Toxin secretion and trafficking by Mycobacterium tuberculosis

Abstract

The tuberculosis necrotizing toxin (TNT) is the major cytotoxicity factor of Mycobacterium tuberculosis (Mtb) in macrophages. TNT is the C-terminal domain of the outer membrane protein CpnT and gains access to the cytosol to kill macrophages infected with Mtb. However, molecular mechanisms of TNT secretion and trafficking are largely unknown. A comprehensive analysis of the five type VII secretion systems of Mtb revealed that the ESX-4 system is required for export of CpnT and surface accessibility of TNT. Furthermore, the ESX-2 and ESX-4 systems are required for permeabilization of the phagosomal membrane in addition to the ESX-1 system. Thus, these three ESX systems need to act in concert to enable trafficking of TNT into the cytosol of Mtb-infected macrophages. These discoveries establish new molecular roles for the two previously uncharacterized type VII secretion systems ESX-2 and ESX-4 and reveal an intricate link between toxin secretion and phagosomal permeabilization by Mtb.

Fig. 1. ESX motifs are required for surface accessibility of CpnT in M. tuberculosis.

a The cpnT locus of Mtb. The positions of the three putative ESX motifs labeled Y1, Y2, and Y3 of CpnT are indicated. b Immunoblot of Mtb whole-cell lysates probed with antibodies for CpnT (anti-TNT antibody) and IFT. LpqH was used as a loading control. c Detection of surface-accessible CpnT of the indicated Mtb strains using fluorescence microscopy. The Mtb strains were labeled with the metabolic dye DMN-trehalose (green) and stained with a polyclonal antibody against the TNT domain and Alexa Fluor-594 secondary antibody (red). The yellow color indicates co-localization of TNT with the bacterial cell. d Surface accessibility of CpnT by flow cytometry. The indicated Mtb strains were stained with a polyclonal antibody against the TNT domain and Alexa Fluor-488 secondary antibody. The mean fluorescence of Mtb cells is displayed in histograms. e Secretion of TNT into the cytosol of Mtb-infected macrophages. The indicated Mtb strains were labeled with the metabolic dye DMN-Trehalose (green) and used to infect THP-1 macrophages at an MOI of 10:1. The macrophages were permeabilized with Triton X-100 48 h after infection, stained with an anti-TNT antibody and Alexa Fluor-594 secondary antibody (red). The macrophage nuclei were stained with DAPI. f Quantification of TNT-positive macrophages from images shown in e. Macrophages were scored as TNT-positive when distinct red punctae were observed as compared with the Mtb cpnT operon deletion mutant (ΔcpnT_op). Data are represented as mean ± SEM of at least two independent experiments (n ≥ 2). Shown are representative images/blots of at least two independent experiments (n ≥ 2). Asterisks indicate significant differences (*p value ≤ 0.05, **p value ≤ 0.01, ***p value ≤ 0.001, ****p value ≤ 0.0001, calculated using the one-way ANOVA with Dunnett’s correction) compared with the Mtb mc²6206 strain. Source data are provided in the Source Data file.

Fig. 2. The ESX-4 system is essential for CpnT export and surface accessibility in M. tuberculosis.

a Detection of surface-accessible CpnT in Mtb using fluorescence microscopy. The Mtb strains were labeled with DMN-trehalose (green) and stained with polyclonal anti-TNT and Alexa Fluor-594 secondary antibodies (red). b Quantification of TNT-positive Mtb cells from images shown in a. Mtb cells were scored as TNT-positive when a red signal was observed as compared with Mtb ΔcpnT_op. c Surface accessibility of CpnT in Mtb by flow cytometry. The Mtb strains were stained with polyclonal anti-TNT and Alexa Fluor-488 secondary antibodies. The mean fluorescence of Mtb cells is displayed in histograms. d Surface-accessibility of CpnT of the Mtb H37Rv esx-3 deletion mutant and the control strains by fluorescence microscopy. The staining procedure was performed as in a. e Quantification of TNT-positive Mtb cells from images shown in d. Mtb cells were scored as TNT-positive when a red signal was observed as compared to Mtb ΔcpnT. f Depletion of a functional ESX-5 system in Mtb Erdman. The wt and eccD₅ Tet-OFF mutant strains were grown in the presence/absence of anhydrotetracycline hydrochloride (ATc; 100 ng/ml). Proteins from the whole-cell lysates and culture filtrates were analyzed by immunoblotting to detect EccD₅, PPE41, and CpnT with specific antibodies. g Surface-accessibility of CpnT in Mtb Erdman depleted of a functional Esx-5 system using fluorescence microscopy. The staining procedure was performed as in a. The wt and ΔcpnT strains in the H37Rv background were used as positive and negative controls, respectively. h TNT-positive Mtb cells from images shown in g were quantified as described in e. The Δesx-1 strain has a deletion of the RD1 region and lacks the panCD genes. Data are represented as mean ± SEM of at least two independent experiments (n ≥ 2) and representative images/blots are shown. Asterisks indicate significant differences (*p value ≤ 0.05, **p value ≤ 0.01, *** value ≤ 0.001, ****p value ≤ 0.0001, calculated using the one-way ANOVA with Dunnett’s correction) compared with the corresponding wt strain. Source data are provided in the Source Data file.

