant constructs in (C). Data are shown as mean ± SD (n = 12 cells).
(E) Western blot analysis of nuclear and cytoplasmic fractions from HEK293T cells transfected with wild-type and mutant MmpE-EGFP confirmed their nuclear localization. MmpE-EGFP was detected using an anti-GFP antibody, and histone H3 and β-actin served as nuclear and cytoplasmic markers, respectively.
All experiments were performed in three biologically independent replicates. Statistical significance was assessed by two-tailed unpaired Student’s t-tests, with *P < 0.05 considered statistically significant.
Deletion of NLSs does not alter the phosphatase activity of MmpE.
(A) Domain architecture of MmpE. Schematic representation of MmpE with the annotated functional domains, including a Tat signal peptide (1–41 aa, twin-arginine translocation motif), a purple acid phosphatase domain (68–149 aa), a calcineurin-like phosphoesterase domain (211–389 aa) and two nuclear localization signals (NLS1: 20-22 aa; NLS2: 460–462 aa).
(B) Phylogenetic and structural conservation of MmpE. Neighbor-joining phylogenetic tree of MmpE homologs across Mycobacterium species (1,000 bootstrap replicates; values ≥50% shown). Species abbreviations: M. tuberculosis (Mtu), M. bovis BCG (Mbb), M. orygis (Mory), M. kubicae (Mku), M. paraterrae (Mpaa), M. farcinogenes (Mfg), M. mucogenicum (Mmuc), M. vicinigordonae (Mgor), M. lentiflavum (Mlw), M. avium (Mav), M. manitobense (Mman).
(C) Prediction of metal ion-binding residues in MmpE. Structural modeling and visualization of MmpE were performed using PyMOL. Conserved residues located in the putative metal binding pocket are shown as sticks and colored according to atom type. The predicted metal coordination site is highlighted, and key residues potentially involved in metal ion binding are labeled. Surface representation is included to illustrate the spatial accessibility of the binding pocket.
(D-F) Phosphatase activity assays of MmpEΔTat under various conditions. Phosphatase activity of MmpEΔTat (lacking the N-terminal Tat signal peptide) was measured using p-nitrophenyl phosphate (p-NPP) as the substrate in the presence of increasing concentrations (0–500 μM) of Fe³⁺ and Zn²⁺ (D). Phosphatase activity of increasing concentrations of MmpEΔTat and the mutant MmpEΔTat-H348AN359A was measured in the absence or presence of 50 μM Fe³⁺ (E). Phosphatase activity of increasing concentrations of MmpEΔTat and the double-NLS deletion mutant MmpEΔTat/ΔNLS1-2 was measured under standard conditions (F).
Data represent mean ± SD of three biologically independent experiments, analyzed using two-tailed unpaired Student’s t-tests (*P < 0.05, **P < 0.01, ***P < 0.001).
The nuclear translocation and phosphatase activity of MmpE are essential for M. bovis BCG survival in macrophage cells.
(A–B) Intracellular survival of M. bovis BCG strains in human THP-1 macrophages (A) and murine RAW264.7 macrophages (B). Strains include wild-type BCG (WT), MmpE deletion mutant (ΔMmpE), wild-type MmpE complemented strain (Comp-MmpE), and NLS2-deleted complement strain (Comp-MmpEΔNLS2).
(C–D) Intracellular survival of M. bovis BCG strains in THP-1 (C) and RAW264.7 (D) macrophages. Strains include ΔMmpE, Comp-MmpE, and NLS-deficient complement strains (Comp-MmpEΔNLS1 and Comp-MmpEΔNLS1-2).
(E) Intracellular survival of metal ion binding site mutants in THP-1 cells. Strains include Comp-MmpE and a phosphatase-deficient mutant (Comp-MmpE-H348AN359H).
(F) Inflammatory cytokine expression in infected THP-1 cells. mRNA levels of IL-1α, IL-1β, and IL-6 were quantified by qRT-PCR 20 hpi with the indicated BCG strains. Data represent mean ± SD from three independent biological replicates. Statistical analysis was performed using two-tailed unpaired Student’s t-tests (*P < 0.05, **P < 0.01, ***P < 0.001).
MmpE modulates host transcription network involved in inflammation response and lysosomal maturation.
