Department of Microbiology and Immunology, University of North Carolina, Chapel Hill, NC 27599, USA.
J Bacteriol. 2011 Feb;193(4):854-61. doi: 10.1128/JB.01271-10. Epub 2010 Dec 10.
The exported proteins of Mycobacterium tuberculosis that are localized at the bacterial cell surface or secreted into the environment are ideally situated to interact with host factors and to function in virulence. In this study, we constructed a novel β-lactamase reporter transposon and used it directly in M. tuberculosis for genome-wide identification of exported proteins. From 177 β-lactam-resistant transposon mutants, we identified 111 different exported proteins. The majority of these proteins have no known function, and for nearly half of the proteins, our demonstration that they are exported when fused to a β-lactamase reporter is the first experimental proof of their extracytoplasmic localization. The transposon mutants in our banked library were of further value as a collection of mutants lacking individual exported proteins. By individually testing each of 111 mutants for growth in macrophages, six attenuated mutants with insertions in mce1A, mce1B, mce2F, rv0199, ctaC, and lppX were identified. Given that much of the M. tuberculosis genome encodes proteins of unknown function, our library of mapped transposon mutants is a valuable resource for efforts in functional genomics. This work also demonstrates the power of a β-lactamase reporter transposon that could be applied similarly to other bacterial pathogens.
结核分枝杆菌（Mycobacterium tuberculosis）位于细菌细胞表面或分泌到环境中的分泌蛋白最适合与宿主因子相互作用，并在毒力中发挥作用。在这项研究中，我们构建了一种新型的β-内酰胺酶报告转座子，并直接在结核分枝杆菌中用于全基因组鉴定分泌蛋白。从 177 个β-内酰胺抗性转座子突变体中，我们鉴定出了 111 种不同的分泌蛋白。这些蛋白中的大多数没有已知的功能，对于近一半的蛋白，我们证明它们与β-内酰胺酶报告基因融合后可以分泌出来，这是它们细胞外定位的第一个实验证据。我们的文库中存储的转座子突变体作为缺乏单个分泌蛋白的突变体集合具有进一步的价值。通过单独测试 111 个突变体在巨噬细胞中的生长情况，鉴定出了六个插入 mce1A、mce1B、mce2F、rv0199、ctaC 和 lppX 的衰减突变体。鉴于结核分枝杆菌基因组的大部分编码未知功能的蛋白，我们映射的转座子突变体文库是功能基因组学努力的宝贵资源。这项工作还证明了β-内酰胺酶报告转座子的强大功能，它可以类似地应用于其他细菌病原体。
