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• W2916874117 abstract "•MbcTA is a RES-Xre toxin-antitoxin system in M. tuberculosis (Mtb)•MbcT is a NAD+ phosphorylase•MbcT-catalyzed NAD+ depletion leads to Mtb cell death•MbcT activity synergizes with antibiotics to reduce Mtb burden in infected mice Toxin-antitoxin (TA) systems regulate fundamental cellular processes in bacteria and represent potential therapeutic targets. We report a new RES-Xre TA system in multiple human pathogens, including Mycobacterium tuberculosis. The toxin, MbcT, is bactericidal unless neutralized by its antitoxin MbcA. To investigate the mechanism, we solved the 1.8 Å-resolution crystal structure of the MbcTA complex. We found that MbcT resembles secreted NAD+-dependent bacterial exotoxins, such as diphtheria toxin. Indeed, MbcT catalyzes NAD+ degradation in vitro and in vivo. Unexpectedly, the reaction is stimulated by inorganic phosphate, and our data reveal that MbcT is a NAD+ phosphorylase. In the absence of MbcA, MbcT triggers rapid M. tuberculosis cell death, which reduces mycobacterial survival in macrophages and prolongs the survival of infected mice. Our study expands the molecular activities employed by bacterial TA modules and uncovers a new class of enzymes that could be exploited to treat tuberculosis and other infectious diseases. Toxin-antitoxin (TA) systems regulate fundamental cellular processes in bacteria and represent potential therapeutic targets. We report a new RES-Xre TA system in multiple human pathogens, including Mycobacterium tuberculosis. The toxin, MbcT, is bactericidal unless neutralized by its antitoxin MbcA. To investigate the mechanism, we solved the 1.8 Å-resolution crystal structure of the MbcTA complex. We found that MbcT resembles secreted NAD+-dependent bacterial exotoxins, such as diphtheria toxin. Indeed, MbcT catalyzes NAD+ degradation in vitro and in vivo. Unexpectedly, the reaction is stimulated by inorganic phosphate, and our data reveal that MbcT is a NAD+ phosphorylase. In the absence of MbcA, MbcT triggers rapid M. tuberculosis cell death, which reduces mycobacterial survival in macrophages and prolongs the survival of infected mice. Our study expands the molecular activities employed by bacterial TA modules and uncovers a new class of enzymes that could be exploited to treat tuberculosis and other infectious diseases. Toxin-antitoxin (TA) systems are widespread in prokaryotes and play a central role in the response and adaptation of bacteria to various stress conditions, including starvation, phage attack, or antibiotic treatment (Hall et al., 2017Hall A.M. Gollan B. Helaine S. Toxin-antitoxin systems: reversible toxicity.Curr. Opin. Microbiol. 2017; 36: 102-110Crossref PubMed Scopus (63) Google Scholar, Harms et al., 2018Harms A. Brodersen D.E. Mitarai N. Gerdes K. Toxins, targets, and triggers: an overview of toxin-antitoxin biology.Mol. Cell. 2018; 70: 768-784Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar, Lobato-Márquez et al., 2016Lobato-Márquez D. Díaz-Orejas R. García-del Portillo F. Toxin-antitoxins and bacterial virulence.FEMS Microbiol. Rev. 2016; 40: 592-609Crossref PubMed Scopus (117) Google Scholar, Page and Peti, 2016Page R. Peti W. Toxin-antitoxin systems in bacterial growth arrest and persistence.Nat. Chem. Biol. 2016; 12: 208-214Crossref PubMed Scopus (464) Google Scholar). TA systems encode a toxic protein, which targets an essential physiological process in the bacterial cell, together with a toxin-neutralizing “antidote” or antitoxin. Under favorable growth conditions, toxin activity is blocked by the presence of the antitoxin. When faced with antibiotic or environmental stress, the antitoxin is rapidly degraded, which allows the toxin to become activated, thereby reducing the bacterial growth rate (Deter et al., 2017Deter H.S. Jensen R.V. Mather W.H. Butzin N.C. Mechanisms for differential protein production in toxin – antitoxin systems.Toxins (Basel). 