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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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Sequence analysis of the protein sequence of mycosin-1. (A) Sequence alignment of mycosin-1 with the B. amyloliquifaciens subtilisin BPN protein. The conserved amino acid residues are indicated in bold. The catalytic residues (D-90, H-131, and S-332, mycosin-1 numbering) are in open boxes; the oxyanion hole (N-237) is in a closed box. The mycosin-1 signal peptide cleavage site (A-21/I-22) is shaded. The arrows below the BPN sequence bracket the propeptide sequence; arrows above mycosin-1 indicate the predicted propeptide. Overlines (numbered 1–5) above the mycosin-1 sequence indicate hydrophobic regions and correspond to domains 1–5 shown in (B). (B) Hydrophobicity plot of mycosin-1.
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Figure 2: Sequence analysis of the protein sequence of mycosin-1. (A) Sequence alignment of mycosin-1 with the B. amyloliquifaciens subtilisin BPN protein. The conserved amino acid residues are indicated in bold. The catalytic residues (D-90, H-131, and S-332, mycosin-1 numbering) are in open boxes; the oxyanion hole (N-237) is in a closed box. The mycosin-1 signal peptide cleavage site (A-21/I-22) is shaded. The arrows below the BPN sequence bracket the propeptide sequence; arrows above mycosin-1 indicate the predicted propeptide. Overlines (numbered 1–5) above the mycosin-1 sequence indicate hydrophobic regions and correspond to domains 1–5 shown in (B). (B) Hydrophobicity plot of mycosin-1.

Mentions: A 73 kDa GST-mycosin-1 fusion protein was detected in the insoluble (cell lysate) fraction of E. coli transformed with pGEX-P1 (Fig. 1). Most of the GST-mycosin-1 fusion protein was insoluble and significant amounts were lost during purification, consistent with the high degree of hydrophobicity predicted from the amino acid sequence (see Fig. 2). Addition of detergents to solubilize the fusion protein increased the amount of GST-mycosin-1 in the soluble fraction and enhanced purification yields. The purified product was used to generate antiserum against mycosin-1.


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)

Sequence analysis of the protein sequence of mycosin-1. (A) Sequence alignment of mycosin-1 with the B. amyloliquifaciens subtilisin BPN protein. The conserved amino acid residues are indicated in bold. The catalytic residues (D-90, H-131, and S-332, mycosin-1 numbering) are in open boxes; the oxyanion hole (N-237) is in a closed box. The mycosin-1 signal peptide cleavage site (A-21/I-22) is shaded. The arrows below the BPN sequence bracket the propeptide sequence; arrows above mycosin-1 indicate the predicted propeptide. Overlines (numbered 1–5) above the mycosin-1 sequence indicate hydrophobic regions and correspond to domains 1–5 shown in (B). (B) Hydrophobicity plot of mycosin-1.
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Related In: Results  -  Collection

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

Figure 2: Sequence analysis of the protein sequence of mycosin-1. (A) Sequence alignment of mycosin-1 with the B. amyloliquifaciens subtilisin BPN protein. The conserved amino acid residues are indicated in bold. The catalytic residues (D-90, H-131, and S-332, mycosin-1 numbering) are in open boxes; the oxyanion hole (N-237) is in a closed box. The mycosin-1 signal peptide cleavage site (A-21/I-22) is shaded. The arrows below the BPN sequence bracket the propeptide sequence; arrows above mycosin-1 indicate the predicted propeptide. Overlines (numbered 1–5) above the mycosin-1 sequence indicate hydrophobic regions and correspond to domains 1–5 shown in (B). (B) Hydrophobicity plot of mycosin-1.
Mentions: A 73 kDa GST-mycosin-1 fusion protein was detected in the insoluble (cell lysate) fraction of E. coli transformed with pGEX-P1 (Fig. 1). Most of the GST-mycosin-1 fusion protein was insoluble and significant amounts were lost during purification, consistent with the high degree of hydrophobicity predicted from the amino acid sequence (see Fig. 2). Addition of detergents to solubilize the fusion protein increased the amount of GST-mycosin-1 in the soluble fraction and enhanced purification yields. The purified product was used to generate antiserum against mycosin-1.

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