The Antigens of <i>Mycobacterium bovis</i>, Strain BCG, Studied by Crossed Immunoelectrophoresis: a Reference System

Closs, O; Harboe, Morten; Axelsen, N. H.; Bunch‐Christensen, K.; Magnusson, M.

doi:10.1111/j.1365-3083.1980.tb00065.x

Cited by 190 publications

(147 citation statements)

References 34 publications

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“…The repertoire of proteins in the culture filtrates has been studied for a long time using various techniques [10,11]. The sensitivity and sophistication of analytical techniques have increased and several hundred different proteins can be revealed by two dimensional polyacrylamide gel electrophoresis [12±14].…”

Section: The Mechanism For Transporting Proteins Across the Outer Permentioning

confidence: 99%

“…Comparisons of culture filtrate preparations and sonicates of washed bacilli using immunological techniques have shown that the majority of protein species that can be found in the culture filtrate are also represented in the soluble pool of proteins inside the mycobacterial envelope including the cytoplasm [10,11]. Only a few proteins were originally found in the culture filtrates without being equally represented in the sonicated preparations, most notably antigen 85 which was later designated the antigen 85 complex [15].…”

Section: The Mechanism For Transporting Proteins Across the Outer Permentioning

confidence: 99%

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Liberation of Soluble Proteins from Live and Dead Mycobacterial Cells and the Implications for Pathogenicity of Tubercle Bacilli

Wiker¹

2001

Scand J Immunol

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Soluble proteins liberated from live M. tuberculosis are translocated through the cytoplasmic membrane to à periplasmic space'. For further export of proteins across the outer permeability barrier, it is necessary to postulate an excretion mechanism possibly involving some kind of porin. Observations of the repertoire of proteins in culture filtrates after liquid culture of M. tuberculosis show that a large repertoire of various kinds of proteins cross the outer permeability barrier of tubercle bacilli indicating that the excretion mechanism has a wide range of specificities for proteins. Culture filtrates of tubercle bacilli almost always contain both truly secreted proteins and cytoplasmatically-derived proteins. It is questionable whether cytoplasmic proteins can cross an intact cytoplasmic membrane. The simplest explanation for the appearance of cytoplasmic proteins in culture filtrates of tubercle bacilli would be that they are released after disintegration of the cytoplasmic membrane in dying or dead bacilli. Tubercle bacilli armed with secreted factors that may specifically inhibit innate and adaptive immune responses, excrete these from the periplasmic space of live bacilli. Unspecific in its character, the excretion mechanism also liberates proteins that are essential for building and maintaining the cell wall, thereby reducing the effectiveness of this process. This may be part of the explanation why M. tuberculosis and other pathogenic mycobacteria grow so slowly. Finally, it may be postulated that dormant or latent tubercle bacilli use their repertoire of secreted proteins to control their intracellular habitat and that bacterial cytoplasmic proteins would not be liberated from such bacilli. The consequence would be that only immune responses to secreted proteins would be effective for elimination of the dormant stage of infection. In a situation with active infection there will be considerable growth and turnover of bacilli with liberation of all kinds of immunogenic substances from the bacilli. In this situation immunity against cytoplasmic proteins would also be effective and immunity to cytoplasmic proteins should also be effective for control of the reactivation of latent disease because as soon as the bacilli start to grow there will also be a subpopulation of dead bacilli on the arena. THE ENVELOPE OF MYCOBACTERIACompared to many other bacterial genera, the outer permeability barrier of mycobacteria is extremely tight and particularly so for M. tuberculosis [1,2]. This is owing to the unusual structure of the mycobacterial cell wall which is highly impermeable to hydrophilic solutes. Hydrophobic solutes can penetrate the barrier [1]. Antibiotics of the most hydrophobic types for example, are also the most effective drugs against mycobacteria [3,4].Considering the extreme rigidity and impermeability of the outer permeability barrier [5] of M. tuberculosis, it is a paradox that liquid cultures contain excessive amounts of soluble proteins which are much larger than the small solutes referred to...

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Section: The Mechanism For Transporting Proteins Across the Outer Permentioning

confidence: 99%

Section: The Mechanism For Transporting Proteins Across the Outer Permentioning

confidence: 99%

Liberation of Soluble Proteins from Live and Dead Mycobacterial Cells and the Implications for Pathogenicity of Tubercle Bacilli

Wiker¹

2001

Scand J Immunol

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“…(1) anti (a)-MPT64, a polyclonal antibody reacting with a secreted protein isolated from the early M. tuberculosis culture fluid [21,22]; (2) a-ManLAM, a polyclonal antibody reacting with mannose-capped lipoarabinomannan, a cell wall component from M. bovis, bacillus Calmette-Guérin (BCG) Copenhagen strain [23,24]; and (3) a-BCG, a polyclonal antibody reacting with multiple antigens of sonicate and culture fluid of M. bovis BCG Copenhagen strain (Dako A/ S, Copenhagen, Denmark) [25,26].…”