2017; 9: 1-13Crossref Scopus (21) Google Scholar, Hall et al., 2017Hall A.M. Gollan B. Helaine S. Toxin-antitoxin systems: reversible toxicity.Curr. Opin. Microbiol. 2017; 36: 102-110Crossref PubMed Scopus (63) Google Scholar). TA systems are classified in four families (I–IV) based on the nature of the antitoxin and the associated mechanism of toxin inhibition (Harms et al., 2018Harms A. Brodersen D.E. Mitarai N. Gerdes K. Toxins, targets, and triggers: an overview of toxin-antitoxin biology.Mol. Cell. 2018; 70: 768-784Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar). Most studies have focused on type II TA systems, which are composed of a protein antitoxin and toxin pair. Strikingly, type II TA systems are highly abundant in the tuberculosis (TB) bacillus, Mycobacterium tuberculosis (Mtb), in which they are thought to contribute to pathogenicity and persistence (Keren et al., 2011Keren I. Minami S. Rubin E. Lewis K. Characterization and transcriptome analysis of Mycobacterium tuberculosis persisters.MBiol. 2011; 2 (e00100-11)PubMed Google Scholar, Ramage et al., 2009Ramage H.R. Connolly L.E. Cox J.S. Comprehensive functional analysis of Mycobacterium tuberculosis toxin-antitoxin systems: Implications for pathogenesis, stress responses, and evolution.PLoS Genet. 2009; 5: e1000767Crossref PubMed Scopus (369) Google Scholar, Sala et al., 2014Sala A. Bordes P. Genevaux P. Multiple toxin-antitoxin systems in Mycobacterium tuberculosis.Toxins (Basel). 2014; 6: 1002-1020Crossref PubMed Scopus (172) Google Scholar, Slayden et al., 2018Slayden R.A. Dawson C.C. Cummings J.E. Toxin antitoxin systems and regulatory mechanisms in Mycobacterium tuberculosis.Pathog. Dis. 2018; 76https://doi.org/10.1093/femspd/fty039Crossref PubMed Scopus (47) Google Scholar). Among the ∼80 TA system-encoding operons identified in the Mtb genome, three antitoxin-encoding genes are essential for viability, as evidenced by saturating transposon mutagenesis studies (DeJesus et al., 2017DeJesus M.A. Gerrick E.R. Xu W. Park S.W. Long J.E. Boutte C.C. Rubin E.J. Schnappinger D. Ehrt S. Fortune S.M. et al.Comprehensive essentiality analysis of the Mycobacterium tuberculosis genome via saturating transposon mutagenesis.MBio. 2017; 8 (e02133-16)Crossref PubMed Scopus (309) Google Scholar). This suggests that the cognate toxins of these essential antitoxins are lethal to Mtb, and such TA systems could be exploited for the development of novel anti-TB therapies. Here, we focus on the Mtb type II TA module Rv1989c-Rv1990c, in which the antitoxin-encoding gene (Rv1990c) is essential, whereas the cognate toxin-encoding gene (Rv1989c) is dispensable for bacterial growth (DeJesus et al., 2017DeJesus M.A. Gerrick E.R. Xu W. Park S.W. Long J.E. Boutte C.C. Rubin E.J. Schnappinger D. Ehrt S. Fortune S.M. et al.Comprehensive essentiality analysis of the Mycobacterium tuberculosis genome via saturating transposon mutagenesis.MBio. 2017; 8 (e02133-16)Crossref PubMed Scopus (309) Google Scholar) (Figure S1A). This TA pair was previously identified by in silico genomic analysis of prokaryotic TA loci and classified as a so-called COG5654-COG5642 TA system (Makarova et al., 2009Makarova K.S. Wolf Y.I. Koonin E.V. Comprehensive comparative-genomic analysis of Type 2 toxin-antitoxin systems and related mobile stress response systems in prokaryotes.Biol. Direct. 