(A) Volcano plot of differentially expressed genes (DEGs). DEGs in MmpE deleted strain (KommpE) compared to wild-type BCG (BCG/wt) were visualized in a volcano plot. DEGs were defined as genes with |log2(fold change)| ≥ 1 and P < 0.05. The x-axis represents log2(fold change), and the y-axis shows -log10(P-value). Genes with significant upregulation and downregulation are highlighted.
(B) GO enrichment analysis of DEGs in KommpE-infected THP-1 cells versus BCG/wt. The circular plot shows enriched biological process and molecular function terms. Outer rings represent GO terms, with red and green dots indicating upregulated and downregulated DEGs, respectively. The inner ring color reflects z-scores (blue: low; pink: high). The table lists selected GO terms with their IDs and descriptions.
(C) KEGG enrichment analysis of DEGs in KommpE-infected THP-1 cells versus BCG/wt. The bar plot displays selected significantly enriched pathways. Bar length represents the number of DEGs associated with each pathway, and color indicates statistical significance based on –log₁₀(P-value).
(D) Interaction network of DEGs related to immune regulation. Network construction was performed using STRING v12.0 and visualized with Cytoscape.
(E) Heatmap of immune-related DEGs in KommpE-infected and BCG/wt-infected THP-1 cells. Log₂fold change values are shown across three biological replicates. Red and blue indicate upregulation and downregulation, respectively.
(F-G) Quantitative RT-PCR analysis of gene expression in infected THP-1 cells. THP-1 cells were infected with BCG/wt or KommpE strains for 20 hpi. mRNA levels of cytokine genes (F); mRNA levels of genes involved in lysosomal acidification and biogenesis (G).
Data are presented as mean ±SD of three biologically independent experiments, analyzed using two-tailed unpaired Student’s t-tests (*P < 0.05, **P < 0.01, and ***P < 0.001).
MmpE suppresses the expression of VDR and inhibits activation of the PI3K–Akt–mTOR signaling pathway.
(A) Genomic distribution of potential MmpE-binding regions in HEK293T cells.
(B) Distribution of the chromosomal location of MmpE-specific ChIP-seq signals (fold-enrichment > 9).
(C) Biotype distribution of potential MmpE-binding regions in HEK293T cells.
(D) The number and position of the potential MmpE-binding sites along 20 kb from the nearest TSS of the protein-coding genes.
(E) Heatmap of transcription factors identified by MmpE ChIP-seq. Colors represent expression changes, with red indicating upregulation and blue indicating downregulation.
(F) MmpE-binding motifs identified by de novo motif analysis of ChIP-seq peak sequences using HOMER. Motif enrichment P-values were calculated using TOMTOM. “% of targets” indicates the proportion of MmpE-bound regions containing each motif. Letter height reflects the frequency of each nucleotide at the corresponding position in the consensus motif.
(G-H) ChIP-PCR and qPCR analyses of MmpE binding to the VDR promoter region. Chromatin was immunoprecipitated from HEK293T cells transfected with control vector (CK), EGFP, or MmpE–EGFP constructs. PCR was performed using primers targeting the GAPDH region (negative control) and the VDR promoter region, and products were analyzed by agarose gel electrophoresis (G). ChIP enrichment for VDR and GAPDH was quantified in HEK293T cells using qPCR, with enrichment calculated by the 2−ΔCt method(H).
(I-J) EMSA analysis showing the binding of MmpE to the human VDR promoter. The formation of DNA–protein complexes was observed with increasing concentrations of MmpE protein. Arrows indicate the positions of free DNA and DNA–protein complexes on the native polyacrylamide gel (I). Quantitative analysis of DNA–protein complexes in each lane (I) was performed by densitometry (J).
Data are presented as mean ±SD of three biologically independent experiments. Statistical significance was determined by a two-tailed unpaired Student’s t-test (***P < 0.001).
Nuclear translocation of MmpE is essential for mycobacterial survival in mice.
(A) Bacterial burden in lungs of infected mice. Specific pathogen-free (SPF) C57BL/6 mice (n = 6 per group) were intratracheally infected with 1.0 × 107 colony-forming units (CFU) of M. bovis BCG strains, including wild-type (WT), MmpE deleted (ΔMmpE), MmpE-complemented (Comp-MmpE), or MmpE-complemented with NLS-deleted variants (Comp-MmpEΔNLS1-2). Bacterial titers in lung homogenates were quantified by CFU assays at 0,14,28, and 56 days post-infection.
