Composition, formation, and regulation of the cytosolic c-ring, a dynamic component of the type III secretion injectisome.
Composition, formation, and regulation of the cytosolic c-ring, a dynamic component of the type III secretion injectisome.
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DOI：
10.1371/journal.pbio.1002039
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发表时间：
2015-01
期刊：
影响因子：
9.8
通讯作者：
Armitage JP
中科院分区：
文献类型：
--
作者：
Diepold A;Kudryashev M;Delalez NJ;Berry RM;Armitage JP
注射体是一种膜复合物，一些细菌可以通过它将效应蛋白注射到宿主细胞中。本研究表明，胞质c环结构与注射体的其余部分具有动态关系，可能对分泌的调节有影响。许多革兰氏阴性病原体使用III型分泌注射体将效应蛋白转运到真核宿主细胞中。虽然远端“针复合体”的结构有很好的文献记载，但功能重要的细胞质复合体的组成和作用仍然不太清楚。利用功能性荧光融合，我们发现c环是该系统的重要且保守的细胞质成分，由~22个SctQ拷贝（小肠结肠炎耶尔森菌中的YscQ）组成，它们需要YscQC （YscQ内部翻译起始位点的产物）的存在才能协同组装。光激活定位显微镜（PALM）显示，在体内，YscQ既存在于自由移动的细胞质中，也存在于稳定的注射结合状态中。值得注意的是，光漂白后荧光恢复（FRAP）显示注射小体和细胞质之间的YscQ交换，在注射小体分泌时，交换时间为68±8秒。相比之下，分泌素SctC （YscC）和主要出口器官成分SctV （YscV）的交换最小。在非分泌条件下，YscQ的交换速率降低到t½= 134±16秒，揭示了c环交换与注射活性之间的相关性，这表明c环稳定性可能在调节III型分泌中起作用。c环的稳定取决于功能性atp酶SctN （YscN）的存在。这些数据为注射体的形成和组成提供了新的见解，并呈现了III型分泌的新方面，即c环亚基的交换，这与分泌有关。III型分泌系统，也称为注射体，是许多革兰氏阴性细菌的关键毒力因子，负责将细菌蛋白直接传播到宿主细胞中。虽然该系统的一些元素已被很好地表征，但参与底物识别和处理的细胞质成分尚未被很好地理解。其中一个主要的问题是c环的作用，一个重要的但神秘的细胞质注射成员。我们使用荧光显微镜分析了活的小肠结肠炎细菌c环的结构和行为。我们发现，在体内，c环在其自身c端片段的额外拷贝的帮助下协同组装，并具有高度动态的结构，c环亚基在工作注射体和细胞质池之间交换。分泌和非分泌注射体的交换速率不同，取决于III型分泌atp酶的功能，表明复合物的稳定性在功能时发生改变。这种动态行为提出了c环是III型分泌系统靶向蛋白递送的调节剂的可能性，并使c环成为开发新型抗毒药物的可行靶点。
The injectisome is a membrane complex through which some bacteria can inject effector proteins into host cells. This study reveals that the cytosolic C-ring structure has a dynamic relationship to the rest of the injectisome, with implications for the regulation of secretion. Many gram-negative pathogens employ a type III secretion injectisome to translocate effector proteins into eukaryotic host cells. While the structure of the distal “needle complex” is well documented, the composition and role of the functionally important cytosolic complex remain less well understood. Using functional fluorescent fusions, we found that the C-ring, an essential and conserved cytosolic component of the system, is composed of ~22 copies of SctQ (YscQ in Yersinia enterocolitica), which require the presence of YscQC, the product of an internal translation initiation site in yscQ, for their cooperative assembly. Photoactivated localization microscopy (PALM) reveals that in vivo, YscQ is present in both a free-moving cytosolic and a stable injectisome-bound state. Notably, fluorescence recovery after photobleaching (FRAP) shows that YscQ exchanges between the injectisome and the cytosol, with a t½ of 68 ± 8 seconds when injectisomes are secreting. In contrast, the secretin SctC (YscC) and the major export apparatus component SctV (YscV) display minimal exchange. Under non-secreting conditions, the exchange rate of YscQ is reduced to t½ = 134 ± 16 seconds, revealing a correlation between C-ring exchange and injectisome activity, which indicates a possible role for C-ring stability in regulation of type III secretion. The stabilization of the C-ring depends on the presence of the functional ATPase SctN (YscN). These data provide new insights into the formation and composition of the injectisome and present a novel aspect of type III secretion, the exchange of C-ring subunits, which is regulated with respect to secretion. The type III secretion system, also known as the injectisome, is a key virulence factor in many gram-negative bacteria, and is responsible for the transmission of bacterial proteins directly into host cells. While some elements of the system are well characterized, the cytosolic components involved in substrate recognition and handling are not well understood. One of the major questions is the role of the C-ring, an essential yet enigmatic cytosolic injectisome member. We used fluorescence microscopy to analyze the architecture and behavior of the C-ring in live Y. enterocolitica bacteria, a human pathogen. We found that in vivo, the C-ring assembles cooperatively with the help of additional copies of its own C-terminal fragment and has a highly dynamic structure, with C-ring subunits exchanging between the working injectisomes and a cytosolic pool. The rate of exchange is different between secreting and non-secreting injectisomes and depends on the function of the type III secretion ATPase, indicating that the stability of the complex is altered when functioning. This dynamic behaviour raises the possibility that the C-ring is a regulator of targeted protein delivery by the type III secretion system and makes the C-ring a viable target for the development of novel anti-virulence drugs.
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