2009; 4: 19Crossref PubMed Scopus (322) Google Scholar). It was predicted to encode a RES domain-containing toxin and a cognate antitoxin with a XRE-like HTH domain, typically found in phage repressor proteins (Wood et al., 1990Wood H.E. Devine K.M. McConnell D.J. Characterisation of a repressor gene (xre) and a temperature-sensitive allele from the Bacillus subtilis prophage, PBSX.Gene. 1990; 96: 83-88Crossref PubMed Scopus (48) Google Scholar) (Figure S1A). According to a SMART search for analysis of protein domain architectures, the three conserved polar groups (R-E-S) that are predicted to form an active site in Rv1989c are Arg47, Glu69, and Ser126 (Letunic and Bork, 2018Letunic I. Bork P. 20 years of the SMART protein domain annotation resource.Nucleic Acids Res. 2018; 46: D493-D496Crossref PubMed Scopus (1050) Google Scholar). COG5654 or RES domains are widely spread in bacteria and often found in conjunction with various other conserved domains. Interestingly, a plasmid-encoded RES-Xre locus from the legume symbiont Sinorhizobium meliloti was reported to function as an active TA system (Milunovic et al., 2014Milunovic B. DiCenzo G.C. Morton R.A. Finan T.M. Cell growth inhibition upon deletion of four toxin-antitoxin loci from the megaplasmids of Sinorhizobium meliloti.J. Bacteriol. 2014; 196: 811-824Crossref PubMed Scopus (33) Google Scholar). The Rv1989c-Rv1990c TA system is particularly interesting because it is significantly upregulated in a variety of stress conditions, including in Mtb persister cells (Keren et al., 2011Keren I. Minami S. Rubin E. Lewis K. Characterization and transcriptome analysis of Mycobacterium tuberculosis persisters.MBiol. 2011; 2 (e00100-11)PubMed Google Scholar), during hypoxic stress (Rustad et al., 2008Rustad T.R. Harrell M.I. Liao R. Sherman D.R. The enduring hypoxic response of Mycobacterium tuberculosis.PLoS ONE. 2008; 3: 1-8Crossref Scopus (371) Google Scholar), under starvation (Gupta et al., 2017Gupta A. Venkataraman B. Vasudevan M. Gopinath Bankar K. Co-expression network analysis of toxin-antitoxin loci in Mycobacterium tuberculosis reveals key modulators of cellular stress.Sci. Rep. 2017; 7: 5868Crossref PubMed Scopus (48) Google Scholar), and within host macrophages (Homolka et al., 2010Homolka S. Niemann S. Russell D.G. Rohde K.H. Functional genetic diversity among Mycobacterium tuberculosis complex clinical isolates: Delineation of conserved core and lineage-specific transcriptomes during intracellular survival.PLoS Pathog. 2010; 6: 1-17Crossref Scopus (202) Google Scholar). A BLASTp search predicts Rv1989c-Rv1990c-like TA systems in multiple mycobacterial species of the M. tuberculosis complex (Tortoli et al., 2017Tortoli E. Fedrizzi T. Meehan C.J. Trovato A. Grottola A. Giacobazzi E. Serpini G.F. Tagliazucchi S. Fabio A. Bettua C. et al.The new phylogeny of the genus Mycobacterium : The old and the news.Infect. Genet. Evol. 2017; 56: 19-25Crossref PubMed Scopus (92) Google Scholar), with orthologs detected in a limited number of strains of opportunistic non-tuberculous mycobacteria (e.g., M. avium). Homologs of this TA system are also present in environmental prokaryotes, such as Gordonia spp (Figure S1B). This is in line with our previous suggestion that the Rv1989c-Rv1990c TA pair was most likely acquired through horizontal gene transfer with environmental bacteria (Becq et al., 2007Becq J. Gutierrez M.C. Rosas-Magallanes V. Rauzier J. Gicquel B. Neyrolles O. Deschavanne P. Contribution of horizontally acquired genomic islands to the evolution of the Tubercle bacilli.Mol. Biol. Evol. 2007; 24: 1861-1871Crossref PubMed Scopus (116) Google Scholar). To uncover the mechanism of action of the Rv1989c toxin, we used a combination of biochemical, structural biology, and microbiological methods. We show that Rv1989c encodes a novel NAD+ phosphorylase, an enzymatic activity that has never been described thus far, and reveal a synergistic protective