Mycobacterium tuberculosis Toxin CpnT Is an ESX-5 Substrate and Requires Three Type VII Secretion Systems for Intracellular Secretion

Abstract

CpnT, a NAD⁺ glycohydrolase, is the only known toxin that is secreted by Mycobacterium tuberculosis CpnT is composed of two domains; the C-terminal domain is the toxin, whereas the N-terminal domain is required for secretion. CpnT shows characteristics of type VII secretion (T7S) substrates, including a predicted helix-turn-helix domain followed by a secretion motif (YxxxE). Disruption of this motif indeed abolished CpnT secretion. By analyzing different mutants, we established that CpnT is specifically secreted by the ESX-5 system in Mycobacterium marinum under axenic conditions and during macrophage infection. Surprisingly, intracellular secretion of CpnT was also dependent on both ESX-1 and ESX-4. These secretion defects could be partially rescued by coinfection with wild-type bacteria, indicating that secreted effectors are involved in this process. In summary, our data reveal that three different type VII secretion systems have to be functional in order to observe intracellular secretion of the toxin CpnT.IMPORTANCE For decades, it was believed that the intracellular pathogen M. tuberculosis does not possess toxins. Only fairly recently it was discovered that CpnT is a potent secreted toxin of M. tuberculosis, causing necrotic cell death in host cells. However, until now the secretion pathway remained unknown. In our study, we were able to identify CpnT as a substrate of the mycobacterial type VII secretion system. Pathogenic mycobacteria have up to five different type VII secretion systems, called ESX-1 to ESX-5, which play distinct roles for the pathogen during growth or infection. We were able to elucidate that CpnT is exclusively secreted by the ESX-5 system in bacterial culture. However, to our surprise we discovered that, during infection studies, CpnT secretion relies on intact ESX-1, ESX-4, and ESX-5 systems. We elucidate for the first time the intertwined interplay of three different and independent secretion systems to secrete one substrate during infection.

Keywords: Mycobacterium; Mycobacterium tuberculosis; exotoxins; protein secretion; secretion systems.

FIG 1

CpnT is a type VII substrate secreted in an ESX-5-dependent manner. (A) Genetic organization of the esxF-esxE-cpnT-rv3902c (IFT) operon and CpnT domain organization. (B) Structural modeling of CpnT with the N-terminus of EspB as a template (PDB accession number 4WJ2; M. smegmatis); confidence of 83% of the N-terminus domain (amino acids 48 to 276). The N-terminus of EspB is displayed in blue, and CpnT in light green; the secretion signal motif is marked in red (YxxxE). Root-mean-square deviation (RMSD) for the structural alignment is 1.57 Å (402 atoms out of 972 aligned atoms). (C) Secretion analysis of M. marinum strains, WT, ESX-1-deficient (ESX-1⁻, M^VU), ESX-4-deficient (ESX-4⁻, ΔeccC₄), and ESX-5-deficient (ESX-5⁻, ΔeccC₅ + mspA) expressing CpnT-HA or WT expressing _AxxxeCpnT-HA. Whole-cell lysate and supernatant preparations were immunoblotted for CpnT detection (anti-HA antibody); immunodetection of GroEL was used as the lysis control, and PE_PGRS, as a control for efficient secretion. Complementation of the ESX-5-deficient strain (ESX-5^– + eccC₅, ΔeccC₅ + eccC₅) restored PE_PGRS secretion.

FIG 2

Surface localization of CpnT within macrophages. (A) Fluorescence microscopy of infected RAW 264.7 macrophages with WT, WT+ CpnT, and WT+ _AxxxECpnT. All bacterial strains carried L5::gfp for fluorescence detection. Macrophages were infected at an MOI of 2 and fixed at 24 h postinfection. Cells were permeabilized with 0.2% Triton X-100 and immuno-labeled with anti-HA antibody (CpnT-HA). Additional dyes were used to label the nuclei (Hoechst) and the actin filaments (Phalloidin). The scale bar represents 10 μm. Images are a representation of five biological replicates. (B) The fluorescence quantification of the anti-HA signal per infected RAW macrophage was calculated using automated image analysis software (CellProfiler). Analysis was performed on one representative experiment out of three independent experiments, with a total number of 40 to 46 infected macrophages. One-way ANOVA and multiple comparisons using Dunnet’s statistical test were performed for statistical significance. ns, P > 0.05; ****, P < 0.0001. (C) Confocal microscopy of macrophages infected with WT+ CpnT strain. Infection conditions and labeling procedure were identical to those for panel A. The scale bar represents 10 μm.

