Identification of a nitroimidazo-oxazine-specific protein involved in PA-824 resistance in
            <i>Mycobacterium tuberculosis</i>

Manjunatha, Ujjini H.; Boshoff, Helena I.; Dowd, Cynthia S.; Zhang, Liang; Albert, Thomas; Norton, Jason E.; Daniels, Lacy; Dick, Thomas; Pang, Siew Siew; Barry, Clifton E.

doi:10.1073/pnas.0508392103

Cited by 311 publications

(291 citation statements)

References 32 publications

(33 reference statements)

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“…Spontaneously arising PDIM-deficient variants have been described previously (1,7,8,11,15,(19)(20)(21) and are probably more common than has been reported or realized. Although spontaneous PDIM deficiency has most frequently been associated with the H37Rv strain, it has also recently been described for the M. tuberculosis Erdman strain and the clinical isolate HN878 (7,20).…”

Section: Discussionmentioning

confidence: 99%

Spontaneous Phthiocerol Dimycocerosate-Deficient Variants of Mycobacterium tuberculosis Are Susceptible to Gamma Interferon-Mediated Immunity

et al. 2011

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Onset of the adaptive immune response in mice infected with Mycobacterium tuberculosis is accompanied by slowing of bacterial replication and establishment of a chronic infection. Stabilization of bacterial numbers during the chronic phase of infection is dependent on the activity of the gamma interferon (IFN-␥)-inducible nitric oxide synthase (NOS2). Previously, we described a differential signature-tagged mutagenesis screen designed to identify M. tuberculosis "counterimmune" mechanisms and reported the isolation of three mutants in the H37Rv strain background containing transposon insertions in the rv0072, rv0405, and rv2958c genes. These mutants were impaired for replication and virulence in NOS2 ؊/؊ mice but were growth-proficient and virulent in IFN-␥ ؊/؊ mice, suggesting that the disrupted genes were required for bacterial resistance to an IFN-␥-dependent immune mechanism other than NOS2. Here, we report that the attenuation of these strains is attributable to an underlying transposon-independent deficiency in biosynthesis of phthiocerol dimycocerosate (PDIM), a cell wall lipid that is required for full virulence in mice. We performed whole-genome resequencing of a PDIM-deficient clone and identified a spontaneous point mutation in the putative polyketide synthase PpsD that results in a G44C amino acid substitution. We demonstrate by complementation with the wild-type ppsD gene and reversion of the ppsD gene to the wild-type sequence that the ppsD(G44C) point mutation is responsible for PDIM deficiency, virulence attenuation in NOS2 ؊/؊ and wild-type C57BL/6 mice, and a growth advantage in vitro in liquid culture. We conclude that PDIM biosynthesis is required for M. tuberculosis resistance to an IFN-␥-mediated immune response that is independent of NOS2.Pathogenic mycobacteria possess a unique array of complex cell wall-associated lipids. The most abundant of these lipids, the phthiocerol dimycocerosates (PDIMs) (Fig. 1), are among the best characterized (23). PDIMs contain long-chain diols esterified by methyl-branched fatty acid chains. As early as 1974, it was recognized that a spontaneously arising PDIMdeficient variant of the laboratory strain H37Rv was attenuated in a guinea pig model of infection (11). Shortly thereafter, it was shown that the in vivo survival of an avirulent Mycobacterium tuberculosis strain was enhanced by coating the bacteria with cholesterol oleate and purified PDIM (16). A genetic link between PDIM production and virulence was not established until a quarter century later, when a large chromosomal locus was identified as playing an essential role in the biosynthesis and export of PDIM (3,4,6). Transposon insertions within the fadD26, fadD28, mmpL7, and drrC genes, and in the putative transcriptional promoter region upstream of the fadD26 gene, were identified in strains deficient in surface-localized PDIM. The fadD26 and fadD28 mutants apparently fail to synthesize PDIM, whereas the mmpL7 and drrC mutants produce PDIM but accumulate it intracellularly, thus implicating these ...

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Section: Discussionmentioning

confidence: 99%

Spontaneous Phthiocerol Dimycocerosate-Deficient Variants of Mycobacterium tuberculosis Are Susceptible to Gamma Interferon-Mediated Immunity

et al. 2011

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“…This discrepancy in complementation led us to investigate whether the attenuation of the complemented strains might be due to a deficiency of the cell wall-associated lipid phthiocerol dimycocerosate (PDIM). Loss of PDIM results in impaired replication of Mtb in mouse lungs (24,25), and mutations that abolish synthesis of this lipid have been show to occur spontaneously (26)(27)(28). Lipid profile analysis revealed that both WT and ΔpckA strains expressed similar levels of PDIM.…”

Section: Pepck Is Essential For Growth and Survival Of Mtb During Botmentioning

confidence: 99%

Gluconeogenic carbon flow of tricarboxylic acid cycle intermediates is critical for Mycobacterium tuberculosis to establish and maintain infection