effect of toxin activity and antibiotic treatment in a mouse model of Mtb infection. We first expressed Rv1989c and Rv1990c from different inducible promoters in Escherichia coli. Induction of Rv1989c inhibited E. coli growth on agar plates, unless Rv1990c was co-expressed (Figure S2A). In contrast, wild-type (WT) Mtb expressing Rv1989c from a tetracycline-inducible promoter on an integrated plasmid (Ehrt et al., 2005Ehrt S. Guo X.V. Hickey C.M. Ryou M. Monteleone M. Riley L.W. Schnappinger D. Controlling gene expression in mycobacteria with anhydrotetracycline and Tet repressor.Nucleic Acids Res. 2005; 33: e21Crossref PubMed Scopus (272) Google Scholar) did not show impaired growth (Figures 1A and 1B ). We hypothesized that the quantity of antitoxin protein expressed from the chromosomally encoded Rv1990c gene was sufficient to neutralize the amount of toxin expressed from both the chromosomal Rv1989c gene and the plasmid-encoded copy of Rv1989c. To test our hypothesis, we constructed a Mtb knockout (KO) mutant with a deletion of the entire Rv1989c-Rv1990c operon (MtbΔTA) by homologous recombination, as outlined in Figures S2B–S2E. Indeed, induction of an ectopic copy of the toxin gene in the MtbΔTA strain completely abolished mycobacterial growth, both on agar medium and in liquid culture (Figures 1A and 1B). Further, MtbΔTA displayed a substantial decrease in colony-forming units (CFUs) after induction of the toxin gene, with a loss of more than 3-Log10 in CFUs over only 4 days, suggesting bactericidal activity of the toxin (Figure 1C). We then tested the viability of MtbΔTA cells following ATc-induced expression of Rv1989c by flow cytometry analysis (Figures 1D and 1E) and fluorescence microscopy (Figure 1F) of bacteria labeled with LIVE/DEAD BacLight stains. Addition of ATc to a culture of MtbΔTA carrying an empty vector had no effect on the proportion of cells permeable to propidium iodine (PI). In contrast, for MtbΔTA cells expressing Rv1989c, the proportion of PI-permeable cells increased from 15% in the absence ATc to 57% in the presence of ATc after 4 days incubation, indicative of the bactericidal activity of Rv1989c. To assess the expression level of the Rv1989c gene in our experimental setup, we compared Rv1989c mRNA levels in WT Mtb versus in the Rv1989c-inducible MtbΔTA strain by real-time qPCR (Figure S2F). Rv1989c mRNA level in the Rv1989c-induced MtbΔTA strain was ≈2-fold higher than that in WT Mtb during exponential growth, and ≈2-fold lower than that in Mtb grown under starvation, a natural stress condition known to induce the TA system (Gupta et al., 2017Gupta A. Venkataraman B. Vasudevan M. Gopinath Bankar K. Co-expression network analysis of toxin-antitoxin loci in Mycobacterium tuberculosis reveals key modulators of cellular stress.Sci. Rep. 2017; 7: 5868Crossref PubMed Scopus (48) Google Scholar). Thus, the absence of toxicity in an Mtb WT strain and the real-time qPCR analysis shows that in our experimental setup the Mtb cell death we observed by CFU counting and viability analysis in combination with flow cytometry is not due to an overwhelming production of the toxin. Taken together, these results establish that Rv1989c-Rv1990c can function as a bactericidal TA system in Mtb. Hence, we named the Rv1989c-Rv1990c system mycobacterial cidal toxin (MbcT) and antitoxin (MbcA). To elucidate the molecular basis of MbcT activity, we solved the high-resolution crystal structure of the MbcTA complex (Figures 2A and S3A; Table 1). The complex adopts a donut-like structure composed of three heterotetrameric MbcTA complexes ([MbcTA]2). The oligomerization state and overall shape of the heterododecameric complex were validated by light scattering and by small-angle X-ray scattering (SAXS) (Figures