Enninga Jost, Mounier Joëlle, Sansonetti Philippe, Tran Van Nhieu Guy
INSERM U389, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris Cedex 15, France.
Nat Methods. 2005 Dec;2(12):959-65. doi: 10.1038/nmeth804. Epub 2005 Nov 18.
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.
III型分泌（T3S）系统是许多革兰氏阴性菌的关键特征，它能将T3S效应蛋白直接转运到真核细胞中。在真核细胞中，T3S效应蛋白发挥多种作用，如细胞侵袭或调节宿主免疫反应。在不破坏各种效应蛋白功能的情况下，研究它们在时空上的协同分泌一直很困难。在此，我们开发了一种新方法，以弗氏志贺菌T3S为模型，通过多维延时显微镜研究细菌对单个效应蛋白的转运。我们证明，对带有四半胱氨酸基序标签的效应蛋白IpaB和IpaC进行直接荧光标记，可在不丧失功能的情况下原位实现。通过研究单个细菌中IpaB和IpaC排出的T3S动力学，我们发现，IpaB和IpaC的整个库在与宿主细胞接触时同时释放，且每种效应蛋白的50%在240秒内分泌。该方法使人们能够对T3S过程中的时空事件进行前所未有的分析。
