Glycine and alanine dehydrogenase activities are catalyzed by the same protein in<i>Mycobacterium smegmatis</i>: upregulation of both activities under microaerophilic adaptation

Usha, Veeraraghavan; Jayaraman, Ramesh; Toro, Juan Carlos; Hoffner, Sven; Das, Kaveri

doi:10.1139/w01-126

Cited by 27 publications

(28 citation statements)

References 19 publications

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“…Expression of ald is upregulated in M. tuberculosis upon nutrient starvation (7) and in M. marinum during persistence within the granulomas of infected frogs (10). L-Alanine dehydrogenase production and activity were also increased when M. tuberculosis and M. smegmatis were shifted from aerobic to anaerobic growth (21,31,34). In contrast, Western blotting has failed to detect L-alanine dehydrogenase in BCG, even though the ald gene has been identified by DNA hybridization (2).…”

Section: Discussionmentioning

confidence: 99%

Mycobacterium bovis BCG Vaccines Exhibit Defects in Alanine and Serine Catabolism

et al. 2003

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Mycobacterium bovis BCG is the only accepted vaccine for the prevention of tuberculosis (TB) in humans.BCG is a live vaccine, and induction of immunity to TB requires productive infection of the host by BCG. However, BCG is not a satisfactory vaccine, because it fails to protect against pulmonary TB in adults. In this study, we found that BCG strains cannot utilize many naturally occurring amino acids as the sole nitrogen source for growth. This defect is caused, at least partially, by the lack of functional metabolic enzymes. All BCG strains are unable to catabolize L-alanine or D-alanine due to a frameshift mutation in the L-alanine dehydrogenase gene (ald). Some BCG strains, such as BCG-Pasteur and BCG-Frappier, cannot catabolize L-serine, apparently due to inadequate expression of L-serine deaminase (sdaA). We also found that undegraded alanine and serine inhibit the growth of BCG through blockage of glutamine synthetase. These results suggest that BCG strains are limited in nitrogen metabolic capacity and predict defects that may restrict multiplication and persistence of the live vaccine within the host.Bacille Calmette-Guérin (BCG), an attenuated strain of Mycobacterium bovis isolated in 1921, is currently administered to over 100 million newborns per year as the only available vaccine against tuberculosis (TB) (13). BCG is only efficacious as a live vaccine; killed BCG does not provide protection in animal models, and killed Mycobacterium tuberculosis in human trials provided only weak and transient protection. In clinical trials of BCG vaccination, the observed efficacy has ranged from no protection to 80% fewer cases of TB (11, 13). A better understanding of this variable efficacy should help guide BCG programs and suggest avenues for the development of novel anti-TB vaccines.Several hypotheses have been generated to explain the vaccine trial data (3). The most prominent is that exposure to environmental mycobacteria partially sensitizes the host against mycobacteria and thereby provides heterologous immunity that obscures the potential benefits of BCG vaccination (14, 15). To support this, a recent study showed that the multiplication of the Danish strain of BCG was inhibited in animals previously sensitized with environmental mycobacteria. Consequently BCG vaccination elicited only a transient immune response and afforded no protection against a subsequent TB challenge (8). A second hypothesis involves differences in the vaccine strains used in clinical trials (9). After their introduction in 1921, BCG vaccines were maintained by in vitro passage in a variety of vaccine laboratories for 4 to 5 decades, resulting in phenotypic and genetic differences between BCG strains (6, 27). The capability of each of these BCG vaccines to protect against TB is unknown, because most clinical trials were performed with vaccine strains that have not been preserved. Notably, among genetic changes in BCG strains after 1921, one observes deletions in regulatory genes (which are postulated to govern the capacity to survi...

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Section: Discussionmentioning

confidence: 99%

Mycobacterium bovis BCG Vaccines Exhibit Defects in Alanine and Serine Catabolism

et al. 2003

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“…Expression of the ald gene as well as the synthesis and activity of Ald were observed to be increased under oxygen-limiting conditions in M. tuberculosis and Mycobacterium smegmatis (2)(3)(4)(10)(11)(12)(13)(14). Other studies reported that expression of the ald gene was upregulated in Mycobacterium marinum during persistence within its host granulomas and in M. tuberculosis under nutrient starvation conditions (5,15).…”

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confidence: 89%

“…The enzyme forms a hexamer of identical subunits with the N-terminal catalytic domain and the C-terminal NAD(H)-binding domain (6, 7). Ald was shown to be one of the major antigens found in culture filtrates of Mycobacterium tuberculosis (8,9).Expression of the ald gene as well as the synthesis and activity of Ald were observed to be increased under oxygen-limiting conditions in M. tuberculosis and Mycobacterium smegmatis (2)(3)(4)(10)(11)(12)(13)(14). Other studies reported that expression of the ald gene was upregulated in Mycobacterium marinum during persistence within its host granulomas and in M. tuberculosis under nutrient starvation conditions (5, 15).…”

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confidence: 99%

Regulation Mechanism of the ald Gene Encoding Alanine Dehydrogenase in Mycobacterium smegmatis and Mycobacterium tuberculosis by the Lrp/AsnC Family Regulator AldR