S3B–S3D; Table 2). MbcA folds into a single structured domain consisting of eight α helices, whereas Mb cT exhibits a β sandwich fold formed by six β strands arranged in two opposing antiparallel β sheets that are flanked and connected by nine α helices (Figure 2B). The lateral side of the substrate-binding pocket of MbcT is formed by a stretch of 11 amino acids arranged in a kinked loop pointing inward (α2-β2 loop) with the side chain of Arg47 extending from the tip of the loop. The main interactions in the MbcTA complex are between residues of the MbcA C terminus and residues, mostly arginines (R27, R33, R43, R47, R72), lining a deep central cleft in MbcT (Figure 2B). To validate the role of the C terminus of MbcA in sterically blocking access to the toxin active site, we designed a truncated MbcA version lacking the last ten C-terminal amino acids (residues 104–113). As expected, this variant was not able to neutralize the toxic effect of MbcT in a MtbΔTA background (Figure S3E).Table 1Crystallographic Data Collection, Phasing, and Refinement StatisticsNative DatasetS-SAD DatasetData collectionSpace groupP63P63Cell dimensions (Å,°)105.3, 105.3, 108.7105.3, 105.3, 108.790.0, 90.0, 12090.0, 90.0, 120Wavelength (Å)0.9762.479Resolution (Å)9.99–1.80 (1.86–1.80)aValues in parentheses indicate the highest-resolution shells and their statistics91.70–2.51 (2.61–2.51)Rmerge (%)0.0562 (1.364)0.123 (0.667)I/σ(I)21.6 (1.5)42.5 (7.3)Completeness (%)98.8 (90.2)89.3 (70.3)Redundancy10.0 (7.5)57.6 (49.2)CC1/20.999 (0.497)0.999 (0.920)Total number of reflections620,194 (42,332)1,225,773 (92,688)Unique reflections62,157 (5,639)21,269 (1,882)RefinementRwork (%)16.23 (27.26)–Rfree (%)21.11 (31.28)–No. atomsTotal4,865–Macromolecules4,569–Ligands18–Waters278–No. protein residues589–B-factorsbValues from PHENIX (Adams et al., 2010) (Å2)Macromolecules42.4–Solvent62.8–RMSDbValues from PHENIX (Adams et al., 2010)Bond lengths (Å)0.007–Bond angles (°)0.990–RamachandrancValues from MOLPROBITY (Chen et al., 2010) (%)Most favored99.0–Allowed1.0–Outliers0.0–a Values in parentheses indicate the highest-resolution shells and their statisticsb Values from PHENIX (Adams et al., 2010Adams P.D. Afonine P.V. Bunkóczi G. Chen V.B. Davis I.W. Echols N. Headd J.J. Hung L.-W. Kapral G.J. Grosse-Kunstleve R.W. et al.PHENIX: a comprehensive Python-based system for macromolecular structure solution.Acta Crystallogr. D Biol. Crystallogr. 2010; 66: 213-221Crossref PubMed Scopus (16524) Google Scholar)c Values from MOLPROBITY (Chen et al., 2010Chen V.B. Arendall W.B. Headd J.J. Keedy D.A. Immormino R.M. Kapral G.J. Murray L.W. Richardson J.S. Richardson D.C. MolProbity : all-atom structure validation for macromolecular crystallography.Acta Crystallogr. Sect. D Biol. Crystallogr. 2010; 66: 12-21Crossref PubMed Scopus (9877) Google Scholar) Open table in a new tab Table 2SAXS Data Collection and Derived ParametersMbcTAData collectionInstrumentP12 at EMBL/DESY, storage ring PETRA III, GermanyBeam geometry0.2 × 0.12 mm2Wavelength (Å)1.24q-range (Å−1)0.008–0.47Exposure time (ms)20 × 50Concentration range (mg mL−1)0.6–7.1Temperature (K)283Structural parametersaReported for MbcTA at 0.6 mg mL−1I(0) (arbitrary units) (from P(r))31,320 ± 10Rg (from P(r)) (Å)41 ± 1I(0) (arbitrary units) (from Guinier)31,340 ± 30Rg (Å) (from Guinier)41 ± 1Dmax (Å)114Porod volume (103 Å3)262Molecular mass determinationaReported for MbcTA at 0.6 mg mL−1MMPOROD (from Porod volume) (kDa)154 ± 15MMsaxs (from I(0), kDa)110 ± 20MMDAM (from bead model, kDa)170 ± 35Calculated monomeric MM from sequence (kDa)197.2Software employedPrimary data reductionAutomated radial averagingData processingPRIMUSAb initio analysisDAMMINValidation and averagingSASRES, DAMAVERComputation of model intensitiesCRYSOLSASBDB entry codeSASDD33a Reported for MbcTA at 0.6 mg mL−1 Open table in a new tab The closest structural relatives to MbcT are ADP-ribosyltransferases (ARTs), in particular bacterial ART toxins and poly (ADP-ribose) polymerases (PARPs) (Aravind et al., 2015Aravind L. Zhang D. de Souza R.F. Anand S. Iyer L.M. The natural history of ADP-ribosyltransferases and the ADP-ribosylation system.Curr. Top. Microbiol. Immunol. 