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Mycosin-1, a subtilisin-like serine protease of Mycobacterium tuberculosis, is cell wall-associated and expressed during infection of macrophages.

Dave JA, Gey van Pittius NC, Beyers AD, Ehlers MR, Brown GD - BMC Microbiol. (2002)

Bottom Line: The gene mycP1 (encoding mycosin-1) was found to be situated 3700 bp (four ORF's) from the RD1 deletion region in the genome of the attenuated vaccine strain M. bovis BCG (bacille de Calmette et Guérin) and was selected for further analyses due to the absence of expression in this organism.The protein is expressed after infection of macrophages and is subjected to proteolytic processing.Although proteolytically active mycosin-1 could not be generated recombinantly, serine protease activity containing features typical of the subtilisins was detected in M. tuberculosis culture filtrates.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medical Biochemistry, University of Cape Town Medical School, Observatory 7925, Cape Town, South Africa. joeldave@xsinet.co.za

ABSTRACT

Background: Exported proteases are commonly associated with virulence in bacterial pathogens, yet there is a paucity of information regarding their role in Mycobacterium tuberculosis. There are five genes (mycP1-5) present within the genome of Mycobacterium tuberculosis H37Rv that encode a family of secreted, subtilisin-like serine proteases (the mycosins). The gene mycP1 (encoding mycosin-1) was found to be situated 3700 bp (four ORF's) from the RD1 deletion region in the genome of the attenuated vaccine strain M. bovis BCG (bacille de Calmette et Guérin) and was selected for further analyses due to the absence of expression in this organism.

Results: Full-length, 50 kDa mycosin-1 was observed in M. tuberculosis cellular lysates, whereas lower-molecular-weight species were detected in culture filtrates. A similar lower-molecular-weight species was also observed during growth in macrophages. Mycosin-1 was localized to the membrane and cell wall fractions in M. tuberculosis by Western blotting, and to the cell envelope by electron microscopy. Furthermore, M. tuberculosis culture filtrates were shown to contain a proteolytic activity inhibited by mixed serine/cysteine protease inhibitors and activated by Ca2+, features typical of the subtilisins.

Conclusions: Mycosin-1 is an extracellular protein that is membrane- and cell wall-associated, and is shed into the culture supernatant. The protein is expressed after infection of macrophages and is subjected to proteolytic processing. Although proteolytically active mycosin-1 could not be generated recombinantly, serine protease activity containing features typical of the subtilisins was detected in M. tuberculosis culture filtrates.

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Degradation of 125I-fibrinogen by M. tuberculosis culture filtrates and inhibition by class-specific protease inhibitors. Culture filtrates were concentrated 20-fold and incubated with 125I-fibrinogen for 16 h. Class specific protease inhibitors (defined in the text) were added to the protease assays, which were performed as described in the Methods. Extent of 125I-fibrinogen degradation was quantified by measurement of TCA-soluble counts. Results are expressed as percent degradation relative to control (no inhibitor) and are the means ± standard deviations of four independent experiments.
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Figure 6: Degradation of 125I-fibrinogen by M. tuberculosis culture filtrates and inhibition by class-specific protease inhibitors. Culture filtrates were concentrated 20-fold and incubated with 125I-fibrinogen for 16 h. Class specific protease inhibitors (defined in the text) were added to the protease assays, which were performed as described in the Methods. Extent of 125I-fibrinogen degradation was quantified by measurement of TCA-soluble counts. Results are expressed as percent degradation relative to control (no inhibitor) and are the means ± standard deviations of four independent experiments.

Mentions: To examine this proteolytic activity further, 125I-fibrinogen was incubated with M. tuberculosis GSH-3052 culture filtrate in the presence of diverse, class-specific protease inhibitors. This analysis revealed that the proteolytic activity was significantly inhibited by serine and cysteine protease inhibitors (Fig. 6), with strongest inhibition obtained with chymostatin (77 ± 14%), ALLN (69 ± 20%), ALLM (66 ± 22%), and PMSF (66 ± 30%). Some subtilisins, including mycosin-1 (Fig. 2A), contain a cysteine residue near the active site histidine, which renders these enzymes susceptible to some cysteine protease inhibitors [5,6]. Moderate inhibition was also noted with 3,4-dichloroisocoumarin (38 ± 8%) and EDTA (33 ± 9%), whereas inhibition by other inhibitors was variable and generally less than 20%. Proteolytic activity was enhanced 38% by the addition of 2.5 mM Ca2+ but was unaffected by 0.1 mM Zn2+ (results not shown). The results suggest that the predominant protease activity in M. tuberculosis culture filtrates comprise one or more serine and/or cysteine proteases that are partially Ca2+ dependent. Attempts at purifying this activity failed (data not shown).