Jeong

Hyun

2015

J Bacteriol

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In the presence of alanine, AldR, which belongs to the Lrp/AsnC family of transcriptional regulators and regulates ald encoding alanine dehydrogenase in Mycobacterium smegmatis, changes its quaternary structure from a homodimer to an octamer with an open-ring conformation. Four AldR-binding sites (O2, O1, O4, and O3) with a consensus sequence of GA/T-N 2 -NWW/WWN-N 2 -A/TC were identified upstream of the M. smegmatis ald gene by means of DNase I footprinting analysis. O2, O1, and O4 are required for the induction of ald expression by alanine, while O3 is directly involved in the repression of ald expression. In addition to O3, both O1 and O4 are also necessary for full repression of ald expression in the absence of alanine, due to cooperative binding of AldR dimers to O1, O4, and O3. Binding of a molecule of the AldR octamer to the ald control region was demonstrated to require two AldR-binding sites separated by three helical turns between their centers and one additional binding site that is in phase with the two AldR-binding sites. The cooperative binding of AldR dimers to DNA requires three AldR-binding sites that are aligned with a periodicity of three helical turns. The aldR gene is negatively autoregulated independently of alanine. Comparative analysis of ald expression of M. smegmatis and Mycobacterium tuberculosis in conjunction with sequence analysis of both ald control regions led us to suggest that the expression of the ald genes in both mycobacterial species is regulated by the same mechanism. IMPORTANCEIn mycobacteria, alanine dehydrogenase (Ald) is the enzyme required both to utilize alanine as a nitrogen source and to grow under hypoxic conditions by maintaining the redox state of the NADH/NAD ؉ pool. Expression of the ald gene was reported to be regulated by the AldR regulator that belongs to the Lrp/AsnC (feast/famine) family, but the underlying mechanism was unknown. This study revealed the regulation mechanism of ald in Mycobacterium smegmatis and Mycobacterium tuberculosis. Furthermore, a generalized arrangement pattern of cis-acting regulatory sites for Lrp/AsnC (feast/famine) family regulators is suggested in this study. N AD(H)-dependent alanine dehydrogenase (Ald; EC 1.4.1.1) catalyzes the oxidative deamination of L-alanine to pyruvate and reversibly catalyzes the reductive amination of pyruvate to L-alanine. Ald is required for mycobacteria to utilize alanine as a nitrogen source via the oxidative deamination reaction (1-3). It was also proposed that Ald helps mycobacteria maintain the redox state of the NADH/NAD ϩ pool under respiration-inhibiting conditions, such as hypoxic conditions, by regenerating NAD ϩ from NADH via its reductive amination reaction (2, 4, 5). The enzyme forms a hexamer of identical subunits with the N-terminal catalytic domain and the C-terminal NAD(H)-binding domain (6, 7). Ald was shown to be one of the major antigens found in culture filtrates of Mycobacterium tuberculosis (8,9).Expression of the ald gene as well as the synthesis and activity of Ald we...

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“…Granuloma-specific activation of the M. marinum ald gene encoding alanine͞glycine dehydrogenase (30,31) is of particular interest. In M. tuberculosis this enzyme catalyzes the formation of alanine and͞or glycine (31,32) and is a major secreted antigen that is not secreted by the nonpathogenic vaccine strain M. bovis, bacillus Calmette-Guérin (33).…”

Section: Marinum Causes a Silent Long-term Infection In Frogs Withmentioning

confidence: 99%

“…In M. tuberculosis this enzyme catalyzes the formation of alanine and͞or glycine (31,32) and is a major secreted antigen that is not secreted by the nonpathogenic vaccine strain M. bovis, bacillus Calmette-Guérin (33). Also of particular interest is the granuloma induction of the M. tuberculosis pknB homologue encoding a protein serine͞threonine kinase (PSTK) (30,34).…”

Section: Marinum Causes a Silent Long-term Infection In Frogs Withmentioning

confidence: 99%

Complex pattern of Mycobacterium marinum gene expression during long-term granulomatous infection

Chan

Knaak

Satkamp

et al. 2002

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

107

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During latent infection of humans with Mycobacterium tuberculosis, bacteria persist in the asymptomatic host within granulomas, organized collections of differentiated macrophages, and other immune cells. The mechanisms for persistence remain poorly understood, as is the metabolic and replicative state of the microbes within granulomas. We analyzed the gene expression profile of Mycobacterium marinum, the cause of fish and amphibian tuberculosis, during its persistence in granulomas. We identified genes expressed specifically when M. marinum persists within granulomas. These granuloma-activated genes were not activated in vitro in response to various conditions postulated to be operant in tuberculous granulomas, suggesting that their granuloma-specific activation was caused by complex conditions that could not be mimicked in vitro. In addition to the granuloma-activated genes, the bacteria resident in granulomas expressed a wide range of metabolic and synthetic genes that are expressed during logarithmic growth in laboratory medium. Our results suggest a dynamic host-pathogen interaction in the granuloma, where metabolically active bacteria are kept in check by the host immune system and where the products of granuloma-specific bacterial genes may thwart the host's attempt to completely eradicate the bacteria.

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Glycine and alanine dehydrogenase activities are catalyzed by the same protein inMycobacterium smegmatis: upregulation of both activities under microaerophilic adaptation

Cited by 27 publications

References 19 publications

Mycobacterium bovis BCG Vaccines Exhibit Defects in Alanine and Serine Catabolism

Mycobacterium bovis BCG Vaccines Exhibit Defects in Alanine and Serine Catabolism

Regulation Mechanism of the ald Gene Encoding Alanine Dehydrogenase in Mycobacterium smegmatis and Mycobacterium tuberculosis by the Lrp/AsnC Family Regulator AldR

Complex pattern of Mycobacterium marinum gene expression during long-term granulomatous infection

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