Genome-wide gene expression tuning reveals diverse vulnerabilities of M. tuberculosis.

Antibacterial agents target the products of essential genes but rarely achieve complete target inhibition. Thus, the all-or-none definition of essentiality afforded by traditional genetic approaches fails to discern the most attractive bacterial targets: those whose incomplete inhibition results in major fitness costs. In contrast, gene "vulnerability" is a continuous, quantifiable trait that relates the magnitude of gene inhibition to the effect on bacterial fitness. We developed a CRISPR interference-based functional genomics method to systematically titrate gene expression in Mycobacterium tuberculosis (Mtb) and monitor fitness outcomes. We identified highly vulnerable genes in various processes, including novel targets unexplored for drug discovery. Equally important, we identified invulnerable essential genes, potentially explaining failed drug discovery efforts. Comparison of vulnerability between the reference and a hypervirulent Mtb isolate revealed incomplete conservation of vulnerability and that differential vulnerability can predict differential antibacterial susceptibility. Our results quantitatively redefine essential bacterial processes and identify high-value targets for drug development.

An NAD+ Phosphorylase Toxin Triggers Mycobacterium tuberculosis Cell Death.

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.

A dose-response model for statistical analysis of chemical genetic interactions in CRISPRi screens.

An important application of CRISPR interference (CRISPRi) technology is for identifying chemical-genetic interactions (CGIs). Discovery of genes that interact with exposure to antibiotics can yield insights to drug targets and mechanisms of action or resistance. The objective is to identify CRISPRi mutants whose relative abundance is suppressed (or enriched) in the presence of a drug when the target protein is depleted, reflecting synergistic behavior. Different sgRNAs for a given target can induce a wide range of protein depletion and differential effects on growth rate. The effect of sgRNA strength can be partially predicted based on sequence features. However, the actual growth phenotype depends on the sensitivity of cells to depletion of the target protein. For essential genes, sgRNA efficiency can be empirically measured by quantifying effects on growth rate. We observe that the most efficient sgRNAs are not always optimal for detecting synergies with drugs. sgRNA efficiency interacts in a non-linear way with drug sensitivity, producing an effect where the concentration-dependence is maximized for sgRNAs of intermediate strength (and less so for sgRNAs that induce too much or too little target depletion). To capture this interaction, we propose a novel statistical method called CRISPRi-DR (for Dose-Response model) that incorporates both sgRNA efficiencies and drug concentrations in a modified dose-response equation. We use CRISPRi-DR to re-analyze data from a recent CGI experiment in Mycobacterium tuberculosis to identify genes that interact with antibiotics. This approach can be generalized to non-CGI datasets, which we show via an CRISPRi dataset for E. coli growth on different carbon sources. The performance is competitive with the best of several related analytical methods. However, for noisier datasets, some of these methods generate far more significant interactions, likely including many false positives, whereas CRISPRi-DR maintains higher precision, which we observed in both empirical and simulated data.

Large-scale chemical-genetics yields new M. tuberculosis inhibitor classes.

New antibiotics are needed to combat rising levels of resistance, with new Mycobacterium tuberculosis (Mtb) drugs having the highest priority. However, conventional whole-cell and biochemical antibiotic screens have failed. Here we develop a strategy termed PROSPECT (primary screening of strains to prioritize expanded chemistry and targets), in which we screen compounds against pools of strains depleted of essential bacterial targets. We engineered strains that target 474 essential Mtb genes and screened pools of 100-150 strains against activity-enriched and unbiased compound libraries, probing more than 8.5 million chemical-genetic interactions. Primary screens identified over tenfold more hits than screening wild-type Mtb alone, with chemical-genetic interactions providing immediate, direct target insights. We identified over 40 compounds that target DNA gyrase, the cell wall, tryptophan, folate biosynthesis and RNA polymerase, as well as inhibitors that target EfpA. Chemical optimization yielded EfpA inhibitors with potent wild-type activity, thus demonstrating the ability of PROSPECT to yield inhibitors against targets that would have eluded conventional drug discovery.

Chemical-genetic interaction mapping links carbon metabolism and cell wall structure to tuberculosis drug efficacy.

