Secretion of type III effectors into host cells in real time
Secretion of type III effectors into host cells in real time
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DOI：
10.1038/nmeth804
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发表时间：
2005-12-01
期刊：
影响因子：
48
通讯作者：
Van Nhieu, GT
中科院分区：
文献类型：
--
作者：
Enninga, J;Mounier, J;Van Nhieu, GT
III型分泌（T3S）系统是许多革兰氏阴性细菌的关键特征，其将T3S效应蛋白直接转运到真核细胞中。在那里，T3S效应子发挥许多作用，例如细胞侵袭或调节宿主免疫应答。研究时空协调分泌的各种效应一直很难不破坏他们的功能。在这里，我们开发了一种新的方法，使用福氏志贺菌T3S作为模型，通过多维延时显微镜研究细菌易位的个别效应。我们证明，直接荧光标记的四半胱氨酸基序标记的效应IpaB和IpaC是可能的原位功能没有损失。研究IpaB和IpaC从单个细菌排出的T3S动力学，我们发现IpaB和IpaC的整个库在宿主细胞接触时同时释放，并且每种效应物的50%在240秒内分泌。这种方法允许一个前所未有的分析T3S期间的时空事件。
Type III secretion (T3S) systems are key features of many gram-negative bacteria that translocate T3S effector proteins directly into eukaryotic cells. There, T3S effectors exert many effects, such as cellular invasion or modulation of host immune responses. Studying spatiotemporal orchestrated secretion of various effectors has been difficult without disrupting their functions. Here we developed a new approach using Shigella flexneri T3S as a model to investigate bacterial translocation of individual effectors via multidimensional time-lapse microscopy. We demonstrate that direct fluorescent labeling of tetracysteine motif-tagged effectors IpaB and IpaC is possible in situ without loss of function. Studying the T3S kinetics of IpaB and IpaC ejection from individual bacteria, we found that the entire pools of IpaB and IpaC were released concurrently upon host cell contact, and that 50% of each effector was secreted in 240 s. This method allows an unprecedented analysis of the spatiotemporal events during T3S.