Stepwise visualization of membrane pore formation by suilysin, a bacterial cholesterol-dependent cytolysin.
Stepwise visualization of membrane pore formation by suilysin, a bacterial cholesterol-dependent cytolysin.
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DOI：
10.7554/elife.04247
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发表时间：
2014-12-02
期刊：
影响因子：
7.7
通讯作者：
Hoogenboom BW
中科院分区：
文献类型：
--
作者：
Leung C;Dudkina NV;Lukoyanova N;Hodel AW;Farabella I;Pandurangan AP;Jahan N;Pires Damaso M;Osmanović D;Reboul CF;Dunstone MA;Andrew PW;Lonnen R;Topf M;Saibil HR;Hoogenboom BW
膜攻击复合物/穿孔素/胆固醇依赖性溶细胞素（MACPF/CDC）蛋白是一个主要的成孔蛋白超家族，在细菌的免疫防御中起毒力因子和效应子的作用。在结合到膜上时，它们从可溶性单体形式转化为低聚的膜插入孔。使用实时原子力显微镜（AFM），电子显微镜（EM）和原子结构拟合，我们已经绘制了细菌CDC的结构和组装途径在前所未有的细节和准确性，专注于猪链球菌的suilysin。我们发现，猪溶素组装是一个非合作的过程，终止前的蛋白质插入到膜。由此产生的环形孔和动力学捕获的弧形组件都被视为覆盖膜，也可以通过其脂质的喷射来观察。膜插入需要单体亚基的协同构象变化，由于亚基结构和包装的大变化，孔径显著扩大。DOI：http://dx.doi.org/10.7554/eLife.04247.001许多致病细菌分泌有毒蛋白质，在我们的细胞上钻孔杀死它们。胆固醇依赖性溶细胞素（CDC）是这类毒素的一个家族，由引起肺炎、脑膜炎和败血症的细菌产生。细菌释放CDC毒素作为单一蛋白质分子，可以结合到宿主细胞周围的膜上。结合到细胞膜上后，毒素分子聚集成环，在宿主细胞膜上形成大孔。这个过程有几个阶段，但我们对分子水平上发生的事情的理解是不完整的。Leung等人研究了猪溶血素，这是一种由细菌产生的CDC毒素，对养猪业有很大影响，因为它会导致仔猪脑膜炎。这种细菌还可以通过接触受污染的猪或猪肉引起人类严重疾病。Leung等人使用了一种称为电子显微镜的技术来获得毒素插入膜之前和之后毒素结构的原子尺度快照。此外，使用另一种称为原子力显微镜的技术收集了该过程的实时电影。实验表明，猪溶素在膜上形成一次生长一个分子的组装体，而不是通过合并较大的分子组装体。这导致膜上环状和弧形毒素组装体的混合物。猪溶素的弧是不完整的环组装，但它们仍然能够在细胞膜上穿孔。为了插入细胞膜，弧形和环形的毒素分子在形状上发生了巨大的变化。了解CDC如何在膜中组装将指导进一步的工作，以开发新的疫苗，这些疫苗可以靶向这些蛋白质，以减少细菌感染造成的损害。DOI：http://dx.doi.org/10.7554/eLife.04247.002网站
Membrane attack complex/perforin/cholesterol-dependent cytolysin (MACPF/CDC) proteins constitute a major superfamily of pore-forming proteins that act as bacterial virulence factors and effectors in immune defence. Upon binding to the membrane, they convert from the soluble monomeric form to oligomeric, membrane-inserted pores. Using real-time atomic force microscopy (AFM), electron microscopy (EM), and atomic structure fitting, we have mapped the structure and assembly pathways of a bacterial CDC in unprecedented detail and accuracy, focussing on suilysin from Streptococcus suis. We show that suilysin assembly is a noncooperative process that is terminated before the protein inserts into the membrane. The resulting ring-shaped pores and kinetically trapped arc-shaped assemblies are all seen to perforate the membrane, as also visible by the ejection of its lipids. Membrane insertion requires a concerted conformational change of the monomeric subunits, with a marked expansion in pore diameter due to large changes in subunit structure and packing. DOI: http://dx.doi.org/10.7554/eLife.04247.001 Many disease-causing bacteria secrete toxic proteins that drill holes into our cells to kill them. Cholesterol-dependent cytolysins (CDCs) are a family of such toxins, and are produced by bacteria that cause pneumonia, meningitis, and septicaemia. The bacteria release CDC toxins as single protein molecules, which can bind to the membrane that surrounds the host cell. After binding to the membrane, the toxin molecules assemble in rings to form large pores in the host membrane. There are several stages to this process, but our understanding of what happens at the molecular level is incomplete. Leung et al. studied suilysin, a CDC toxin produced by a bacterium that has a big impact on the pig farming industry because it causes meningitis in piglets. The bacterium can also cause serious diseases in humans through exposure to contaminated pigs or pig meat. Leung et al. used a technique called electron microscopy to obtain atomic-scale snapshots of the toxin structures before and after the toxins were inserted into the membrane. In addition, real-time movies of the process were gathered using another technique called atomic force microscopy. The experiments show that suilysin forms assemblies on the membrane that grow by one molecule at a time, rather than by the merging of larger assemblies of molecules. This results in a mixture of ring-shaped and arc-shaped toxin assemblies on the membrane. The arcs of suilysin are incomplete ring assemblies, but they are still able to make holes in the cell membrane. In order to insert into the membrane, the toxin molecules in the arcs and rings undergo a dramatic change in shape. Understanding how CDCs assemble in membranes will guide further work into the development of new vaccines that can target these proteins to reduce the damage caused by bacterial infections. DOI: http://dx.doi.org/10.7554/eLife.04247.002