Marrero¹,

Rhee

Schnappinger³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

311

315

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Metabolic adaptation to the host niche is a defining feature of the pathogenicity of Mycobacterium tuberculosis (Mtb). In vitro, Mtb is able to grow on a variety of carbon sources, but mounting evidence has implicated fatty acids as the major source of carbon and energy for Mtb during infection. When bacterial metabolism is primarily fueled by fatty acids, biosynthesis of sugars from intermediates of the tricarboxylic acid cycle is essential for growth. The role of gluconeogenesis in the pathogenesis of Mtb however remains unaddressed. Phosphoenolpyruvate carboxykinase (PEPCK) catalyzes the first committed step of gluconeogenesis. We applied genetic analyses and 13 C carbon tracing to confirm that PEPCK is essential for growth of Mtb on fatty acids and catalyzes carbon flow from tricarboxylic acid cycle-derived metabolites to gluconeogenic intermediates. We further show that PEPCK is required for growth of Mtb in isolated bone marrow-derived murine macrophages and in mice. Importantly, Mtb lacking PEPCK not only failed to replicate in mouse lungs but also failed to survive, and PEPCK depletion during the chronic phase of infection resulted in mycobacterial clearance. Mtb thus relies on gluconeogenesis throughout the infection. PEPCK depletion also attenuated Mtb in IFNγ-deficient mice, suggesting that this enzyme represents an attractive target for chemotherapy.carbon metabolism | gluconeogenesis | metabolomics | microbial pathogenesis | phosphoenolpyruvate carboxykinase

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“…The reduction of these compounds is probably mediated by the nitroreductase Rv3547 as mutants lacking this enzyme (H37Rv-T2) are also cross-resistant. 16 The MAC (minimum anaerobicidal concentration) values for compounds 4-6 were equivalent. 30…”

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confidence: 97%

“…14,15 Prior work has shown that PA-824 acts as a prodrug requiring bioreductive activation before exerting its biological effect. 16 One serious issue with the clinical use of both PA-824 and OPC-67683 lies in their low water solubility. PA-824 is only soluble at 10.2μg/mL 17 (Table 1) and the in vivo activity of OPC-67683 requires formulation in 5% gum arabic.…”

mentioning

confidence: 99%

Synthesis and antitubercular activity of 7-(R)- and 7-(S)-methyl-2-nitro-6-(S)-(4-(trifluoromethoxy)benzyloxy)-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazines, analogues of PA-824

Manjunatha

Goodwin

et al. 2008

Bioorganic & Medicinal Chemistry Letters

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Nitroimidazoles such as PA-824 and OPC-67683 are currently in clinical development as members of a promising new class of therapeutics for tuberculosis. While the antitubercular activity of these compounds is high, they both suffer from poor water solubility thus complicating development. We determined the single-crystal X-ray structure of PA-824 and found a close packing of the nitroimidazoles facilitated by a pseudoaxial conformation of the p-trifluoromethoxybenzyl ether. To attempt to disrupt this tight packing by destabilizing the axial preference of this side chain, we prepared the two diastereomers of the 7-methyl-nitroimidazo-oxazine. Determination of the crystal structure of the 7-(S)-methyl derivative (5, cis) revealed that the benzylic side chain remained pseudoaxial while the 7-(R)-methyl derivative (6, trans) adopted the desired pseudoequatorial conformation. Both derivatives displayed similar activities against Mycobacterium tuberculosis, but neither showed improved aqueous solubility, suggesting that inherent lattice stability is not likely to be a major factor in limiting solubility. Conformational analysis revealed that all three compounds have similar energetically accessible conformations in solution. Additionally, these results suggest that the nitroreductase that initially recognizes PA-824 is somewhat insensitive to substitutions at the 7-position. KeywordsMycobacterium tuberculosis; PA-824; solubility Despite decades of research, tuberculosis continues to be a major public health threat. The WHO estimates that, in 2005, 1.6 million people died from tuberculosis (TB) which is caused *Corresponding author: Department of Chemistry, George Washington University, 725 21st St. NW, Corcoran Hall 107, Washington DC 20052, Tel: 202-994-8405 Fax: 202-994- Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. by the microorganism Mycobacterium tuberculosis (Mtb). 1 Current therapy for TB lasts for several months and is called directly-observed therapy short-course (DOTS). This regimen calls for an intensive phase of chemotherapy using four drugs for 2 months, followed by a continuation phase using two drugs for 4-6 months. 2 Rifampin is inarguably the most important drug in the DOTS regimen as it allowed shortening the course of therapy to 6 months from the standard 12-18 months of previous regimens. The potency of rifampin is thought to be due in part to its action against both aerobically-growing and anaerobically-adapted, nonreplicating mycobacteria. 3 NIH Public AccessNitroimidazoles are a class of compounds widely used clinically for the management of a...

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Identification of a nitroimidazo-oxazine-specific protein involved in PA-824 resistance in Mycobacterium tuberculosis

Cited by 311 publications

References 32 publications

Spontaneous Phthiocerol Dimycocerosate-Deficient Variants of Mycobacterium tuberculosis Are Susceptible to Gamma Interferon-Mediated Immunity

Spontaneous Phthiocerol Dimycocerosate-Deficient Variants of Mycobacterium tuberculosis Are Susceptible to Gamma Interferon-Mediated Immunity

Gluconeogenic carbon flow of tricarboxylic acid cycle intermediates is critical for Mycobacterium tuberculosis to establish and maintain infection

Synthesis and antitubercular activity of 7-(R)- and 7-(S)-methyl-2-nitro-6-(S)-(4-(trifluoromethoxy)benzyloxy)-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazines, analogues of PA-824

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