Purification, characterization, and genetic analysis of Mycobacterium tuberculosis urease, a potentially critical determinant of host-pathogen interaction

Clemens, Daniel L.; Lee, Bai‐Yu; Horwitz, Marcus A.

doi:10.1128/jb.177.19.5644-5652.1995

Cited by 100 publications

(84 citation statements)

References 39 publications

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“…Indeed, previous results have shown that at least gelatinase (Imboden et al, 1996), fibrinogen and elastin hydrolase (Dave et al, 1996), urease (Clemens et al, 1995) or phospholipase (Wheeler & Ratledge, 1992) activities can be induced or strongly stimulated according to the culture medium. Cultivating the micro-organisms on media containing various substrates mimicking the environment of infected cells would complement the present approach.…”

Section: Discussionmentioning

confidence: 99%

Extracellular enzyme activities potentially involved in the pathogenicity of Mycobacterium tuberculosis

et al. 1998

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T o evaluate the potential contribution of extracellular enzymes to the pathogenicity of mycobacteria, the presence of selected enzyme activities was investigated in the culture filtrates of the obligate human pathogen Mycobacterium tuberculosis, M. bovis BCG, the opportunistic pathogens M. kansasii and M. fortuitum, and the non-pathogenic species M. phlei and M. smegmatis. For M. tuberculosis and M. bovis, 22 enzyme activities were detected in the culture filtrates and/or cell surfaces, of which eight were absent from the culture fluids of non-pathogens: alanine dehydrogenase, glutamine synthetase, nicotinamidase, isonicotinamidase, superoxide dismutase, catalase, peroxidase and alcohol dehydrogenase. These activities, which correspond to secreted enzymes, formed a significant part (up to 92 O/ O) of the total enzyme activities of the bacteria and were absent from the culture fluids and the cell surfaces of the non-pathogenic species M. smegmatis and M. phlei. The extracellular location of superoxide dismutase and glutamine synthetase seemed to be restricted to the obligate pathogens examined. The difference in the enzyme profiles was not attributable to the growth rates of the two groups of bacteria. The presence of the eight enzyme activities in the outermost compartments of obligate pathogens and their absence in those of non-pathogens provides further evidence that these enzymes may be involved in the pathogenicity of mycobacteria. In addition, the eight enzyme activities were demonstrated in the cell extract of M. smegmatis. Stepwise erosion of the cell surface of M. smegmatis to expose internal capsular constituents showed that the various enzyme activities, with the possible exception of superoxide dismutase, were located more deeply in the cell envelope of this bacterium. This suggests that the molecular architecture of the mycobacterial envelopes may play an important role in the pathogenicity of these organisms.

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Section: Discussionmentioning

confidence: 99%

Extracellular enzyme activities potentially involved in the pathogenicity of Mycobacterium tuberculosis

et al. 1998

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“…ureC is involved in the maintenance of neutral intraphagosomal pH within BCG-containing vacuoles (21). Hence, lack of this enzyme allows phagosomal acidification, thereby creating an optimal pH milieu for cytolytic functions of Hly.…”

Section: Figurementioning

confidence: 99%

Increased vaccine efficacy against tuberculosis of recombinant Mycobacterium bovis bacille Calmette-Guerin mutants that secrete listeriolysin

Grode

Seiler

Baumann

et al. 2005

Journal of Clinical Investigation

497

428

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The tuberculosis vaccine Mycobacterium bovis bacille Calmette-Guérin (BCG) was equipped with the membraneperforating listeriolysin (Hly) of Listeria monocytogenes, which was shown to improve protection against Mycobacterium tuberculosis. Following aerosol challenge, the Hly-secreting recombinant BCG (hly + rBCG) vaccine was shown to protect significantly better against aerosol infection with M. tuberculosis than did the parental BCG strain. The isogenic, urease C-deficient hly + rBCG (∆ureC hly + rBCG) vaccine, providing an intraphagosomal pH closer to the acidic pH optimum for Hly activity, exhibited still higher vaccine efficacy than parental BCG. ∆ureC hly + rBCG also induced profound protection against a member of the M. tuberculosis Beijing/W genotype family while parental BCG failed to do so consistently. Hly not only promoted antigen translocation into the cytoplasm but also apoptosis of infected macrophages. We concluded that superior vaccine efficacy of ∆ureC hly + rBCG as compared with parental BCG is primarily based on improved cross-priming, which causes enhanced T cell-mediated immunity.

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“…Latex beads were coated with urease from Bacillus, which is closely related to mycobacterial urease (9). Beads were washed twice with 25 mM morpholineethanesulfonic acid buffer (pH 5.5), and pellets were resuspended in the same buffer containing 500 g of either Bacillus pasteuri urease (ϳ200 U/mg of protein; Sigma) or bovine serum albumin (BSA; control)/ml.…”

Section: Reagentsmentioning

confidence: 99%

Mycobacterium bovisBCG Urease Attenuates Major Histocompatibility Complex Class II Trafficking to the Macrophage Cell Surface

et al. 2004

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We have previously shown that Mycobacterium tuberculosis attenuates cell surface expression of major histocompatibility complex class II molecules in response to gamma interferon (IFN-␥) by a mechanism dependent on intracellular sequestration of ␣,␤ dimers. In this study we examined whether intracellular alkalinization due to mycobacterial urease could account for the defect in intracellular trafficking of class II molecules. Phagocytosis of wild-type Mycobacterium bovis BCG was associated with secretion of ammonia intracellularly, which increased substantially upon addition of exogenous urea to the culture medium. Increased intracellular ammonia, due to urea degradation by the bacterium, correlated with inhibition of class II surface expression. Conversely, no ammonia was detected in cells infected with a urease-negative mutant strain of M. bovis BCG, which also displayed a reduced effect on surface expression of class II molecules. A direct cause-effect relationship between urease and class II molecule trafficking was established with experiments where cells ingesting beads coated with purified urease showed an increased ammonia level and decreased surface expression of class II in response to IFN-␥. In contrast to BCG, infection of macrophages with Mycobacterium smegmatis, which expresses relatively greater urease activity in cell-free culture, had a marginal effect on both the intracellular level of ammonia and class II expression. The limited effect of M. smegmatis was consistent with a failure to resist intracellular killing, suggesting that urease alone is not sufficient to resist macrophage microbicidal mechanisms and that this is required for a more distal effect on cell regulation. Our results demonstrate that alkalinization of critical intracellular organelles by pathogenic mycobacteria expressing urease contributes significantly to the intracellular retention of class II dimers.

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Purification, characterization, and genetic analysis of Mycobacterium tuberculosis urease, a potentially critical determinant of host-pathogen interaction

Cited by 100 publications

References 39 publications

Extracellular enzyme activities potentially involved in the pathogenicity of Mycobacterium tuberculosis

Extracellular enzyme activities potentially involved in the pathogenicity of Mycobacterium tuberculosis

Increased vaccine efficacy against tuberculosis of recombinant Mycobacterium bovis bacille Calmette-Guerin mutants that secrete listeriolysin

Mycobacterium bovisBCG Urease Attenuates Major Histocompatibility Complex Class II Trafficking to the Macrophage Cell Surface

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