Current chemotherapy against Mycobacterium tuberculosis (Mtb), an important human pathogen, requires a multidrug regimen lasting several months. While efforts have been made to optimize therapy by exploiting drug­drug synergies, testing new drug combinations in relevant host environments remains arduous. In particular, host environments profoundly affect the bacterial metabolic state and drug efficacy, limiting the accuracy of predictions based on in vitro assays alone. In this study, we utilized conditional Mtb knockdown mutants of essential genes as an experimentally tractable surrogate for drug treatment and probe the relationship between Mtb carbon metabolism and chemical­genetic interactions (CGIs). We examined the antitubercular drugs isoniazid, rifampicin, and moxifloxacin and found that CGIs are differentially responsive to the metabolic state, defining both environment-independent and -dependent interactions. Specifically, growth on the in vivo­relevant carbon source, cholesterol, reduced rifampicin efficacy by altering mycobacterial cell surface lipid composition. We report that a variety of perturbations in cell wall synthesis pathways restore rifampicin efficacy during growth on cholesterol, and that both environment-independent and cholesterol-dependent in vitro CGIs could be leveraged to enhance bacterial clearance in the mouse infection model. Our findings present an atlas of chemical­genetic­environmental interactions that can be used to optimize drug­drug interactions, as well as provide a framework for understanding in vitro correlates of in vivo efficacy.

Re-discovery of PF-3845 as a new chemical scaffold inhibiting phenylalanyl-tRNA synthetase in Mycobacterium tuberculosis.

Mycobacteria tuberculosis (Mtb) remains the deadliest pathogenic bacteria worldwide. The search for new antibiotics to treat drug-sensitive as well as drug-resistant tuberculosis has become a priority. The essential enzyme phenylalanyl-tRNA synthetase (PheRS) is an antibacterial drug target because of the large differences between bacterial and human PheRS counterparts. In a high-throughput screening of 2148 bioactive compounds, PF-3845, which is a known inhibitor of human fatty acid amide hydrolase (FAAH), was identified inhibiting Mtb PheRS at Ki ~0.73 ± 0.06 µM. The inhibition mechanism was studied with enzyme kinetics, protein structural modelling and crystallography, in comparison to a PheRS inhibitor of the noted phenyl-thiazolylurea-sulfonamide class. The 2.3-Å crystal structure of Mtb PheRS in complex with PF-3845 revealed its novel binding mode, in which a trifluoromethyl-pyridinylphenyl group occupies the Phe pocket while a piperidine-piperazine urea group binds into the ATP pocket through an interaction network enforced by a sulfate ion. It represents the first non-nucleoside bi-substrate competitive inhibitor of bacterial PheRS. PF-3845 inhibits the in vitro growth of Mtb H37Rv at ~24 µM, and the potency of PF-3845 increased against Mtb pheS-FDAS, suggesting on target activity in mycobacterial whole cells.  PF-3845 does not inhibit human cytoplasmic or mitochondrial PheRS in biochemical assay, which can be explained from the crystal structures. Further medicinal chemistry efforts focused on the piperidine-piperazine urea moiety may result in the identification of a selective antibacterial lead compound.

Rediscovery of PF-3845 as a new chemical scaffold inhibiting phenylalanyl-tRNA synthetase in Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) remains the deadliest pathogenic bacteria worldwide. The search for new antibiotics to treat drug-sensitive as well as drug-resistant tuberculosis has become a priority. The essential enzyme phenylalanyl-tRNA synthetase (PheRS) is an antibacterial drug target because of the large differences between bacterial and human PheRS counterparts. In a high-throughput screening of 2148 bioactive compounds, PF-3845, which is a known inhibitor of human fatty acid amide hydrolase, was identified inhibiting Mtb PheRS at Ki ∼ 0.73 ± 0.06 µM. The inhibition mechanism was studied with enzyme kinetics, protein structural modeling, and crystallography, in comparison to a PheRS inhibitor of the noted phenyl-thiazolylurea-sulfonamide class. The 2.3-Å crystal structure of Mtb PheRS in complex with PF-3845 revealed its novel binding mode, in which a trifluoromethyl-pyridinylphenyl group occupies the phenylalanine pocket, whereas a piperidine-piperazine urea group binds into the ATP pocket through an interaction network enforced by a sulfate ion. It represents the first non-nucleoside bisubstrate competitive inhibitor of bacterial PheRS. PF-3845 inhibits the in vitro growth of Mtb H37Rv at ∼24 µM, and the potency of PF-3845 increased against an engineered strain Mtb pheS-FDAS, suggesting on target activity in mycobacterial whole cells. PF-3845 does not inhibit human cytoplasmic or mitochondrial PheRS in biochemical assay, which can be explained from the crystal structures. Further medicinal chemistry efforts focused on the piperidine-piperazine urea moiety may result in the identification of a selective antibacterial lead compound.