2015; 384: 3-32PubMed Google Scholar, Palazzo et al., 2017Palazzo L. Mikoč A. Ahel I. ADP-ribosylation: new facets of an ancient modification.FEBS J. 2017; 284: 2932-2946Crossref PubMed Scopus (98) Google Scholar, Simon et al., 2014Simon N.C. Aktories K. Barbieri J.T. Novel bacterial ADP-ribosylating toxins: structure and function.Nat. Rev. Microbiol. 2014; 12: 599-611Crossref PubMed Scopus (148) Google Scholar) (Figure S4A). ARTs catalyze the transfer of an ADP-ribose group from an NAD+ donor molecule to a substrate (proteins, DNA, or RNA) and release free nicotinamide (NAA). Bacterial ART toxins are classified into two major groups based on conserved active-site motifs distributed across three regions. The diphtheria toxin (ARTD) group has an H-Y/Y-E motif, also found in PARPs, whereas the cholera toxin (ARTC) group contains an R-S-E motif (Aravind et al., 2015Aravind L. Zhang D. de Souza R.F. Anand S. Iyer L.M. The natural history of ADP-ribosyltransferases and the ADP-ribosylation system.Curr. Top. Microbiol. Immunol. 2015; 384: 3-32PubMed Google Scholar, Simon et al., 2014Simon N.C. Aktories K. Barbieri J.T. Novel bacterial ADP-ribosylating toxins: structure and function.Nat. Rev. Microbiol. 2014; 12: 599-611Crossref PubMed Scopus (148) Google Scholar) (Figure 2C). The structural hallmark of ARTs is a central cleft bearing a conserved NAD+-binding pocket (Aravind et al., 2015Aravind L. Zhang D. de Souza R.F. Anand S. Iyer L.M. The natural history of ADP-ribosyltransferases and the ADP-ribosylation system.Curr. Top. Microbiol. Immunol. 2015; 384: 3-32PubMed Google Scholar, Han and Tainer, 2002Han S. Tainer J.A. The ARTT motif and a unified structural understanding of substrate recognition in ADP-ribosylating bacterial toxins and eukaryotic ADP-ribosyltransferases.Int. J. Med. Microbiol. 2002; 291: 523-529Crossref PubMed Scopus (77) Google Scholar). An NAD+-binding pocket is also present in NAD+ glycohydrolases (NADases), such as the bacterial exotoxins TNT (Sun et al., 2015Sun J. Siroy A. Lokareddy R.K. Speer A. Doornbos K.S. Cingolani G. Niederweis M. The tuberculosis necrotizing toxin kills macrophages by hydrolyzing NAD.Nat. Struct. Mol. Biol. 2015; 22: 672-678Crossref PubMed Scopus (88) Google Scholar), SPN (Ghosh et al., 2010Ghosh J. Anderson P.J. Chandrasekaran S. Caparon M.G. Characterization of Streptococcus pyogenes beta-NAD+ glycohydrolase: re-evaluation of enzymatic properties associated with pathogenesis.J. Biol. Chem. 2010; 285: 5683-5694Crossref PubMed Scopus (39) Google Scholar), and Tse6 (Whitney et al., 2015Whitney J.C. Quentin D. Sawai S. LeRoux M. Harding B.N. Ledvina H.E. Tran B.Q. Robinson H. Goo Y.A. Goodlett D.R. et al.An interbacterial NAD(P)+ glycohydrolase toxin requires elongation factor Tu for delivery to target cells.Cell. 2015; 163: 607-619Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar), but the overall structural homology of MbcT with NADases is less obvious (Figure S4A). Structural superimposition with selected ARTs and NADases suggests that MbcT could consume NAD+ as well, and pinpoints Arg27 in region 1, and Tyr28 and Tyr58 in region 2, as potential NAD+-binding residues (Figures 2C and 2D). Yet, the region-3 residue, which is thought to confer substrate recognition and specificity, is replaced by a glycine (Gly152) in MbcT (Figure 2C). To