Biosynthesis and Recycling of Nicotinamide Cofactors in Mycobacterium tuberculosis
AN ESSENTIAL ROLE FOR NAD IN NONREPLICATING BACILLI*
Helena I. M. Boshoff
1,
Xia Xu,
Kapil Tahlan,
Cynthia S. Dowd2,
Kevin Pethe
,
Luis R. Camacho,
Tae-Ho Park¶,
Chang-Soo Yun¶,
Dirk Schnappinger||,
Sabine Ehrt||,
Kerstin J. Williams**, and
Clifton E. Barry, III
From the
Tuberculosis Research Section, Laboratory of Clinical Infectious Diseases, NIAID, National Institutes of Health, Bethesda, Maryland 20892, Novartis Institute for Tropical Diseases, Singapore 138670, Singapore, ¶Center for Anti-Infective Agents Research, Drug Discovery Division, Korea Research Institute of Chemical Technology, 100 Jang-Dong, Daejeon, 305-600, Korea, the ||Department of Microbiology and Immunology, Weill Cornell Medical College, New York, New York 10065, and the **Department of Microbiology, Centre for Molecular Microbiology and Infection, 3.40 Flowers Building, Imperial College London, South Kensington, London SW7 2AZ, United Kingdom
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.
Received for publication, January 28, 2008
, and in revised form, May 19, 2008.
* This work was supported, in whole or in part, by the National Institutes of Health Intramural Research Program of the Division of Intramural Research, NIAID (to C. E. B.). This work was also supported by a grant from the Bill and Melinda Gates Foundation and the Wellcome Trust through the Grand Challenges in Global Health Initiative. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1 and Fig. S1.
2 Present address: Dept. of Chemistry, George Washington University, 725 21st St. NW, Washington, D. C. 20052.
1 To whom correspondence should be addressed: 33 North Dr., Bldg. 33, Rm. 2W20G, Bethesda, MD 20892. Fax: 301-402-0993; E-mail: HBOSHOFF{at}niaid.nih.gov.
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