Three-dimensional electron microscopy reconstruction and cysteine-mediated crosslinking provide a model of the type III secretion system needle tip complex.
Three-dimensional electron microscopy reconstruction and cysteine-mediated crosslinking provide a model of the type III secretion system needle tip complex.
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DOI：
10.1111/mmi.12843
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发表时间：
2015-01
影响因子：
3.6
通讯作者：
Blocker AJ
中科院分区：
文献类型：
--
作者：
Cheung M;Shen DK;Makino F;Kato T;Roehrich AD;Martinez-Argudo I;Walker ML;Murillo I;Liu X;Pain M;Brown J;Frazer G;Mantell J;Mina P;Todd T;Sessions RB;Namba K;Blocker AJ
III型分泌系统存在于许多革兰氏阴性细菌中。它们通过与真核细胞接触而被激活，并将毒力蛋白注入其中。宿主细胞检测需要位于设备外部注射针头尖端的蛋白质复合物。志贺氏菌尖端复合物（TC）由IpaD（一种亲水性蛋白）和IpaB（一种疏水性蛋白）组成，IpaB后来形成宿主膜中的注射孔的一部分。在这里，我们使用标记和交联方法来显示来自Δ ipaB菌株的TC含有5个IpaD亚基，而来自野生型的TC也可以含有1个IpaB和4个IpaD亚基。电子显微镜，然后单粒子和螺旋图像分析被用来重建的三维图像的TC在2000年的分辨率。对接的IpaD晶体结构，所观察到的交联约束，揭示了TC组织是不同的，从所有以前提出的模型。我们的研究结果提出了TC组装和功能的新机制。TC是这些分泌系统中唯一被疾病保护抗体靶向的位点。通过提出这些作用方式，我们的工作将允许改进预防和治疗策略。
Type III secretion systems are found in many Gram-negative bacteria. They are activated by contact with eukaryotic cells and inject virulence proteins inside them. Host cell detection requires a protein complex located at the tip of the device's external injection needle. The Shigella tip complex (TC) is composed of IpaD, a hydrophilic protein, and IpaB, a hydrophobic protein, which later forms part of the injection pore in the host membrane. Here we used labelling and crosslinking methods to show that TCs from a ΔipaB strain contain five IpaD subunits while the TCs from wild-type can also contain one IpaB and four IpaD subunits. Electron microscopy followed by single particle and helical image analysis was used to reconstruct three-dimensional images of TCs at ∼20 Å resolution. Docking of an IpaD crystal structure, constrained by the crosslinks observed, reveals that TC organisation is different from that of all previously proposed models. Our findings suggest new mechanisms for TC assembly and function. The TC is the only site within these secretion systems targeted by disease-protecting antibodies. By suggesting how these act, our work will allow improvement of prophylactic and therapeutic strategies.