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The tuberculosis necrotizing toxin kills macrophages by hydrolyzing NAD

Nature Structural & Molecular Biology volume 22, pages 672–678 (2015)Cite this article

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Abstract

Mycobacterium tuberculosis (Mtb) induces necrosis of infected cells to evade immune responses. Recently, we found that Mtb uses the protein CpnT to kill human macrophages by secreting its C-terminal domain, named tuberculosis necrotizing toxin (TNT), which induces necrosis by an unknown mechanism. Here we show that TNT gains access to the cytosol of Mtb-infected macrophages, where it hydrolyzes the essential coenzyme NAD+. Expression or injection of a noncatalytic TNT mutant showed no cytotoxicity in macrophages or in zebrafish zygotes, respectively, thus demonstrating that the NAD+ glycohydrolase activity is required for TNT-induced cell death. To prevent self-poisoning, Mtb produces an immunity factor for TNT (IFT) that binds TNT and inhibits its activity. The crystal structure of the TNT–IFT complex revealed a new NAD+ glycohydrolase fold of TNT, the founding member of a toxin family widespread in pathogenic microorganisms.

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Figure 1: TNT is a new β-NAD+ glycohydrolase of M. tuberculosis.
Figure 2: M. tuberculosis produces the endogenous immunity factor IFT, which binds to and inactivates TNT.
Figure 3: Atomic structure of the TNT–IFT complex.
Figure 4: Identification of TNT residues involved in NAD+ hydrolysis and cytotoxicity in macrophages.
Figure 5: M. tuberculosis secretes TNT into the macrophage cytosol.
Figure 6: TNT is toxic in zebrafish.

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Acknowledgements

We thank L. DeLucas for providing the Rv3902c structure before its publication, J. Pascal for stimulating discussions, Y. Wang for initial purification of IFT, the scientific staff at National Synchrotron Light Source beamlines X6A and X29 for beam time, the University of Alabama at Birmingham Zebrafish Core for generous support of the zebrafish experiments, F. Wolschendorf for use of his microscope, and P. Patel and U. Tak for technical assistance. Some work was carried out at the Sidney Kimmel Cancer Center X-ray Crystallography and Molecular Interaction Facility, which is supported in part by the US National Cancer Institute Cancer Center Support Grant P30 CA56036. This work was supported by the US National Institutes of Health grants GM100888 (G.C.), AI63432 (M.N.), AI083632 (M.N.) and AI074805 (M.N.).

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  1. Axel Siroy

    Present address: Present address: Institute of Nanoscopy, University of Maastricht, Maastricht, the Netherlands.,

Authors and Affiliations

  1. Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama, USA

    Jim Sun, Axel Siroy, Alexander Speer, Kathryn S Doornbos & Michael Niederweis

  2. Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania, USA

    Ravi K Lokareddy & Gino Cingolani

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Contributions

J.S., A. Siroy, A. Speer, K.S.D., G.C. and M.N. conceived and designed experiments and analyzed data; J.S., A. Siroy, R.K.L., K.S.D., A. Speer and G.C. performed experiments; J.S., G.C. and M.N. wrote and edited the paper.

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Integrated supplementary information

Supplementary Figure 1 TNT reduces cellular NAD+ levels by degradation.

(a) Global gene expression after induction of tnt in E. coli was determined by deep sequencing. Gene expression changes were compared to E. coli containing an empty vector and are plotted as a ratio (log2-fold changes) against the significance of the difference in expression (Log Odds; −log10 q-value). Only genes with expression ratios larger than four-fold (log2 ratios ≥ 2 or ≤ −2) and log Odds > 1.3 (q-values < 0.05) were included. (b) The amount of NAD+ extracted from 5 × 108 E. coli or 1 × 106 Jurkat cells 2 h or 24 h post-induction of tnt expression, respectively, was measured by a colorimetric enzyme coupling assay. Data represent the means ± s.d. of three independent experiments. (c) NAD+ hydrolysis by TNT releases ADP-ribose (and nicotinamide) as determined by thin-layer chromatography using radiolabeled NAD+, cyclic ADP-ribose, and ADP-ribose as standards as described in Methods.

Supplementary Figure 2 Expression and purification of TNT–IFT complex.

(a) Schematic flow chart of the purification procedure with accompanying yield at various steps. (b) Fractions were taken from each step of the purification process and analyzed in Coomassie-stained SDS-polyacrylamide gels. The final protein complex after gel filtration chromatography (pooled fractions) was used for X-ray crystallization. (c) Fractions from purification of free TNT were analyzed by Coomassie staining. TNT was separated from the TNT-IFT complex by heating at 70°C for 10 min. TNT was recovered from the soluble fraction while IFT remained in the precipitate after heating. WCL, whole cell lysate; SN, soluble.

