Mycosin-1, a subtilisin-like serine protease of Mycobacterium tuberculosis, is cell wall-associated and expressed during infection of macrophages
Mycosin-1, a subtilisin-like serine protease of Mycobacterium tuberculosis, is cell wall-associated and expressed during infection of macrophages
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DOI：
10.1186/1471-2180-2-30
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发表时间：
2002-01-01
期刊：
影响因子：
4.2
通讯作者：
Brown, Gordon D.
中科院分区：
文献类型：
--
作者：
Dave, Joel A.;Gey van Pittius, Nicolaas Claudius;Brown, Gordon D.
背景：出口的蛋白酶通常与细菌病原体的毒力有关，但关于它们在结核分枝杆菌中的作用的信息很少。结核分枝杆菌H37Rv的基因组中存在五个基因(mycP1-5)，它们编码一类分泌型枯草杆菌素样丝氨酸蛋白酶(霉菌素)。该基因位于卡介苗减毒活疫苗株基因组Rd1缺失区3700bp4个开放阅读框(ORF)上，由于在该菌中未见表达，故选择该基因进行进一步分析。结果：在结核分枝杆菌细胞裂解液中检测到全长50 kDa的mycoin-1，而在培养滤液中检测到较低分子量的菌丝素-1。在巨噬细胞的生长过程中也观察到了类似的低分子物种。Western blotting检测到Mycoin-1定位于结核分枝杆菌的细胞膜和细胞壁部分，电子显微镜下定位于细胞膜。此外，结核分枝杆菌培养滤液还具有被丝氨酸/半胱氨酸蛋白酶抑制剂抑制并被钙离子激活的蛋白分解活性，这是枯草杆菌的典型特征。结论：霉菌素-1是一种胞外蛋白，与膜和细胞壁相关，并被释放到培养上清中。该蛋白在感染巨噬细胞后表达，并经过蛋白水解性处理。虽然不能重组产生具有蛋白分解活性的霉菌素-1，但在结核分枝杆菌培养滤液中检测到含有典型枯草杆菌毒素特征的丝氨酸蛋白酶活性。
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 Guerin) 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.