investigate the functional importance of the putative NAD+-binding site of MbcT and the potential catalytic function of the RES motif (R47-E69-S126), we substituted single residues of MbcT to alanine and assessed the effect on growth inhibition of MtbΔTA. Non-toxic MbcT-R27A, MbcT-R47A, and MbcT-Y58A mutants did not affect the growth of MtbΔTA or E. coli, thus establishing the crucial role of these individual residues for MbcT-catalyzed growth inhibition (Figures S4B and S4C). Surprisingly, Ser126 was not essential for toxicity, whereas MbcT-Y28A and MbcT-E69A retained limited toxin activity. Taken together, these results suggest that MbcT toxicity involves NAD+, but that the catalytic mechanism underlying toxin activity is divergent from that of ART enzymes and NADases. To identify substrates of MbcT and explore its NAD+-binding activity in vitro, we sought to purify the WT, recombinant MbcT protein. To overcome cell toxicity, we co-expressed full-length WT MbcT with a His-tagged, C-terminal truncation of MbcA (MbcAΔ112–113). WT MbcT is only weakly associated with His-MbcAΔ112–113, allowing for subsequent isolation of WT MbcT by salt-induced dissociation of the His-MbcAΔ112–113-MbcT complex (Figure S5A). In addition to WT MbcT, we also purified the MbcT active-site mutant R27E (MbcT-R27E) from E. coli as a control (Figures S5B and S5C). This variant of MbcT was non-toxic to MtbΔTA cells (Figure S5D). We then incubated recombinant protein with different bacterial cell fractions in the presence of 32P-labeled NAD+ to probe for ADP-ribosylation of cellular protein but did not detect 32P-ADP-ribose-incorporation into the protein fractions (Figure S5E). MbcT also did not modify nucleic acid substrates, in contrast to the mycobacterial DNA-modifying TA toxin DarT (Jankevicius et al., 2016Jankevicius G. Ariza A. Ahel M. Ahel I. The toxin-antitoxin system DarTG catalyzes reversible ADP-ribosylation of DNA.Mol. Cell. 2016; 64: 1109-1116Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar) (Figure S5F). In addition to NAD+ degradation and ADP-ribose (Appr) production, we observed the appearance of an unknown reaction product, dependent on the MbcT concentration (Figure 3A). Interestingly, supplementing the MbcT reaction buffer with sodium phosphate markedly enhanced NAD+ degradation into NAA and the hitherto unknown reaction product (Figure 3B), whereas the MbcT R27E mutant or the MbcTA complex did not trigger NAD+-turnover (Figures 3B and S5F). We performed high resolution mass spectrometry and nuclear magnetic resonance experiments, which identified the additional reaction product as ADP-ribose-1″-phosphate (Appr1p; [M-H]− m/z = 638.0301) (Figure 3C). To our knowledge, MbcT represents the first reported enzyme with NAD+ phosphorylase activity (Figure 3D). A kinetic analysis of MbcT activity, based on NAD+ consumption at saturating orthophosphate conditions, yielded a Km of 110 ± 8 μM (Figure 3E). The turnover number of MbcT for NAD+ phosphorolysis (kcat) was 167 ± 3 s−1 (Figures S6A and S6B). By contrast, MbcT-R27E did not show any detectable NAD+ turnover establishing the essentiality of Arg27 for NAD+ phosphorolysis (Figure 3F). With a catalytic efficiency (kcat/Km) of 1.5 × 106 M−1s−1, MbcT is one of the most effective NAD+-degrading toxins characterized to date, more potent than diphtheria toxin (5 × 105 M−1s−1) (Perikh and Schramm, 2004Perikh S.L. Schramm V.L. Transition state structure for ADP-ribosylation of eukariotic elongation factor 2 catalyzed by diphtheria toxin.Biochemistry. 