Depletion of the DarG antitoxin in Mycobacterium tuberculosis triggers the DNA-damage response and leads to cell death.

Of the ~80 putative toxin-antitoxin (TA) modules encoded by the bacterial pathogen Mycobacterium tuberculosis (Mtb), three contain antitoxins essential for bacterial viability. One of these, Rv0060 (DNA ADP-ribosyl glycohydrolase, DarGMtb ), functions along with its cognate toxin Rv0059 (DNA ADP-ribosyl transferase, DarTMtb ), to mediate reversible DNA ADP-ribosylation (Jankevicius et al., 2016). We demonstrate that DarTMtb -DarGMtb form a functional TA pair and essentiality of darGMtb is dependent on the presence of darTMtb , but simultaneous deletion of both darTMtb -darGMtb does not alter viability of Mtb in vitro or in mice. The antitoxin, DarGMtb , forms a cytosolic complex with DNA-repair proteins that assembles independently of either DarTMtb or interaction with DNA. Depletion of DarGMtb alone is bactericidal, a phenotype that is rescued by expression of an orthologous antitoxin, DarGTaq , from Thermus aquaticus. Partial depletion of DarGMtb triggers a DNA-damage response and sensitizes Mtb to drugs targeting DNA metabolism and respiration. Induction of the DNA-damage response is essential for Mtb to survive partial DarGMtb -depletion and leads to a hypermutable phenotype.

Tissue Distribution of Doxycycline in Animal Models of Tuberculosis.

Doxycycline, an FDA-approved tetracycline, is used in tuberculosis in vivo models for the temporal control of mycobacterial gene expression. In these models, animals are infected with recombinant Mycobacterium tuberculosis carrying genes of interest under transcriptional control of the doxycycline-responsive TetR-tetO unit. To minimize fluctuations of plasma levels, doxycycline is usually administered in the diet. However, tissue penetration studies to identify the minimum doxycycline content in food achieving complete repression of TetR-controlled genes in tuberculosis (TB)-infected organs and lesions have not been conducted. Here, we first determined the tetracycline concentrations required to achieve silencing of M. tuberculosis target genes in vitro Next, we measured doxycycline concentrations in plasma, major organs, and lung lesions in TB-infected mice and rabbits and compared these values to silencing concentrations measured in vitro We found that 2,000 ppm doxycycline supplemented in mouse and rabbit feed is sufficient to reach target concentrations in TB lesions. In rabbit chow, the calcium content had to be reduced 5-fold to minimize chelation of doxycycline and deliver adequate oral bioavailability. Clearance kinetics from major organs and lung lesions revealed that doxycycline levels fall below concentrations that repress tet promoters within 7 to 14 days after doxycycline is removed from the diet. In summary, we have shown that 2,000 ppm doxycycline supplemented in standard mouse diet and in low-calcium rabbit diet delivers concentrations adequate to achieve full repression of tet promoters in infected tissues of mice and rabbits.

Glyoxylate detoxification is an essential function of malate synthase required for carbon assimilation in Mycobacterium tuberculosis.

