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Boshoff, Helena I. M.,Xu, Xia,Tahlan, Kapil,Dowd, Cynthia S.,Pethe, Kevin,Camacho, Luis R.,Park, Tae-Ho,Yun, Chang-Soo,Schnappinger, Dirk,Ehrt, Sabine,Williams, Kerstin J.,Barry III, Clifton E. American Society for Biochemistry and Molecular Bi 2008 The Journal of biological chemistry Vol.283 No.28
<P>Despite the presence of genes that apparently encode NAD salvage-specific enzymes in its genome, it has been previously thought that Mycobacterium tuberculosis can only synthesize NAD de novo. Transcriptional analysis of the de novo synthesis and putative salvage pathway genes revealed an up-regulation of the salvage pathway genes in vivo and in vitro under conditions of hypoxia. [14C]Nicotinamide incorporation assays in M. tuberculosis isolated directly from the lungs of infected mice or from infected macrophages revealed that incorporation of exogenous nicotinamide was very efficient in in vivo-adapted cells, in contrast to cells grown aerobically in vitro. Two putative nicotinic acid phosphoribosyltransferases, PncB1 (Rv1330c) and PncB2 (Rv0573c), were examined by a combination of in vitro enzymatic activity assays and allelic exchange studies. These studies revealed that both play a role in cofactor salvage. Mutants in the de novo pathway died upon removal of exogenous nicotinamide during active replication in vitro. Cell death is induced by both cofactor starvation and disruption of cellular redox homeostasis as electron transport is impaired by limiting NAD. Inhibitors of NAD synthetase, an essential enzyme common to both recycling and de novo synthesis pathways, displayed the same bactericidal effect as sudden NAD starvation of the de novo pathway mutant in both actively growing and nonreplicating M. tuberculosis. These studies demonstrate the plasticity of the organism in maintaining NAD levels and establish that the two enzymes of the universal pathway are attractive chemotherapeutic targets for active as well as latent tuberculosis.</P>
PA-824 Kills Nonreplicating Mycobacterium tuberculosis by Intracellular NO Release
Singh, R.,Manjunatha, U.,Boshoff, H. I. M.,Ha, Y. H.,Niyomrattanakit, P.,Ledwidge, R.,Dowd, C. S.,Lee, I. Y.,Kim, P.,Zhang, L.,Kang, S.,Keller, T. H.,Jiricek, J.,Barry, C. E. American Association for the Advancement of Scienc 2008 Science Vol.322 No.5906
<P>Bicyclic nitroimidazoles, including PA-824, are exciting candidates for the treatment of tuberculosis. These prodrugs require intracellular activation for their biological function. We found that Rv3547 is a deazaflavin-dependent nitroreductase (Ddn) that converts PA-824 into three primary metabolites; the major one is the corresponding des-nitroimidazole (des-nitro). When derivatives of PA-824 were used, the amount of des-nitro metabolite formed was highly correlated with anaerobic killing of Mycobacterium tuberculosis (Mtb). Des-nitro metabolite formation generated reactive nitrogen species, including nitric oxide (NO), which are the major effectors of the anaerobic activity of these compounds. Furthermore, NO scavengers protected the bacilli from the lethal effects of the drug. Thus, these compounds may act as intracellular NO donors and could augment a killing mechanism intrinsic to the innate immune system.</P>
Woong Park, Sae,Klotzsche, Marcus,Wilson, Daniel J.,Boshoff, Helena I.,Eoh, Hyungjin,Manjunatha, Ujjini,Blumenthal, Antje,Rhee, Kyu,Barry III, Clifton E.,Aldrich, Courtney C.,Ehrt, Sabine,Schnappinger Public Library of Science 2011 PLoS pathogens Vol.7 No.9
<▼1><P>In the search for new drug targets, we evaluated the biotin synthetic pathway of <I>Mycobacterium tuberculosis (Mtb)</I> and constructed an <I>Mtb</I> mutant lacking the biotin biosynthetic enzyme 7,8-diaminopelargonic acid synthase, BioA. In biotin-free synthetic media, <I>ΔbioA</I> did not produce wild-type levels of biotinylated proteins, and therefore did not grow and lost viability. <I>ΔbioA</I> was also unable to establish infection in mice. Conditionally-regulated knockdown strains of <I>Mtb</I> similarly exhibited impaired bacterial growth and viability <I>in vitro</I> and in mice, irrespective of the timing of transcriptional silencing. Biochemical studies further showed that BioA activity has to be reduced by approximately 99% to prevent growth. These studies thus establish that <I>de novo</I> biotin synthesis is essential for <I>Mtb</I> to establish and maintain a chronic infection in a murine model of TB. Moreover, these studies provide an experimental strategy to systematically rank the <I>in vivo</I> value of potential drug targets in <I>Mtb</I> and other pathogens.</P></▼1><▼2><P><B>Author Summary</B></P><P>We evaluated the biotin synthetic pathway of <I>Mycobacterium tuberculosis</I> (<I>Mtb</I>) as a new drug target by first generating an <I>Mtb</I> deletion mutant, <I>ΔbioA</I>, in which the biotin biosynthetic enzyme 7,8-diaminopelargonic acid synthase (BioA) has been inactivated. This mutant grew in the presence of biotin or <I>des</I>-thiobiotin, but not with an intermediate of the biotin biosynthesis pathway that requires BioA to be converted into biotin. Without exogenous biotin or <I>des</I>-thiobiotin, <I>ΔbioA,</I> was unable to produce biotinylated proteins, which are required for the biosynthesis of fatty acids, and thus died in biotin-free media. Using a regulatable promoter and different ribosome binding sequences we next constructed tightly controlled TetON mutants, in which expression of BioA could be induced with tetracyclines, but was inhibited in their absence. Characterization of these mutants during infections demonstrated that <I>de novo</I> biotin synthesis is not only required to establish infections but also to maintain bacterial persistence. Inhibition of BioA or other enzymes of the biotin biosynthesis pathways could thus be used to kill <I>Mtb</I> during both acute and chronic infections. Biochemical and immunological analyses of different <I>Mtb</I> mutants indicate that drugs targeting BioA would have to inactive approximately 99% of its activity to be effective.</P></▼2>