Mycosin-1, a subtilisin-like serine protease of Mycobacterium tuberculosis, is cell wall-associated and expressed during infection of macrophages.

Dave JA, Gey van Pittius NC, Beyers AD, Ehlers MR, Brown GD - BMC Microbiol. (2002)

Degradation of 125I-fibrinogen by M. tuberculosis culture filtrates and inhibition by class-specific protease inhibitors. Culture filtrates were concentrated 20-fold and incubated with 125I-fibrinogen for 16 h. Class specific protease inhibitors (defined in the text) were added to the protease assays, which were performed as described in the Methods. Extent of 125I-fibrinogen degradation was quantified by measurement of TCA-soluble counts. Results are expressed as percent degradation relative to control (no inhibitor) and are the means ± standard deviations of four independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC131053&req=5

Figure 6: Degradation of 125I-fibrinogen by M. tuberculosis culture filtrates and inhibition by class-specific protease inhibitors. Culture filtrates were concentrated 20-fold and incubated with 125I-fibrinogen for 16 h. Class specific protease inhibitors (defined in the text) were added to the protease assays, which were performed as described in the Methods. Extent of 125I-fibrinogen degradation was quantified by measurement of TCA-soluble counts. Results are expressed as percent degradation relative to control (no inhibitor) and are the means ± standard deviations of four independent experiments.
Mentions: To examine this proteolytic activity further, 125I-fibrinogen was incubated with M. tuberculosis GSH-3052 culture filtrate in the presence of diverse, class-specific protease inhibitors. This analysis revealed that the proteolytic activity was significantly inhibited by serine and cysteine protease inhibitors (Fig. 6), with strongest inhibition obtained with chymostatin (77 ± 14%), ALLN (69 ± 20%), ALLM (66 ± 22%), and PMSF (66 ± 30%). Some subtilisins, including mycosin-1 (Fig. 2A), contain a cysteine residue near the active site histidine, which renders these enzymes susceptible to some cysteine protease inhibitors [5,6]. Moderate inhibition was also noted with 3,4-dichloroisocoumarin (38 ± 8%) and EDTA (33 ± 9%), whereas inhibition by other inhibitors was variable and generally less than 20%. Proteolytic activity was enhanced 38% by the addition of 2.5 mM Ca2+ but was unaffected by 0.1 mM Zn2+ (results not shown). The results suggest that the predominant protease activity in M. tuberculosis culture filtrates comprise one or more serine and/or cysteine proteases that are partially Ca2+ dependent. Attempts at purifying this activity failed (data not shown).

Bottom Line: The gene mycP1 (encoding mycosin-1) was found to be situated 3700 bp (four ORF's) from the RD1 deletion region in the genome of the attenuated vaccine strain M. bovis BCG (bacille de Calmette et Guérin) and was selected for further analyses due to the absence of expression in this organism.The protein is expressed after infection of macrophages and is subjected to proteolytic processing.Although proteolytically active mycosin-1 could not be generated recombinantly, serine protease activity containing features typical of the subtilisins was detected in M. tuberculosis culture filtrates.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medical Biochemistry, University of Cape Town Medical School, Observatory 7925, Cape Town, South Africa. joeldave@xsinet.co.za

ABSTRACT

Background: Exported proteases are commonly associated with virulence in bacterial pathogens, yet there is a paucity of information regarding their role in Mycobacterium tuberculosis. There are five genes (mycP1-5) present within the genome of Mycobacterium tuberculosis H37Rv that encode a family of secreted, subtilisin-like serine proteases (the mycosins). The gene mycP1 (encoding mycosin-1) was found to be situated 3700 bp (four ORF's) from the RD1 deletion region in the genome of the attenuated vaccine strain M. bovis BCG (bacille de Calmette et Guérin) and was selected for further analyses due to the absence of expression in this organism.

Results: Full-length, 50 kDa mycosin-1 was observed in M. tuberculosis cellular lysates, whereas lower-molecular-weight species were detected in culture filtrates. A similar lower-molecular-weight species was also observed during growth in macrophages. Mycosin-1 was localized to the membrane and cell wall fractions in M. tuberculosis by Western blotting, and to the cell envelope by electron microscopy. Furthermore, M. tuberculosis culture filtrates were shown to contain a proteolytic activity inhibited by mixed serine/cysteine protease inhibitors and activated by Ca2+, features typical of the subtilisins.

Conclusions: Mycosin-1 is an extracellular protein that is membrane- and cell wall-associated, and is shed into the culture supernatant. The protein is expressed after infection of macrophages and is subjected to proteolytic processing. Although proteolytically active mycosin-1 could not be generated recombinantly, serine protease activity containing features typical of the subtilisins was detected in M. tuberculosis culture filtrates.

Show MeSH
Related in: MedlinePlus