The glyoxylate shunt is a metabolic pathway of bacteria, fungi, and plants used to assimilate even-chain fatty acids (FAs) and has been implicated in persistence of Mycobacterium tuberculosis (Mtb). Recent work, however, showed that the first enzyme of the glyoxylate shunt, isocitrate lyase (ICL), may mediate survival of Mtb during the acute and chronic phases of infection in mice through physiologic functions apart from fatty acid metabolism. Here, we report that malate synthase (MS), the second enzyme of the glyoxylate shunt, is essential for in vitro growth and survival of Mtb on even-chain fatty acids, in part, for a previously unrecognized activity: mitigating the toxicity of glyoxylate excess arising from metabolism of even-chain fatty acids. Metabolomic profiling revealed that MS-deficient Mtb cultured on fatty acids accumulated high levels of the ICL aldehyde endproduct, glyoxylate, and increased levels of acetyl phosphate, acetoacetyl coenzyme A (acetoacetyl-CoA), butyryl CoA, acetoacetate, and ß-hydroxybutyrate. These changes were indicative of a glyoxylate-induced state of oxaloacetate deficiency, acetate overload, and ketoacidosis. Reduction of intrabacterial glyoxylate levels using a chemical inhibitor of ICL restored growth of MS-deficient Mtb, despite inhibiting entry of carbon into the glyoxylate shunt. In vivo depletion of MS resulted in sterilization of Mtb in both the acute and chronic phases of mouse infection. This work thus identifies glyoxylate detoxification as an essential physiologic function of Mtb malate synthase and advances its validation as a target for drug development.

Statistical analysis of variability in TnSeq data across conditions using zero-inflated negative binomial regression.

BACKGROUND: Deep sequencing of transposon mutant libraries (or TnSeq) is a powerful method for probing essentiality of genomic loci under different environmental conditions. Various analytical methods have been described for identifying conditionally essential genes whose tolerance for insertions varies between two conditions. However, for large-scale experiments involving many conditions, a method is needed for identifying genes that exhibit significant variability in insertions across multiple conditions. RESULTS: In this paper, we introduce a novel statistical method for identifying genes with significant variability of insertion counts across multiple conditions based on Zero-Inflated Negative Binomial (ZINB) regression. Using likelihood ratio tests, we show that the ZINB distribution fits TnSeq data better than either ANOVA or a Negative Binomial (in a generalized linear model). We use ZINB regression to identify genes required for infection of M. tuberculosis H37Rv in C57BL/6 mice. We also use ZINB to perform a analysis of genes conditionally essential in H37Rv cultures exposed to multiple antibiotics. CONCLUSIONS: Our results show that, not only does ZINB generally identify most of the genes found by pairwise resampling (and vastly out-performs ANOVA), but it also identifies additional genes where variability is detectable only when the magnitudes of insertion counts are treated separately from local differences in saturation, as in the ZINB model.

Mycobacterial genes essential for the pathogen's survival in the host.

Mycobacterium tuberculosis (Mtb) has evolved within the human immune system as both host and reservoir. The study of genes required for its growth and persistence in vivo thus offers linked insights into its pathogenicity and host immunity. Studies of Mtb mutants have implicated metabolic adaptation (consisting of carbon, nitrogen, vitamin, and cofactor metabolism), intrabacterial pH homeostasis, and defense against reactive oxygen and reactive nitrogen species, as key determinants of its pathogenicity. However, the mechanisms of host immunity are complex and often combinatorial. Growing evidence has thus begun to reveal that the determinants of Mtb's pathogenicity may serve a broader and more complex array of functions than the isolated experimental settings in which they were initially found. Here, we review select examples, which exemplify this complexity, highlighting the distinct phases of Mtb's life cycle and the diverse microenvironments encountered therein.

Mycobacterium tuberculosis Thioredoxin Reductase Is Essential for Thiol Redox Homeostasis but Plays a Minor Role in Antioxidant Defense.

Mycobacterium tuberculosis (Mtb) must cope with exogenous oxidative stress imposed by the host. Unlike other antioxidant enzymes, Mtb's thioredoxin reductase TrxB2 has been predicted to be essential not only to fight host defenses but also for in vitro growth. However, the specific physiological role of TrxB2 and its importance for Mtb pathogenesis remain undefined. Here we show that genetic inactivation of thioredoxin reductase perturbed several growth-essential processes, including sulfur and DNA metabolism and rapidly killed and lysed Mtb. Death was due to cidal thiol-specific oxidizing stress and prevented by a disulfide reductant. In contrast, thioredoxin reductase deficiency did not significantly increase susceptibility to oxidative and nitrosative stress. In vivo targeting TrxB2 eradicated Mtb during both acute and chronic phases of mouse infection. Deliberately leaky knockdown mutants identified the specificity of TrxB2 inhibitors and showed that partial inactivation of TrxB2 increased Mtb's susceptibility to rifampicin. These studies reveal TrxB2 as essential thiol-reducing enzyme in Mtb in vitro and during infection, establish the value of targeting TrxB2, and provide tools to accelerate the development of TrxB2 inhibitors.

