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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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Expression of mycosin-1 in mycobacteria in culture and during infection of macrophages. Samples were resolved by SDS-PAGE and analyzed by Western blotting using anti-mycosin-1 antiserum. Lanes: 1, M. smegmatis; 2, M. smegmatis transformed with p19Kpro; 3, M. smegmatis transformed with p19K-P1; 4, M. tuberculosis clinical isolate GSH-3052 cell lysate; 5, M. tuberculosis clinical isolate GSH-3052 culture filtrate after growth in Kirchner's broth for 6 weeks; 6, lysate of uninfected P388D1 macrophages; 7, lysate of M. tuberculosis clinical isolate GSH-3052-infected P388D1 macrophages. Molecular weights (in kDa) are indicated on the left.
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Figure 3: Expression of mycosin-1 in mycobacteria in culture and during infection of macrophages. Samples were resolved by SDS-PAGE and analyzed by Western blotting using anti-mycosin-1 antiserum. Lanes: 1, M. smegmatis; 2, M. smegmatis transformed with p19Kpro; 3, M. smegmatis transformed with p19K-P1; 4, M. tuberculosis clinical isolate GSH-3052 cell lysate; 5, M. tuberculosis clinical isolate GSH-3052 culture filtrate after growth in Kirchner's broth for 6 weeks; 6, lysate of uninfected P388D1 macrophages; 7, lysate of M. tuberculosis clinical isolate GSH-3052-infected P388D1 macrophages. Molecular weights (in kDa) are indicated on the left.

Mentions: As reported previously [3], the purified anti-mycosin-1 antiserum was tested by performing Western blot analyses on cellular lysates of M. smegmatis heterologously expressing mycosin-1. A ~50 kDa immunoreactive band was only detected in M. smegmatis transformed with p19K-P1 (designated M. smegmatis -P1; Fig. 3, lanes 1–3), consistent with the full-length proform of mycosin-1, having a calculated molecular mass of 46 kDa. The detection of the full-length isoform suggests that propeptide cleavage did not occur during expression in M. smegmatis.


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)

Expression of mycosin-1 in mycobacteria in culture and during infection of macrophages. Samples were resolved by SDS-PAGE and analyzed by Western blotting using anti-mycosin-1 antiserum. Lanes: 1, M. smegmatis; 2, M. smegmatis transformed with p19Kpro; 3, M. smegmatis transformed with p19K-P1; 4, M. tuberculosis clinical isolate GSH-3052 cell lysate; 5, M. tuberculosis clinical isolate GSH-3052 culture filtrate after growth in Kirchner's broth for 6 weeks; 6, lysate of uninfected P388D1 macrophages; 7, lysate of M. tuberculosis clinical isolate GSH-3052-infected P388D1 macrophages. Molecular weights (in kDa) are indicated on the left.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Expression of mycosin-1 in mycobacteria in culture and during infection of macrophages. Samples were resolved by SDS-PAGE and analyzed by Western blotting using anti-mycosin-1 antiserum. Lanes: 1, M. smegmatis; 2, M. smegmatis transformed with p19Kpro; 3, M. smegmatis transformed with p19K-P1; 4, M. tuberculosis clinical isolate GSH-3052 cell lysate; 5, M. tuberculosis clinical isolate GSH-3052 culture filtrate after growth in Kirchner's broth for 6 weeks; 6, lysate of uninfected P388D1 macrophages; 7, lysate of M. tuberculosis clinical isolate GSH-3052-infected P388D1 macrophages. Molecular weights (in kDa) are indicated on the left.
Mentions: As reported previously [3], the purified anti-mycosin-1 antiserum was tested by performing Western blot analyses on cellular lysates of M. smegmatis heterologously expressing mycosin-1. A ~50 kDa immunoreactive band was only detected in M. smegmatis transformed with p19K-P1 (designated M. smegmatis -P1; Fig. 3, lanes 1–3), consistent with the full-length proform of mycosin-1, having a calculated molecular mass of 46 kDa. The detection of the full-length isoform suggests that propeptide cleavage did not occur during expression in M. smegmatis.

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