S-nitroso proteome of Mycobacterium tuberculosis: Enzymes of intermediary metabolism and antioxidant defense

Department of Medicine, Cornell University, Итак, New York, United States
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 02/2005; 102(2):467-72. DOI: 10.1073/pnas.0406133102
Source: PubMed


The immune response to Mycobacterium tuberculosis (Mtb) includes expression of nitric oxide (NO) synthase (NOS)2, whose products can kill Mtb in vitro with a molar potency greater than that of many conventional antitubercular agents. However, the targets of reactive nitrogen intermediates (RNIs) in Mtb are unknown. One major action of RNIs is protein S-nitrosylation. Here, we describe, to our knowledge, the first proteomic analysis of S-nitrosylation in a whole organism after treating Mtb with bactericidal concentrations of RNIs. The 29 S-nitroso proteins identified are all enzymes, mostly serving intermediary metabolism, lipid metabolism, and/or antioxidant defense. Many are essential or implicated in virulence, including defense against RNIs. For each of two target enzymes tested, lipoamide dehydrogenase and mycobacterial proteasome ATPase, S-nitrosylation caused enzyme inhibition. Moreover, endogenously biotinylated proteins were driven into mixed disulfide complexes. Targeting of metabolic enzymes and antioxidant defenses by means of protein S-nitrosylation and mixed disulfide bonding may contribute to the antimycobacterial actions of RNIs.

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Available from: Hediye Erdjument-Bromage, May 22, 2014
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    • "In E. coli, nitrate reductase is an important source of species reactive enough to nitrosylate proteins, as evidenced by an 80% reduction in protein S-nitrosylation in narG-deficient as compared to wild-type bacteria (Ralt et al. 1988; Corker 2003; Seth et al. 2012). Rhee et al. (2005) identified 29 mycobacterial proteins that were S-nitrosylated when Mtb was treated with nitrite at low pH, including KatG. In addition, heme peroxidases such as KatG oxidize nitrite to more reactive species, such as nitrogen dioxide (•NO2) and nitryl chloride (NO2Cl) (Klebanoff 1993; van der Vliet et al. 1997; Eiserich et al. 1998; Battistuzzi et al. 2010). "
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    ABSTRACT: When access to molecular oxygen is restricted, Mycobacterium tuberculosis (Mtb) can respire an alternative electron acceptor, nitrate. We found that Mtb within infected primary human macrophages in vitro at physiologic tissue oxygen tensions respired nitrate, generating copious nitrite. A strain of Mtb lacking a functioning nitrate reductase was more susceptible than wild-type Mtb to treatment with isoniazid during infection of macrophages. Likewise, nitrate reductase-deficient Mtb was more susceptible to isoniazid than wild-type Mtb in axenic culture, and more resistant to hydrogen peroxide. These phenotypes were reversed by the addition of exogenous nitrite. Further investigation suggested that nitrite might inhibit the bacterial catalase. To the extent that Mtb itself is the most relevant source of nitrite acting within Mtb, these findings suggest that inhibitors of Mtb's nitrate transporter or nitrate reductase could enhance the efficacy of isoniazid.
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    • "This strategy was used to verify the role of H2O2 or NO2 stimulus on fungal growth and cell signaling. Yeast cells were exposed to different concentrations of H2O2 or NaNO2 (in culture medium mildly acidic, pH 5.5; in this condition NaNO2 releases NO), for 5 h at 37°C [22], [23], [24]. Then yeast cells were washed and incubated for 24 h at 37°C under shaking in fresh culture media. "
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    ABSTRACT: Paracoccidioides brasiliensis, a causative agent of paracoccidioidomycosis (PCM), should be able to adapt to dramatic environmental changes inside the infected host after inhalation of air-borne conidia and transition to pathogenic yeasts. Proteins with antioxidant functions may protect fungal cells against reactive oxygen (ROS) and nitrogen (RNS) species generated by phagocytic cells, thus acting as potential virulence factors. Ras GTPases are involved in stress responses, cell morphology, and differentiation in a range of organisms. Ras, in its activated form, interacts with effector proteins and can initiate a kinase cascade. In lower eukaryotes, Byr2 kinase represents a Ras target. The present study investigated the role of Ras in P. brasiliensis after in vitro stimulus with ROS or RNS. We have demonstrated that low concentrations of H2O2 (0.1 mM) or NO2 (0.1-0.25 µM) stimulated P. brasiliensis yeast cell proliferation and that was not observed when yeast cells were pre-incubated with farnesyltransferase inhibitor. We constructed an expression plasmid containing the Byr2 Ras-binding domain (RBD) fused with GST (RBD-Byr2-GST) to detect the Ras active form. After stimulation with low concentrations of H2O2 or NO2, the Ras active form was observed in fungal extracts. Besides, NO2 induced a rapid increase in S-nitrosylated Ras levels. This alternative posttranslational modification of Ras, probably in residue Cys123, would lead to an exchange of GDP for GTP and consequent GTPase activation in P. brasiliensis. In conclusion, low concentrations of H2O2 or NO2 stimulated P. brasiliensis proliferation through Ras activation.
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    • "of biological activities. In its reduced form, sulfur is used in the biosynthesis of the amino acid cysteine, one of the prime targets for reactive nitrogen intermediates encountered by M. tuberculosis in the intracellular environment [11] . Cysteine is subsequently incorporated into mycothiol, which functions analogously to glutathione [12] and is crucial to M. tuberculosis within the granuloma for regulating the redox balance on encountering free radicals released by host cells. "
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    ABSTRACT: New therapies to control tuberculosis are urgently required because of the inability of the only available vaccine, BCG, to adequately protect against tuberculosis. Here we demonstrate that proteins of the Mycobacterium tuberculosis sulfate-assimilation pathway (SAP) represent major immunogenic targets of the bacillus, as defined by strong T-cell recognition by both mice and humans infected with M. tuberculosis. SAP proteins displayed increased expression when M. tuberculosis was resident within host cells, which may account in part for their ability to stimulate anti-M. tuberculosis host immunity. Vaccination with the first enzyme in this pathway, adenosine-5′-triphosphate sulfurylase, conferred significant protection against murine tuberculosis and boosted BCG-induced protective immunity in the lung. Therefore, we have identified SAP components as a new family of M. tuberculosis antigens, and we have demonstrated that these components are promising candidate for inclusion in new vaccines to control tuberculosis in humans.
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