Critical Impact of Peptidoglycan Precursor Amidation on the Activity of l,d-Transpeptidases from Enterococcus faecium and Mycobacterium tuberculosis.

The bacterial cell wall peptidoglycan contains unusual l- and d-amino acids assembled as branched peptides. Insight into the biosynthesis of the polymer has been hampered by limited access to substrates and to suitable polymerization assays. Here we report the full synthesis of the peptide stem of peptidoglycan precursors from two pathogenic bacteria, Enterococcus faecium and Mycobacterium tuberculosis, and the development of a sensitive post-derivatization assay for their cross-linking by l,d-transpeptidases. Access to series of stem peptides showed that amidation of free carboxyl groups is essential for optimal enzyme activity, in particular the amidation of diaminopimelate (DAP) residues for the cross-linking activity of the l,d-transpeptidase LdtMt2 from M. tuberculosis. Accordingly, construction of a conditional mutant established the essential role of AsnB indicating that this DAP amidotransferase is an attractive target for the development of anti-mycobacterial drugs.

Chemical Genetic Interaction Profiling Reveals Determinants of Intrinsic Antibiotic Resistance in Mycobacterium tuberculosis.

Chemotherapy for tuberculosis (TB) is lengthy and could benefit from synergistic adjuvant therapeutics that enhance current and novel drug regimens. To identify genetic determinants of intrinsic antibiotic susceptibility in Mycobacterium tuberculosis, we applied a chemical genetic interaction (CGI) profiling approach. We screened a saturated transposon mutant library and identified mutants that exhibit altered fitness in the presence of partially inhibitory concentrations of rifampin, ethambutol, isoniazid, vancomycin, and meropenem, antibiotics with diverse mechanisms of action. This screen identified the M. tuberculosis cell envelope to be a major determinant of antibiotic susceptibility but did not yield mutants whose increase in susceptibility was due to transposon insertions in genes encoding efflux pumps. Intrinsic antibiotic resistance determinants affecting resistance to multiple antibiotics included the peptidoglycan-arabinogalactan ligase Lcp1, the mycolic acid synthase MmaA4, the protein translocase SecA2, the mannosyltransferase PimE, the cell envelope-associated protease CaeA/Hip1, and FecB, a putative iron dicitrate-binding protein. Characterization of a deletion mutant confirmed FecB to be involved in the intrinsic resistance to every antibiotic analyzed. In contrast to its predicted function, FecB was dispensable for growth in low-iron medium and instead functioned as a critical mediator of envelope integrity.

Disruption of an M. tuberculosis membrane protein causes a magnesium-dependent cell division defect and failure to persist in mice.

The identification of Mycobacterium tuberculosis genes necessary for persistence in vivo provides insight into bacterial biology as well as host defense strategies. We show that disruption of M. tuberculosis membrane protein PerM (Rv0955) resulted in an IFN-γ-dependent persistence defect in chronic mouse infection despite the mutant's near normal growth during acute infection. The perM mutant required increased magnesium for replication and survival; incubation in low magnesium media resulted in cell elongation and lysis. Transcriptome analysis of the perM mutant grown in reduced magnesium revealed upregulation of cell division and cell wall biosynthesis genes, and live cell imaging showed PerM accumulation at the division septa in M. smegmatis. The mutant was acutely sensitive to ß-lactam antibiotics, including specific inhibitors of cell division-associated peptidoglycan transpeptidase FtsI. Together, these data implicate PerM as a novel player in mycobacterial cell division and pathogenesis, and are consistent with the hypothesis that immune activation deprives M. tuberculosis of magnesium.

Therapeutic Potential of the Mycobacterium tuberculosis Mycolic Acid Transporter, MmpL3.