Supplementary Figure 3 Kinetic analysis and pH dependency of NAD+ hydrolysis by TNT.

(a) 10 nM of purified TNT were incubated with different amounts of NAD+ at pH 7.4. After 1 min, 2.5 min, 5 min, and 10 min, aliquots were quenched by addition of 5 M NaOH and developed for 1 h in the dark. The relative fluorescence of each sample was measured and correlated to a standard concentration curve for NAD+. (b) The observed rate constant (kobs) of TNT enzymatic activity was plotted against NAD+ concentration to determine the kcat. (c) The pH dependency of TNT activity was determined by measuring the initial velocity of NAD+ hydrolysis at a TNT concentration of 10 nM and a substrate concentration of 200 µM. All data represent the means ± s.d. of three independent experiments.

Supplementary Figure 4 Model of the TNT–NAD+ complex.

(a) Docking of NAD+ inside TNT putative active site crevice performed using AutoDock Vina. NAD+ is shown in sticks and TNT as a green "Connolly" surface. (b) Ribbon representation of the docking model in panel (a) with magnified view of TNT residues in the palm-domain predicted to be important in the NAD+ binding and hydrolysis.

Supplementary Figure 5 Binding of TNT-mutant proteins to the antitoxin IFT.

The purified mutant proteins TNTQ822A (a) or TNTH792N Q822K (b) were used in an in vitro pull-down assay with purified MBP-IFT. MBP-IFT alone bound amylose resin while the TNTQ822A or TNTH792N Q822K proteins did not. Incubation of MBP-IFT (1 µg) with equimolar amounts of TNTQ822A or TNTH792N Q822K proteins resulted in binding of both TNT proteins to amylose resin and elution with maltose.

Supplementary Figure 6 Effect of nicotinamide (NM) and nicotinic acid (NA) on cellular NAD+ content and TNT activity.

(a) The amount of NAD+ extracted from 1 × 106 Jurkat cells supplemented with NM (5 mM) or NA (10 µM) or in combination for 3 h and 6 h were quantified by the EnzyFluo assay kit. These baseline NAD+ levels are similar to those previously reported in HEK293 cells and correspond to an NAD+ concentration of 503 µM. (b) NAD+-glycohydrolase activities were determined by the amount of NAD+ remaining after incubation of BSA or TNT (100 ng) with 1 µM NAD+ at 37°C for 30 min in the presence or absence of NM (5 mM) and/or NA (10 µM). Data in (a-b) represent the means ± s.d. of three independent experiments. (c) Jurkat cells expressing tnt were untreated (Ctrl), incubated with 5 mM nicotinamide or 10 µM nicotinic acid, or in combination. Then, tnt expression was induced with 100 ng/mL doxycycline for 2 h. Following removal of doxycycline, cells were incubated in fresh media for 24 h. Cell death was assessed using the 7AAD dye and flow cytometry. 7AAD: 7-amino-actinomycin D, SSC: Side scatter.

Supplementary Figure 7 TNT requires cytosolic localization to induce cell death.

(a) Exogenous purified TNT or TNT-IFT complex (10 µg/mL) was added to THP-1 macrophages and incubated for 24 h. Thereafter, cell death was quantified by 7AAD staining and flow cytometry. (b, c) purified TNT or TNT-IFT complex (250 µg/mL) was adsorbed onto 4 µm latex beads and tested for NAD+ glycohydrolase activity (b) as described in Methods and subsequently used to infect THP-1 macrophages in order to load TNT intracellularly. 48 h post infection, macrophages were assessed for viability with 7AAD staining (c). Data in (b) represent the means ± s.d. of three independent experiments.

Supplementary Figure 8 Plasmid construction for this study.

Plasmid names are shown in dark grey boxes. The oligonucleotide pairs used for PCR amplification and restriction enzymes utilized for cloning are listed in white boxes. The template DNA for PCR is indicated below the oligonucleotide pairs. When chromosomal DNA was used as a template, the originating organism is indicated.

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Sun, J., Siroy, A., Lokareddy, R. et al. The tuberculosis necrotizing toxin kills macrophages by hydrolyzing NAD. Nat Struct Mol Biol 22, 672–678 (2015). https://doi.org/10.1038/nsmb.3064

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