(B) Histopathology of infected lung tissues. Hematoxylin and eosin (H&E)-stained lung sections from mice infected as in (A) show granulomatous inflammation, Scale bars: 200 μm.
(C-D) Pro-inflammatory cytokine expression in the spleen of infected mice. qRT-PCR analysis of cytokine mRNA levels of Il1α (C), Il1β (D)and Il6 (E) in spleen tissues from infected mice (n = 6/group) at 2 to 28 days post-infection.
Data are presented as mean ± SD from six biologically independent experiments. Statistical significance was determined by a two-tailed unpaired Student’s t-test (*P < 0.05, **P < 0.01, ***P < 0.001).
Schematic diagram of nucleomodulin MmpE-mediated immune suppression and mycobacterial survival.
Upon mycobacterial infection, MmpE translocates into the host cell nucleus via its NLS2 motif (RRK⁴⁶⁰−⁴⁶²), where it binds to the promoter region of the VDR gene, repressing transcription of downstream inflammatory genes. Simultaneously, MmpE inhibits the PI3K–Akt–mTOR signaling pathway, leading to impaired phagosome maturation. These combined actions facilitate immune evasion and promote enhanced mycobacterial survival within macrophages and in mice.
Identification of MmpE as a nucleomodulin in Mycobacterium.
(A) MmpE is a mycobacterial nucleomodulin. Subnuclear localization of MmpE-EGFP. Confocal microscopy was used to assess the nuclear distribution of MmpE-EGFP at various time points post-transfection. Nuclei were stained with DAPI (blue); MmpE-EGFP is shown in green. Scale bar: 10 μm.
(B) AlphaFold-predicted structural models of MmpE with NLS truncations. Structural models of MmpE were generated using AlphaFold 2.2.0 to assess the impact of NLS deletions on overall protein conformation. (upper) Full-length MmpE structure shown as a reference. (middle) MmpE structure with NLS deleted. (botten) Predicted structures of MmpE with individual deletions of NLS1, NLS2, or both motifs. Confidence scores (pLDDT) were used to evaluate prediction reliability, and UCSF ChimeraX was used to visualize and compare structural changes.
Identification of MmpE as a conserved Fe3+/Zn2+-metallophosphatase in Mycobacteria.
(A) Sequence and structure-based analyses of MmpE. Sequence and structure-based analyses were performed using UCSF Chimera to investigate the potential function and structural features of MmpE. The structural comparison results, with a Z value >10 and RMSD value <4, indicate high confidence in the predicted structural models.
(B) Clustal Omega sequence alignment of MmpE from various mycobacterial species, highlighting conserved residues (≥90% identity, shown in red). Blue stars indicate the predicted NLSs, and red triangles mark characteristic sequences found in metallophosphatases. Species abbreviations: M. tuberculosis (Mtu), M. bovis BCG (Mbb), M. orygis (Mory), M. kubicae (Mku), M. paraterrae (Mpaa), M. farcinogenes (Mfg), M. mucogenicum (Mmuc), M. vicinigordonae (Mgor), M. lentiflavum (Mlw), M. avium (Mav), M. manitobense (Mman).
The nuclear translocation and phosphatase activity of MmpE are essential for M. bovis BCG survival in macrophage cells.
(A) Detection of mycobacterial MmpE secretion ability. C-terminally 2×Flag-tagged Ag85B (positive control) and MmpE were expressed in M. bovis BCG strains. Immunoblot analysis of bacterial lysates and culture supernatants showing the expression and secretion of Flag-tagged Ag85B and MmpE.
(B) Construction and validation of the MmpE-deleted strain of M. bovis BCG. (left) Schematic diagram of the homologous recombination strategy used to delete mmpE from the M. bovis BCG genome. (right) Wild-type and mutant strains were used as templates to amplify the mmpE gene (600 bp upstream–600 bp downstream) by PCR. Lanes 1 and 3: wild-type genomic DNA; lanes 2 and 4: ΔmmpE genomic DNA.
(C-D) Growth curve analysis of BCG strains. Growth of M. bovis BCG strains, including wild-type BCG (WT), MmpE-deleted (ΔMmpE), MmpE-complemented (Comp-MmpE), and NLS-deleted complemented (Comp-MmpEΔNLS1-2) strains(C), or phosphatase activity site mutation complement (Comp-MmpE-H348AN359H) (D), was measured in 7H9 medium.
