Visualization of the type III secretion sorting platform of Shigella flexneri
Visualization of the type III secretion sorting platform of Shigella flexneri
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DOI：
10.1073/pnas.1411610112
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发表时间：
2015-01-27
影响因子：
11.1
通讯作者：
Liu, Jun
中科院分区：
文献类型：
--
作者：
Hu, Bo;Morado, Dustin R.;Liu, Jun
细菌III型分泌机器被广泛用于将毒力蛋白注入真核宿主细胞。这些分泌机器在进化上与细菌鞭毛相关，并且由大的细胞质复合物、跨膜基体和细胞外针组成。细胞质复合物形成了效应子选择和针组装所必需的分选平台，但它在很大程度上仍未被表征。在这里，我们使用高通量冷冻电子断层扫描（cryo-ET）可视化完整的机器在一个有毒的福氏志贺菌菌株的遗传修饰，以产生能够与宿主细胞相互作用的小细胞。一个高分辨率的原位结构的完整的机器确定subtomography平均揭示了细胞质分选平台，它由一个中心枢纽和六个辐条，与豆荚状结构在每个辐条的末端。野生型和突变机器的分子建模使我们能够提出一个分选平台的模型，其中枢纽主要由Spa 47 ATP酶的六聚体组成，而MxiN蛋白包括辐条，Spa 33蛋白形成豆荚。这些组件之间的多个接触对于将Spa 47 ATP酶与MxiA蛋白输出门的中央通道对齐以形成独特的纳米机器至关重要。志贺氏菌III型分泌机器及其分选平台的分子结构为进一步剖析III型分泌和发病机制提供了结构基础，并且还突出了与细菌鞭毛的主要结构差异。
Bacterial type III secretion machines are widely used to inject virulence proteins into eukaryotic host cells. These secretion machines are evolutionarily related to bacterial flagella and consist of a large cytoplasmic complex, a transmembrane basal body, and an extracellular needle. The cytoplasmic complex forms a sorting platform essential for effector selection and needle assembly, but it remains largely uncharacterized. Here we use high-throughput cryoelectron tomography (cryo-ET) to visualize intact machines in a virulent Shigella flexneri strain genetically modified to produce minicells capable of interaction with host cells. A high-resolution in situ structure of the intact machine determined by subtomogram averaging reveals the cytoplasmic sorting platform, which consists of a central hub and six spokes, with a pod-like structure at the terminus of each spoke. Molecular modeling of wild-type and mutant machines allowed us to propose a model of the sorting platform in which the hub consists mainly of a hexamer of the Spa47 ATPase, whereas the MxiN protein comprises the spokes and the Spa33 protein forms the pods. Multiple contacts among those components are essential to align the Spa47 ATPase with the central channel of the MxiA protein export gate to form a unique nano-machine. The molecular architecture of the Shigella type III secretion machine and its sorting platform provide the structural foundation for further dissecting the mechanisms underlying type III secretion and pathogenesis and also highlight the major structural distinctions from bacterial flagella.