Mycosin-1, a subtilisin-like serine protease of Mycobacterium tuberculosis, is cell wall-associated and expressed during infection of macrophages.
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.
D000818:Animals; D001426:Bacterial Proteins; D015153:Blotting, Western; D002473:Cell Wall / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004925:Escherichia / Q000235:genetics; D005260:Female; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D007106:Immune Sera / Q000276:immunology; D008264:Macrophages / Q000382:microbiology*; D051379:Mice; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D019810:Sodium Azide / Q000494:pharmacology; D020860:Subtilisin / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D013381:Subtilisins / Q000235:genetics*
D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D007106:Immune Sera; D011993:Recombinant Fusion Proteins; D019810:Sodium Azide; D005982:Glutathione Transferase; D013381:Subtilisins; C471626:mycosin-1, Mycobacterium tuberculosis; D020860:Subtilisin