2004; 43: 1204-1212Crossref PubMed Scopus (42) Google Scholar) and the mycobacterial NADase TNT (8.4 × 104 M−1s−1) (Sun et al., 2015Sun J. Siroy A. Lokareddy R.K. Speer A. Doornbos K.S. Cingolani G. Niederweis M. The tuberculosis necrotizing toxin kills macrophages by hydrolyzing NAD.Nat. Struct. Mol. Biol. 2015; 22: 672-678Crossref PubMed Scopus (88) Google Scholar). The high catalytic efficiency of MbcT implies that this enzyme has specifically evolved to carry out NAD+ phosphorolysis. To determine whether MbcT exerts its toxic effect via NAD+ turnover, we measured the levels of NAD+ in MtbΔTA expressing mbcT. We observed rapid depletion of intracellular NAD+ upon induction of mbcT expression, whereas control strains expressing no toxin or the MbcT-R27E inactive mutant exhibited no decrease in intracellular NAD+ levels (Figure 4A). We also exploited the mbcT-inducible system described above to evaluate MbcT toxicity in vivo. First, we showed that, unlike TNT (Sun et al., 2015Sun J. Siroy A. Lokareddy R.K. Speer A. Doornbos K.S. Cingolani G. Niederweis M. The tuberculosis necrotizing toxin kills macrophages by hydrolyzing NAD.Nat. Struct. Mol. Biol. 2015; 22: 672-678Crossref PubMed Scopus (88) Google Scholar), ectopic expression of mbcT in WT Mtb had no deleterious effect on infected human monocyte-derived macrophages (hMDM) (Figure 4B). We then infected hMDM with MtbΔTA transformed with a control vector or a plasmid carrying ATc-inducible mbcT. Induction of mbcT expression 2 days after infection resulted in more than a 10-fold decrease in the intracellular bacterial load (Figure 4C). Next, we infected immune-deficient SCID mice, which are highly sensitive to Mtb infection, with the same MtbΔTA strain. Doxycycline-mediated induction of mbcT after MtbΔTA infection prolonged the survival of infected mice by ∼40% compared to controls without doxycycline (Figure 4D). In addition, we infected immune-competent C57BL/6 mice with the same bacterial strains and induced mbcT expression with doxycycline 21 days after infection. At this stage, the Mtb load in the lungs reaches a plateau. MbcT induction resulted in the potent killing of Mtb (5-fold reduction in CFUs). Further, MbcT enhanced the therapeutic efficacy of the frontline anti-TB drug isoniazid (INH). Treatment with INH alone led to a 10-fold reduction in CFU relative to untreated mice, whereas INH treatment combined with mbcT expression led to a 100-fold reduction in CFUs, indicative of a synergistic effect (Figure 4E). These results indicate that MbcT is highly toxic to Mtb in vivo when not neutralized by MbcA. As such, small inhibitory molecules able to dislocate the MbcTA complex could be promising candidates for the development of novel therapeutics to control Mtb infection. The molecular mechanism underpinning MbcT toxicity, NAD+ phosphorolysis, is unprecedented for TA modules. To our knowledge, MbcTA is also the first TA system that degrades an essential cellular metabolite resulting in rapid cell death. Yet, the biological role of the MbcTA system remains elusive. We did not detect any particular phenotype in our MbcTA-KO mutant in a variety of stress conditions in vitro and in vivo (data not shown), so the relevance of the MbcTA system in the Mtb life cycle is difficult to anticipate. This might be because this system would need to be inactivated together with other TA pairs in order to observe a phenotype, as reported for MazEF TA pairs (Tiwari et al., 2015Tiwari P. Arora G. Singh M. Kidwai S. Narayan O.P. Singh R. MazF ribonucleases promote Mycobacterium tuberculosis drug tolerance and virulence in guinea pigs.Nat. Commun. 2015; 6: 6059Crossref PubMed Scopus (1" @default.
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• W2916874117 date "2019-03-01" @default.
• W2916874117 modified "2023-10-12" @default.
• W2916874117 title "An NAD+ Phosphorylase Toxin Triggers Mycobacterium tuberculosis Cell Death" @default.
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