Fig. 3. The ESX-4 system is involved in EsxF export in M. tuberculosis.

a Detection of surface-accessible EsxF of the indicated Mtb strains using fluorescence microscopy. The Mtb strains were labeled with the metabolic dye DMN-trehalose (green), and stained with a polyclonal antibody against EsxF and Alexa Fluor-594 secondary antibody (red). The yellow color indicates co-localization of EsxF with the bacterial cell. b Quantification of EsxF-positive Mtb cells from images shown in a. Mtb cells were scored as EsxF-positive when a red signal was observed as compared with the Mtb cpnT operon deletion mutant (ΔcpnT_op). Data are represented as mean ± SEM of three independent experiments (n = 3) and representative images are shown. Asterisks indicate significant differences (*p value ≤ 0.05, **p value ≤ 0.01, ***p value ≤ 0.001, ****p value ≤ 0.0001, calculated using the one-way ANOVA with Dunnett’s correction) compared with the Mtb mc²6206 strain. Source data are provided in the Source Data file.

Fig. 4. The ESX-1, ESX-2, and ESX-4 systems are required for TNT trafficking into the cytosol of macrophages infected with M. tuberculosis.

a Secretion of TNT into the cytosol of Mtb-infected macrophages. The indicated Mtb strains were labeled with the metabolic dye DMN-Trehalose (green) and used to infect THP-1 macrophages at an MOI of 10:1. After 48 h of infection, the macrophages were permeabilized with digitonin to enable access of antibodies to the cytoplasm, or with Triton X-100 for access to intracellular compartments. Then, cells were stained with an anti-TNT or anti-Ag85 antibody and with an Alexa Fluor-594 secondary antibody (red). The macrophage nuclei were stained with DAPI. b, c Quantification of TNT-positive macrophages after permeabilization with digitonin (b) or Triton X-100 (c) from images shown in a. Macrophages were scored as TNT-positive when distinct red punctae were observed as compared to the Mtb cpnT operon deletion mutant (ΔcpnT_op). d Cell viability of Mtb-infected macrophages was measured as the total ATP content with a luminescent ATP detection assay kit. e Secretion of TNT into the cytosol of infected macrophages in Mtb H37Rv strains. The macrophage infection and staining procedure were performed as described in a. f Secretion of TNT into the cytosol of infected macrophages in the Mtb H37Rv and Erdman strains. The macrophage infection and staining procedure were performed as described in a. When indicated, bacteria were grown in 7H9 + 100 ng/ml anhydrotetracycline (ATc) to pre-deplete EccD5 prior to infection, then ATc was kept in the culture media during the infection. The Δesx-1 strain has a deletion of the RD1 region and lacks the panCD genes. Data are represented as the mean ± SEM of at least two independent experiments (n ≥ 2) and representative images are shown. Asterisks indicate significant differences (*p value ≤ 0.05, **p value ≤ 0.01, ***p value ≤ 0.001, ****p value ≤ 0.0001, calculated using the one-way ANOVA with Dunnett’s correction) compared with the Mtb mc²6206 strain. Source data are provided in the Source Data file.

Fig. 5. The ESX-1, ESX-2, and ESX-4 systems are required for phagosomal rupture by M. tuberculosis.

a Detection of phagosomal rupture in infected macrophages by using Mtb-specific antibodies. The indicated Mtb strains were labeled with the metabolic dye DMN-Trehalose (green) and used to infect THP-1 macrophages at an MOI of 10:1. After 48 h of infection, the macrophages were permeabilized with digitonin to enable access of antibodies to the cytoplasm, or with Triton X-100 for access to intracellular compartments. Then, cells were stained with an anti-Mtb antibody (αMtb) and with an Alexa Fluor-594 secondary antibody (red). The macrophage nuclei were stained with DAPI. b, c Quantification of αMtb-positive macrophages after permeabilization with digitonin (b) and Triton X-100 (c) from images shown in a. Macrophages were scored as αMtb-positive when distinct red punctae were observed as compared to the Mtb esx-1 mutant treated with digitonin. d Detection of ruptured phagosomes in macrophages infected with Mtb. The indicated Mtb strains were labeled with the metabolic dye DMN-Trehalose (green) and used to infect THP-1 macrophages at an MOI of 10:1. After 48 h of infection, the macrophages were permeabilized with Triton X-100 and stained with an antibody against the phagosome rupture marker Galectin-3 and with an Alexa Fluor-594 secondary antibody (red). e. Quantification of Galectin-3-positive macrophages from images shown in d. Macrophages were scored as Galectin-3-positive when distinct red punctae were observed as compared to the Mtb esx-1 mutant. The Δesx-1 strain has a deletion of the RD1 region and lacks the panCD genes. Data are represented as mean ± SEM of at least two independent experiments (n ≥ 2) and representative images are shown. Asterisks indicate significant differences (*p value ≤ 0.05, **p value ≤ 0.01, ***p value ≤ 0.001, ****p value ≤ 0.0001, calculated using the one-way ANOVA with Dunnett’s correction) compared with the Mtb Δesx-1 strain. Source data are provided in the Source Data file.

Fig. 6. Model of CpnT export and TNT secretion and trafficking by M. tuberculosis.

After phagocytosis Mtb is trapped inside the phagosome and expresses the esx-4 genes and the cpnT operon. The ESX-4 core complex is assembled in the inner membrane (IM). Then, the EsxEF complex is exported (1) by the ESX-4 system and forms a pore in the outer membrane (OM) (2) as shown previously. CpnT is exported through the ESX-4 system, possibly involving the ESX Y3 motif (3). CpnT is then integrated into the outer membrane by an unknown mechanism (4). After translocation of its C-terminal NAD⁺ glycohydrolase domain to the cell surface, possibly via the ESX motif Y3, CpnT/TNT is secreted (5). CpnT/TNT trafficking into the macrophage cytosol requires the permeabilization of the phagosomal membrane by the three type VII secretion systems ESX-1, ESX-2, and ESX-4. TNT access to the cytosol results in NAD(P)⁺ depletion and activation of necroptotic cell death as shown previously. Figure was created using BioRender.com.