FIG 3

EsxE and EsxF are required for CpnT secretion. (A) Secretion analysis of WT+ CpnT, _AxxxECpnT, and ΔesxEF-CpnT strains. Whole-cell lysate (WCL) and supernatant preparations were immunoblotted for CpnT detection (anti-HA antibody); immunodetection of GroEL was used as a lysis control, whereas PE_PGRS was used as a control for secretion. Surface proteins were extracted from intact cells with the detergent Genapol X-080 (Genapol supernatant); nonextracted proteins remained in the Genapol pellet fraction. GroEL was used as a cytosolic control, whereas PE_PGRS was used as a control for protein extraction. (B) Subcellular fractions of WT+ CpnT and WT+ ΔesxEF-CpnT strains were obtained using differential centrifugation steps. Whole-cell lysate (WCL), soluble proteins (S), and pellet fraction (P, insoluble proteins) were collected. Detection of GroEL (cytosolic protein) and EccB₅ (membrane-associated protein) with specific antibodies was used as a control for the supernatant and pellet fraction, respectively.

FIG 4

Secretion of CpnT in macrophages requires functional ESX-1, ESX-4, and ESX-5 systems. (A) Fluorescence microscopy of infected RAW 264.7 macrophages with WT, ESX-1-deficient (ESX-1^–), ESX-4-deficient (ESX-4^–), and ESX-5-deficient (ESX-5^–) strains expressing cpnT. All strains expressed gfp from the L5 bacteriophage integration site. Macrophages were infected at an MOI of 2 and fixed at 24 h postinfection. Cells were permeabilized with 0.2% Triton X-100 and immunolabelled with the anti-HA antibody (CpnT-HA). Additional dyes were used to label the nuclei (Hoechst) and the actin filaments (Phalloidin). The scale bar represents 10 μm. Images are a representation of five biological replicates. (B) The fluorescence quantification of the anti-HA signal per infected RAW macrophage, from microscopy pictures, was calculated using automated image analysis by the software CellProfiler. Analysis was performed on one representative experiment out of three independent experiments with 30 to 46 infected cells. One-way ANOVA and multiple comparisons using Dunnet’s statistical test were performed for statistical significance. ****, P < 0.0001.

FIG 5

The ESX-4 system is not required for phagosomal escape. Detection of cytosolic ubiquinated mycobacteria (Ub⁺). Macrophages were infected with WT, ESX-1-deficient (ESX-1^–), and ESX-4-deficient (ESX-4^–) bacterial strains expressing cpnT. All strains expressed gfp from the L5 bacteriophage integration site. The infection conditions are described in Fig. 4. Cells were permeabilized and immunolabelled with anti-ubiquitin (FK2) antibody; images were obtained by confocal microscopy. WT+ CpnT n = 119, ESX-1^–+ CpnT n = 70, ESX-4^–+ CpnT n = 152 (n = number of bacteria). Colocalized signal of GFP and FK2 represent Ub⁺ bacteria (cytosolic bacteria). The proportion of Ub⁺ bacteria is displayed on the upper-right corner of each picture. The scale bar represents 20 μm.

FIG 6

Coinfection with WT strain rescues CpnT secretion in ESX-1- and ESX-4-deficient strains within macrophages. (A) Fluorescence microscopy of RAW 264.7 cells coinfected with WT and either ESX-1^– or ESX-4^– strains expressing cpnT. WT bacteria expressing cpnT served as a positive control. All strains expressed gfp. Prior to the day of infection, WT cultures were mixed with the respective ESX mutant strain to ensure a heterogeneous culture. Macrophages were infected at an MOI of 5 and fixed at 24 h postinfection. Cells were permeabilized with 0.2% Triton X-100 and immunolabelled with the anti-HA antibody (CpnT-HA). Additional dyes were used to label the nuclei (Hoechst) and the actin filaments (Phalloidin). The scale bar represents 10 μm. (B and C) The fluorescence quantification of the anti-HA signal per infected RAW 264.7 macrophage, from microscopy pictures, was calculated using automated image analysis by the software CellProfiler. Analysis was performed on one representative experiment out of three independent experiments with a total number of infected macrophages between 30 and 60. A paired t test was performed for statistical significance. ***, P < 0.001; *, P < 0.05.

FIG 7

Model of CpnT secretion in mycobacterium-infected macrophages. Bacteria are phagocytosed by the macrophage and trapped in a phagosome to be eventually neutralized. Mycobacteria utilize the ESX-1 secretion system to escape from this phagosome to the cytosol. Once bacteria reach the cytosol, the ESX-4 system could sense the cytosolic environment and then activate the production of virulent factors. CpnT is then secreted by the ESX-5 secretion system to kill the macrophage.