Direct detection of bacterial protein secretion using whole colony proteomics.
Direct detection of bacterial protein secretion using whole colony proteomics.
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DOI：
10.1074/mcp.m112.017533
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发表时间：
2012-09
期刊：
影响因子：
--
通讯作者：
Champion PA
中科院分区：
文献类型：
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作者：
Champion MM;Williams EA;Kennedy GM;Champion PA
细菌利用多种分泌系统将蛋白质转运到细胞膜之外，与环境相互作用。对于细菌病原体来说，这些系统是将细菌蛋白转运到宿主细胞中的关键毒力决定因素。识别出口所需细菌基因的遗传筛选依赖于与已知底物融合的酶或荧光报告基因来监测分泌。然而，它们不能用于分析所有分泌系统，限制了跨细菌的实施。在这里，我们介绍了首次应用改良形式的全菌落 MALDI-TOF MS 来直接检测完整细菌菌落的蛋白质分泌。我们证明该方法能够特异性监测 ESX-1 系统蛋白分泌系统，这是分枝杆菌和革兰氏阳性病原体的主要毒力决定因素，而报告分析难以进行。我们通过从海分枝杆菌转座子插入文库中鉴定 ESAT-6 系统 1 缺陷突变体，验证了该技术作为高通量筛选工具的用途。此外，我们还证明了革兰氏阴性病原体铜绿假单胞菌中常见的 III 型分泌系统的分泌蛋白的检测。该方法将广泛适用于研究其他细菌蛋白质输出系统和鉴定抑制细菌蛋白质分泌的化合物。
Bacteria use a variety of secretion systems to transport proteins beyond their cell membrane to interact with their environment. For bacterial pathogens, these systems are key virulence determinants that transport bacterial proteins into host cells. Genetic screens to identify bacterial genes required for export have relied on enzymatic or fluorescent reporters fused to known substrates to monitor secretion. However, they cannot be used in analysis of all secretion systems, limiting the implementation across bacteria. Here, we introduce the first application of a modified form of whole colony MALDI-TOF MS to directly detect protein secretion from intact bacterial colonies. We show that this method is able to specifically monitor the ESX-1 system protein secretion system, a major virulence determinant in both mycobacterial and Gram-positive pathogens that is refractory to reporter analysis. We validate the use of this technology as a high throughput screening tool by identifying an ESAT-6 system 1-deficient mutant from a Mycobacterium marinum transposon insertion library. Furthermore, we also demonstrate detection of secreted proteins of the prevalent type III secretion system from the Gram-negative pathogen, Pseudomonas aeruginosa. This method will be broadly applicable to study other bacterial protein export systems and for the identification of compounds that inhibit bacterial protein secretion.