In recent years, whole-cell-based screens for novel small molecule inhibitors active against Mycobacterium tuberculosis in culture followed by the whole-genome sequencing of spontaneous resistant mutants have identified multiple chemical scaffolds thought to kill the bacterium through the inactivation of the mycolic acid transporter, MmpL3. Consistent with the fact that MmpL3 is required for the formation of the mycobacterial outer membrane, we have conclusively shown in this study, using conditionally regulated knockdown mutants, that mmpL3 is required for the replication and viability of M. tuberculosis, both under standard laboratory growth conditions and during the acute and chronic phases of infection in mice. Speaking for the vulnerability of this target, silencing mmpL3 had a rapid bactericidal effect on actively replicating cells in vitro and reduced by 3 to 5 logs in less than 4 weeks the bacterial loads of acutely and chronically infected mouse lungs, respectively. Depletion of MmpL3 further rendered M. tuberculosis hypersusceptible to MmpL3 inhibitors. The exquisite vulnerability of MmpL3 at all stages of the infection establishes this transporter as an attractive new target with the potential to improve and shorten current drug-susceptible and drug-resistant tuberculosis chemotherapies.

Inactivation of fructose-1,6-bisphosphate aldolase prevents optimal co-catabolism of glycolytic and gluconeogenic carbon substrates in Mycobacterium tuberculosis.

Metabolic pathways used by Mycobacterium tuberculosis (Mtb) to establish and maintain infections are important for our understanding of pathogenesis and the development of new chemotherapies. To investigate the role of fructose-1,6-bisphosphate aldolase (FBA), we engineered an Mtb strain in which FBA levels were regulated by anhydrotetracycline. Depletion of FBA resulted in clearance of Mtb in both the acute and chronic phases of infection in vivo, and loss of viability in vitro when cultured on single carbon sources. Consistent with prior reports of Mtb's ability to co-catabolize multiple carbon sources, this in vitro essentiality could be overcome when cultured on mixtures of glycolytic and gluconeogenic carbon sources, enabling generation of an fba knockout (Δfba). In vitro studies of Δfba however revealed that lack of FBA could only be compensated for by a specific balance of glucose and butyrate in which growth and metabolism of butyrate were determined by Mtb's ability to co-catabolize glucose. These data thus not only evaluate FBA as a potential drug target in both replicating and persistent Mtb, but also expand our understanding of the multiplicity of in vitro conditions that define the essentiality of Mtb's FBA in vivo.

Post-translational regulation via Clp protease is critical for survival of Mycobacterium tuberculosis.

Unlike most bacterial species, Mycobacterium tuberculosis depends on the Clp proteolysis system for survival even in in vitro conditions. We hypothesized that Clp is required for the physiologic turnover of mycobacterial proteins whose accumulation is deleterious to bacterial growth and survival. To identify cellular substrates, we employed quantitative proteomics and transcriptomics to identify the set of proteins that accumulated upon the loss of functional Clp protease. Among the set of potential Clp substrates uncovered, we were able to unambiguously identify WhiB1, an essential transcriptional repressor capable of auto-repression, as a substrate of the mycobacterial Clp protease. Dysregulation of WhiB1 turnover had a toxic effect that was not rescued by repression of whiB1 transcription. Thus, under normal growth conditions, Clp protease is the predominant regulatory check on the levels of potentially toxic cellular proteins. Our findings add to the growing evidence of how post-translational regulation plays a critical role in the regulation of bacterial physiology.

Mycobacterium tuberculosis exploits asparagine to assimilate nitrogen and resist acid stress during infection.

Mycobacterium tuberculosis is an intracellular pathogen. Within macrophages, M. tuberculosis thrives in a specialized membrane-bound vacuole, the phagosome, whose pH is slightly acidic, and where access to nutrients is limited. Understanding how the bacillus extracts and incorporates nutrients from its host may help develop novel strategies to combat tuberculosis. Here we show that M. tuberculosis employs the asparagine transporter AnsP2 and the secreted asparaginase AnsA to assimilate nitrogen and resist acid stress through asparagine hydrolysis and ammonia release. While the role of AnsP2 is partially spared by yet to be identified transporter(s), that of AnsA is crucial in both phagosome acidification arrest and intracellular replication, as an M. tuberculosis mutant lacking this asparaginase is ultimately attenuated in macrophages and in mice. Our study provides yet another example of the intimate link between physiology and virulence in the tubercle bacillus, and identifies a novel pathway to be targeted for therapeutic purposes.