Section: Gf ¼mentioning

confidence: 99%

A Mouse Model for Slowly Progressive Primary Tuberculosis

et al. 1999

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Mustafa T, Phyu S, Nilsen R, Jonsson R, Bjune G. A Mouse Model for Slowly Progressive Primary Tuberculosis. Scand J Immunol 1999;50:127-136 The progression from primary Mycobacterium tuberculosis infection to disease is usually slow in humans. The aim of this study was to develop and characterize a mouse model for slowly progressive primary tuberculosis, using the intraperitoneal (i.p.) route of infection, and to compare it with our previously described model of latent M. tuberculosis infection. B6D2F1 hybrid mice inoculated with 1.5 × 10 6 colony-forming units (CFUs) of M. tuberculosis H37Rv were followed-up for 70 weeks. Lungs, livers and spleens were examined for bacillary growth, histopathological changes and mycobacterial antigens (MPT64, ManLAM and multiple antigens of M. tuberculosis), by immunohistochemical staining. The infection was found to pass through three distinctive phases. During phase 1, mice were healthy despite development of small granulomas and an increasing number of bacilli in the lungs. During phase 2, mice were unwell but mortality was low. The count of M. tuberculosis and the granuloma size stabilized. The granulomas contained an increasing population of large, vacuolated macrophages. During phase 3, mice became moribund and died rapidly, but the M. tuberculosis count remained relatively stable. The inflammatory infiltrates filled Ϸ 80% of the lung parenchyma and the lesions were not well demarcated. Rapidly progressing inflammation, rather than an increase in the M. tuberculosis count, seems to contribute more to mortality.

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“…The adh-regulated system was used to overexpress the M. bovis BCG ADH and substantial amounts of this enzyme could be produced and purified to apparent homogeneity. This purified ADH was identified as antigen 39 (H. Wiker and M. Harboe, personal communication) in the crossed immunoelectrophoresis reference system [32].…”

Section: Discussionmentioning

confidence: 99%

Overexpression, purification and characterization of Mycobacterium bovis BCG alcohol dehydrogenase

Wilkin¹,

Soetaert²,

Stélandre³

et al. 1999

European Journal of Biochemistry

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A previous study of the effect of zinc deprivation on Mycobacterium bovis BCG pointed out the potential importance of an alcohol dehydrogenase for maintaining the hydrophobic character of the cell envelope. In this report, the effect of the overexpression of the M. bovis BCG alcohol dehydrogenase (ADH) in Mycobacterium smegmatis and M. bovis BCG is described. The purification of the enzyme was performed to apparent homogeneity from overexpressing M. bovis BCG cells and its kinetic parameters were determined. The enzyme showed a strong preference for both aromatic and aliphatic aldehydes while the corresponding alcohols were processed 100±1000-fold less efficiently. The best k cat /K m values were found with benzaldehyde . 3-methoxybenzaldehyde . octanal . coniferaldehyde. A phylogenetic analysis clearly revealed that the M. bovis BCG ADH together with the ADHs from Bacillus subtilis and Helicobacter pylori formed a sister group of the class C medium-chain alcohol dehydrogenases, the plant cinnamyl alcohol dehydrogenases (CADs). Comparison of the kinetic properties of our ADH with some related class C enzymes indicated that the mycobacterial enzyme substrate profile resembled that of the CADs involved in plant defence rather than those implicated in lignification. A possible role for the M. bovis BCG ADH in the biosynthesis of the lipids composing the mycobacterial cell envelope is proposed.Keywords: Mycobacterium bovis BCG; alcohol dehydrogenase; cinnamyl alcohol dehydrogenase.Alcohol dehydrogenases (ADHs) display a wide range of substrate specificities and fulfil several key physiological functions. They can be divided into three major categories. The first category is composed of the NAD(P)-dependent ADHs subdivided into three subgroups according to their metal dependence: (a) the medium-chain zinc-dependent enzymes; (b) the short-chain zinc-independent enzymes; and (c) the iron-activated enzymes. The NAD(P)-independent enzymes form the second category and the third category contains the oxidases which catalyse an essentially irreversible oxidation of alcohols [1].The vaccine strain Mycobacterium bovis BCG grows poorly on zinc-deprived Sauton medium, and forms a thin, pale, unfolded pellicle that gets wet and often sinks. This phenotype suggested a cell envelope with a reduced hydrophobic content. The cell composition and the products excreted into the medium, including aldehydes, can, to some extent, be correlated with a decrease of the specific activity of a soluble zinc-dependent alcohol dehydrogenase. This enzyme was partially purified from BCG cells and characterized as a dimeric NADP-dependent protein of broad specificity with higher affinities towards aldehydes than towards alcohols [2,3].The corresponding gene has been cloned and sequenced [4]. Comparison with other ADHs allowed classification of this enzyme into the zinc-containing, medium-chain alcohol/polyol dehydrogenase family which had been primarily described in eukaryotes [5]. Currently, this family is composed of the tetrameric and the dimeric...

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The Antigens of Mycobacterium bovis, Strain BCG, Studied by Crossed Immunoelectrophoresis: a Reference System

Cited by 190 publications

References 34 publications

Liberation of Soluble Proteins from Live and Dead Mycobacterial Cells and the Implications for Pathogenicity of Tubercle Bacilli

Liberation of Soluble Proteins from Live and Dead Mycobacterial Cells and the Implications for Pathogenicity of Tubercle Bacilli

A Mouse Model for Slowly Progressive Primary Tuberculosis

Overexpression, purification and characterization of Mycobacterium bovis BCG alcohol dehydrogenase

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