Production of avirulent mutants of Mycobacterium bovis with vaccine properties by the use of illegitimate recombination and screening of stationary-phase cultures

Collins, D M; Wilson, Theresa; Campbell, Shannon; Buddle, Bryce M.; Wards, Barry J.; Hotter, Grant S.; Lisle, Geoffrey W. de

doi:10.1099/00221287-148-10-3019

Cited by 44 publications

(42 citation statements)

References 35 publications

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“…Also, methionine sulfoximine augments the therapeutic efficacy of isoniazid in a guinea pig tuberculosis model and inhibits the growth of MTb in human macrophages (6,8). Interestingly, the glnA1 gene encoding MTb GS appears to be essential for bacterial survival (14), and Mycobacterium bovis mutants with GS inactivated by illegitimate recombination were avirulent (15). Collectively, these observations indicate that GS activity is required for MTb pathogenicity, but the precise role of this enzyme is unknown.…”

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confidence: 73%

Adenylylation and Catalytic Properties of Mycobacterium tuberculosis Glutamine Synthetase Expressed in Escherichia coli versus Mycobacteria

Mehta

Pearson

Mahajan

et al. 2004

Journal of Biological Chemistry

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. Here, we demonstrate via electrospray ionization mass spectrometry that GS from pathogenic M. tuberculosis is adenylylated by the Escherichia coli ATase. The adenylyl group can be hydrolyzed by snake venom phosphodiesterase to afford the unmodified enzyme. The site of adenylylation of MTb GS by the E. coli ATase is Tyr-406, as indicated by the lack of adenylylation of the Y406F mutant, and, as expected, is based on amino acid sequence alignments. Using electrospray ionization mass spectroscopy methodology, we found that GS is not adenylylated when obtained directly from MTb cultures that are not supplemented with glutamine. Under these conditions, the highly related but non-pathogenic Mycobacterium bovis BCG yields partially (ϳ25%) adenylylated enzyme. Upon the addition of glutamine to the cultures, the MTb GS becomes significantly adenylylated (ϳ30%), whereas the adenylylation of M. bovis BCG GS does not change. Collectively, the results demonstrate that MTb GS is a substrate for E. coli ATase, but only low adenylylation states are accessible. This parallels the low adenylylation states observed for GS from mycobacteria and suggests the intriguing possibility that adenylylation in the pathogenic versus non-pathogenic mycobacteria is differentially regulated.

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confidence: 73%

Adenylylation and Catalytic Properties of Mycobacterium tuberculosis Glutamine Synthetase Expressed in Escherichia coli versus Mycobacteria

Mehta

Pearson

Mahajan

et al. 2004

Journal of Biological Chemistry

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“…Taken together, these data indicate that the glyoxylate shunt is critical for long-term bacillary survival in infected host tissues but that the requirement for this pathway is partially dependent on the immune status of the host. An essential role for gluconeogenesis in mycobacterial metabolism in vivo has been confirmed by studies showing that disruption of pckA (214), which encodes phosphoenolpyruvate carboxykinase, in virulent Mycobacterium bovis (215) or the vaccine strain M. bovis bacillus Calmette-Guérin (216), resulted in reduced virulence in animal models of infection. Expression of the M. tuberculosis genes pckA and glpX, the latter of which encodes fructose 1,6-bisphosphatase, (215)(216)(217)(218), was found to be upregulated in the lungs of chronically infected mice (216).…”

Section: Microbial Factors Involved In Ltbi Lipid and Energy Metabolismmentioning

confidence: 93%

Latent Tuberculosis Infection: Myths, Models, and Molecular Mechanisms

Dutta

Karakousis

2014

Microbiol Mol Biol Rev

195

174

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SUMMARY The aim of this review is to present the current state of knowledge on human latent tuberculosis infection (LTBI) based on clinical studies and observations, as well as experimental in vitro and animal models. Several key terms are defined, including “latency,” “persistence,” “dormancy,” and “antibiotic tolerance.” Dogmas prevalent in the field are critically examined based on available clinical and experimental data, including the long-held beliefs that infection is either latent or active, that LTBI represents a small population of nonreplicating, “dormant” bacilli, and that caseous granulomas are the haven for LTBI. The role of host factors, such as CD4 + and CD8 + T cells, T regulatory cells, tumor necrosis factor alpha (TNF-α), and gamma interferon (IFN-γ), in controlling TB infection is discussed. We also highlight microbial regulatory and metabolic pathways implicated in bacillary growth restriction and antibiotic tolerance under various physiologically relevant conditions. Finally, we pose several clinically important questions, which remain unanswered and will serve to stimulate future research on LTBI.

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“…The gene encoding PEPCK, pckA, is induced by fatty acids in vitro and during growth of Mtb in mice, suggesting a demand for gluconeogenesis during infection (9)(10)(11). PEPCK was first implicated in mycobacterial pathogenesis because M. bovis deficient in PEPCK did not cause spleen lesions in guinea pigs following s.c. injection (18). In addition, M. bovis bacillus Calmette-Guérin lacking pckA was killed almost 10-fold more than wild-type (WT) bacillus Calmette-Guérin in mouse spleens between day 20 and 35 after i.v.…”

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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.

312

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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Production of avirulent mutants of Mycobacterium bovis with vaccine properties by the use of illegitimate recombination and screening of stationary-phase cultures

Cited by 44 publications

References 35 publications

Adenylylation and Catalytic Properties of Mycobacterium tuberculosis Glutamine Synthetase Expressed in Escherichia coli versus Mycobacteria

Adenylylation and Catalytic Properties of Mycobacterium tuberculosis Glutamine Synthetase Expressed in Escherichia coli versus Mycobacteria

Latent Tuberculosis Infection: Myths, Models, and Molecular Mechanisms

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

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