The Common Structural Architecture of Shigella flexneri and Salmonella typhimurium Type Three Secretion Needles
The Common Structural Architecture of Shigella flexneri and Salmonella typhimurium Type Three Secretion Needles
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DOI：
10.1371/journal.ppat.1003245
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发表时间：
2013-03-01
期刊：
影响因子：
6.7
通讯作者：
Lange, Adam
中科院分区：
文献类型：
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作者：
Demers, Jean-Philippe;Sgourakis, Nikolaos G.;Lange, Adam
第三型分泌系统（T3SS），或注射体，是存在于许多致病性革兰氏阴性菌中的大分子感染机制。它由一个固定在细菌膜上的基底体和一个中空的针组成，通过这个针，效应蛋白被递送到目标宿主细胞中。T3SS针的两种不同架构之前已经被提出。首先，利用固态核磁共振数据生成鼠伤寒沙门氏菌针的原子模型。针亚基蛋白PrgI包括一个刚性延伸的n端片段和一个螺旋-环-螺旋基序，其中n端位于针的外表面。其次，利用高分辨率的7.7埃低温电子显微镜密度图生成了福氏志贺氏菌针的模型。亚基蛋白MxiH包含一个n端α -螺旋，一个环，另一个α -螺旋，一个14个残基长的β -发夹（Q51-Q64）和一个c端α -螺旋，其中n端向内朝向针腔。在本研究中，我们对体外聚合的野生型福氏志贺氏菌针进行了固态核磁共振测量，并确定了MxiH的以下二级结构元素：刚性扩展的n端片段（S2-T11）， α -螺旋（L12-A38），环（E39-P44）和c端α -螺旋（Q45-R83）。在体外和体内功能针上使用免疫金标记，我们定位了MxiH亚基的n端在组装的外部，与进化序列保守模式和突变数据一致。我们生成了一个与两个实验数据兼容的志贺氏菌弗氏针的同源模型：MxiH固态核磁共振化学位移和最先进的低温电镜密度图。这些结果证实了先前解决的鼠伤寒沙门菌PrgI针的固态核磁共振结构，并确定了福氏志贺氏菌和鼠伤寒沙门菌亚基蛋白在其组装状态下采用保守的结构和取向。我们的研究揭示了T3SS针头的共同结构结构，这对于了解T3SS介导的感染和开发治疗方法至关重要。
The Type Three Secretion System (T3SS), or injectisome, is a macromolecular infection machinery present in many pathogenic Gram-negative bacteria. It consists of a basal body, anchored in both bacterial membranes, and a hollow needle through which effector proteins are delivered into the target host cell. Two different architectures of the T3SS needle have been previously proposed. First, an atomic model of the Salmonella typhimurium needle was generated from solid-state NMR data. The needle subunit protein, PrgI, comprises a rigid-extended N-terminal segment and a helix-loop-helix motif with the N-terminus located on the outside face of the needle. Second, a model of the Shigella flexneri needle was generated from a high-resolution 7.7-angstrom cryo-electron microscopy density map. The subunit protein, MxiH, contains an N-terminal alpha-helix, a loop, another alpha-helix, a 14-residue-long beta-hairpin (Q51-Q64) and a C-terminal alpha-helix, with the N-terminus facing inward to the lumen of the needle. In the current study, we carried out solid-state NMR measurements of wild-type Shigella flexneri needles polymerized in vitro and identified the following secondary structure elements for MxiH: a rigid-extended N-terminal segment (S2-T11), an alpha-helix (L12-A38), a loop (E39-P44) and a C-terminal alpha-helix (Q45-R83). Using immunogold labeling in vitro and in vivo on functional needles, we located the N-terminus of MxiH subunits on the exterior of the assembly, consistent with evolutionary sequence conservation patterns and mutagenesis data. We generated a homology model of Shigella flexneri needles compatible with both experimental data: the MxiH solid-state NMR chemical shifts and the state-of-the-art cryoEM density map. These results corroborate the solid-state NMR structure previously solved for Salmonella typhimurium PrgI needles and establish that Shigella flexneri and Salmonella typhimurium subunit proteins adopt a conserved structure and orientation in their assembled state. Our study reveals a common structural architecture of T3SS needles, essential to understand T3SS-mediated infection and develop treatments.