Data are presented as mean ± SD from three biologically independent experiments. Statistical significance was determined by a two-tailed unpaired Student’s t-test.
MmpE modulates host transcription network involved in inflammation response and lysosomal maturation
(A) Biotype distribution of potential MmpE-regulating DEGs genes in THP-1 cells.
(B-C) Quantitative RT-PCR analysis of gene expression in infected THP-1 cells. THP-1 cells were infected with complemented strain (Comp-MmpE) and NLS-deleted complemented (Comp-MmpEΔNLS2, Comp-MmpEΔNLS1-2) strains for 20 h. mRNA levels of cytokine genes (B); mRNA levels of genes involved in lysosomal acidification and biogenesis (C).
Data are presented as mean ± SD from three biologically independent experiments. Statistical significance was determined by a two-tailed unpaired Student’s t-test (*P < 0.05, **P < 0.01, ***P < 0.001).
MmpE suppresses the expression of VDR and inhibits activation of the PI3K–Akt–mTOR signaling pathway.
(A) Interaction network of DEGs associated with MmpE-binding sites. The network was constructed using STRING v12.0 and visualized with Cytoscape, highlighting interactions between genes potentially regulated by MmpE.
(B) KEGG pathway enrichment analysis of MmpE-binding sites identified in EGFP-MmpE-transfected HEK293T cells revealed significant enrichment in multiple signaling pathways, including aldosterone-regulated sodium reabsorption (hsa04925), mTOR signaling (hsa04150), axon guidance (hsa04360), serotonergic synapse (hsa04726), circadian entrainment (hsa04713), inflammatory mediator regulation of TRP channels (hsa04750), cAMP signaling (hsa04024), calcium signaling (hsa04020), glutamatergic synapse (hsa04724), and cGMP–PKG signaling (hsa04022). The bar plot shows selected significantly enriched pathways. Bar length represents the number of MmpE-binding sites associated with each pathway, and the color gradient indicates statistical significance, represented as –log₁₀(P-value).
(C-D) Quantitative RT-PCR analysis of gene expression in infected THP-1 cells. THP-1 cells were infected with wild-type BCG (BCG/wt), MmpE-deleted (KommpE) strains for 20 h. mRNA levels of PI3K-Akt pathway(C); mRNA levels of VDR gene (D).
(E-F) ChIP-PCR and qPCR analyses of VDR promoter binding by MmpE and the phosphatase-deficient mutant MmpE-H348AN359A in HEK293T cells. Chromatin was immunoprecipitated from cells transfected with EGFP control, MmpE-EGFP, or MmpE-H348AN359A-EGFP constructs. PCR was performed using primers targeting the VDR promoter and GAPDH (negative control), and products were analyzed by agarose gel electrophoresis (E). ChIP enrichment for the VDR and GAPDH promoter regions was quantified by qPCR (F), with enrichment calculated using the 2−ΔCt method.
Data are presented as mean ± SD from three biologically independent experiments. Statistical significance was determined by a two-tailed unpaired Student’s t-test (***P < 0.001).
MmpE modulates the transcription of immune-associated genes.
(A) Association between differentially expressed genes and differential peak-related genes. Venn diagram showing the overlap between differential peak-related genes identified by CUT&Tag sequencing and DEGs identified by RNA-seq analysis.
(B) Four-quadrant scatter plot comparing CUT&Tag-seq and RNA-seq results. The log₂(fold change) of peak-related genes from CUT&Tag-seq is plotted against the log₂(fold change) of FPKM values from RNA-seq analysis. The scatter plot demonstrates the correlation between chromatin binding changes and gene expression levels.
(C) GO enrichment analysis of DEGs revealed significant overrepresentation of Biological Process (BP) terms in macrophages infected with KommpE compared to those infected with wild-type BCG (BCG/wt).
(D) Chord plot showing significantly altered genes associated with GO Biological Process pathways in KommpE-infected macrophages compared to BCG/wt infection.
MmpE promotes mycobacterial colonization in the spleen of mice.
Bacterial colonization in the spleen. CFU assays were performed on splenic homogenates from mice infected as described in Figure 6A. Bacterial loads (CFU/g tissue) were quantified at 2, 14, 21, 28, and 56 days post-infection. Data are presented as mean ± SD (n = 3). Statistical significance was determined using a two-tailed unpaired Student’s t-test (P < 0.05, **P < 0.01, and ***P < 0.001).
