The tuberculosis necrotizing toxin kills macrophages by hydrolyzing NAD.

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

Expression of a Subset of Heat Stress Induced Genes of Mycobacterium tuberculosis Is Regulated by 3',5'-Cyclic AMP

Mycobacterium tuberculosis (Mtb) secretes excess of a second messenger molecule, 3',5'-cyclic AMP (cAMP), which plays a critical role in the survival of Mtb in host macrophages. Although Mtb produces cAMP in abundance, its exact role in the physiology of mycobacteria is elusive. In this study we have analyzed the expression of 16 adenylate cyclases (ACs) and kinetics of intracellular cAMP levels in Mtb during in vitro growth under the regular culture conditions, and after exposure to different stress agents. We observed a distinct expression pattern of these ACs which is correlated with intracellular cAMP levels. Interestingly cAMP levels are significantly elevated in Mtb following heat stress, whereas other stress conditions such as oxidative, nitrosative or low pH do not affect intracellular cAMP pool in vitro. A significant increase in expression by >2-fold of five ACs namely Rv1647, Rv2212, Rv1625c, Rv2488c and Rv0386 after heat stress further suggested that cAMP plays an important role in controlling Mtb response to heat stress. In the light of these observations, effect of exogenous cAMP on global gene expression profile was examined by using microarrays. The microarray gene expression analysis demonstrated that cAMP regulates expression of a subset of heat stress-induced genes comprising of dnaK, grpE, dnaJ, and Rv2025c. Further we performed electrophoretic mobility shift assay by using cAMP-receptor protein of Mtb (CRP(M)), which demonstrated that CRP(M) specifically recognizes a sequence (−301) AGCGACCGTCAGCACG (−286) in 5'-untranslated region of dnaK

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Metabolic adaptation of Mycobacterium tuberculosis (M. tuberculosis) to microbicidal intracellular environment of host macrophages is fundamental to its pathogenicity. However, an in-depth understanding of metabolic adjustments through key reaction pathways and networks is limited. To understand how such changes occur, we measured the cellular metabolome of M. tuberculosis subjected to four microbicidal stresses using liquid chromatography-mass spectrometric multiple reactions monitoring (LC-MRM/MS). Overall, 87 metabolites were identified. The metabolites best describing the separation between stresses were identified through multivariate analysis. The coupling of the metabolite measurements with existing genome-scale metabolic model, and using constraint-based simulation led to several new concepts and unreported observations in M. tuberculosis; such as (i) the high levels of released ammonia as an adaptive response to acidic stress was due to increased flux through L-asparaginase rather than urease activity; (ii) nutrient starvation-induced anaplerotic pathway for generation of TCA intermediates from phosphoenolpyruvate using phosphoenolpyruvate kinase; (iii) quenching of protons through GABA shunt pathway or sugar alcohols as possible mechanisms of early adaptation to acidic and oxidative stresses; and (iv) usage of alternate cofactors by the same enzyme as a possible mechanism of rewiring metabolic pathways to overcome stresses. Besides providing new leads and important nodes that can be used for designing intervention strategies, the study advocates the strength of applying flux balance analyses coupled with metabolomics to get a global picture of complex metabolic adjustments.</p