Direct visualization by cryo-EM of the mycobacterial capsular layer: a labile structure containing ESX-1-secreted proteins.
Direct visualization by cryo-EM of the mycobacterial capsular layer: a labile structure containing ESX-1-secreted proteins.
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DOI：
10.1371/journal.ppat.1000794
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发表时间：
2010-03-05
期刊：
影响因子：
6.7
通讯作者：
Peters PJ
中科院分区：
文献类型：
--
作者：
Sani M;Houben EN;Geurtsen J;Pierson J;de Punder K;van Zon M;Wever B;Piersma SR;Jiménez CR;Daffé M;Appelmelk BJ;Bitter W;van der Wel N;Peters PJ
分枝杆菌是一组革兰氏阳性菌，其细胞膜由质膜和含有霉菌酸的革兰氏阴性样外膜组成。此外，分枝杆菌的表面覆盖着一层特征不明确的可提取的非共价连接的聚糖、脂质和蛋白质，统称为胶囊，其出现是一个有争议的问题。通过低温冷冻电镜技术，我们能够证明致病性分枝杆菌产生一个厚胶囊，只有在细胞在无扰动条件下生长时才会出现，并且很容易被温和的洗涤剂去除。这种不稳定的洗涤胶囊层含有阿拉伯甘露聚糖，α-葡聚糖和低聚甘露聚糖覆盖的糖脂。进一步的免疫原性和蛋白质组学分析显示，海洋分枝杆菌胶囊含有大量通过ESX-1途径分泌的蛋白质。最后，细胞感染实验证明了胶囊与细胞结合和抑制促炎细胞因子反应的重要性。总之，这些结果显示分枝杆菌荚膜层作为一个包含esx -1分泌蛋白的不稳定结构的直接可视化。分枝杆菌属含有许多重要的病原体，如结核分枝杆菌。这些细菌高度特征性的细胞包膜在感染过程中起着至关重要的作用。与其他细菌最明显的区别是最近描述的由独特的（糖）脂组成的外膜。然而，在这个膜的顶部分枝杆菌也有一个不明确的荚膜层。本文采用不同的电子显微镜技术和质谱技术研究了这种荚膜层。采用接近天然状态的制备方法，我们发现致病性和非致病性分枝杆菌都有一层不稳定的囊膜覆盖在外膜上。这个荚膜层除了含有阿拉伯半乳聚糖、聚糖和含甘露糖的糖脂外，还令人惊讶地含有大量的海洋分枝杆菌分泌的esx -1蛋白。此外，我们还发现该胶囊在巨噬细胞的结合和细胞因子的诱导中起作用。总的来说，这些结果首次表明该胶囊可以在致病性和非致病性分枝杆菌上可视化。此外，在标准实验室条件下，在搅拌洗涤剂的存在下生长分枝杆菌会促进荚膜脱落并影响细菌的生物学特性。
The cell envelope of mycobacteria, a group of Gram positive bacteria, is composed of a plasma membrane and a Gram-negative-like outer membrane containing mycolic acids. In addition, the surface of the mycobacteria is coated with an ill-characterized layer of extractable, non-covalently linked glycans, lipids and proteins, collectively known as the capsule, whose occurrence is a matter of debate. By using plunge freezing cryo-electron microscopy technique, we were able to show that pathogenic mycobacteria produce a thick capsule, only present when the cells were grown under unperturbed conditions and easily removed by mild detergents. This detergent-labile capsule layer contains arabinomannan, α-glucan and oligomannosyl-capped glycolipids. Further immunogenic and proteomic analyses revealed that Mycobacterium marinum capsule contains high amounts of proteins that are secreted via the ESX-1 pathway. Finally, cell infection experiments demonstrated the importance of the capsule for binding to cells and dampening of pro-inflammatory cytokine response. Together, these results show a direct visualization of the mycobacterial capsular layer as a labile structure that contains ESX-1-secreted proteins. The genus Mycobacterium contains a number of important pathogens, such as Mycobacterium tuberculosis. The highly characteristic cell envelope of these bacteria plays a crucial role in the infection process. The most apparent difference with other bacteria is the recently described outer membrane composed of unique (glyco)lipids. However, on top of this membrane mycobacteria also have an ill-defined capsular layer. In this paper, we studied this capsular layer using different electron microscopy techniques and mass spectrometry. Using close to native state preparation method, we show that both pathogenic and non-pathogenic mycobacteria have a labile capsular layer that covers the outer membrane. This capsular layer, in addition to containing arabinogalactan, glycan and mannose-containing glyco-lipids, also surprisingly contains a large amount of ESX-1-secreted proteins in Mycobacterium marinum. Furthermore, we also show that the capsule plays a role in the binding of macrophages and the induction of cytokines. Collectively, these results show for the first time that the capsule can be visualized on both pathogenic and non-pathogenic mycobacteria. In addition, growing mycobacteria under standard laboratory conditions in the presence of detergent with agitation promotes capsular shedding and influences the biological characteristics of the bacteria.
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