Institut Pasteur, Unité de Pathogénie Microbienne Moléculaire, 75724 Paris, France; INSERM, U786, 75015, Paris, France.
Institut Pasteur, Unité de Pathogénie Microbienne Moléculaire, 75724 Paris, France; INSERM, U786, 75015, Paris, France.
Cell Host Microbe. 2014 Feb 12;15(2):177-89. doi: 10.1016/j.chom.2014.01.005.
Numerous pathogenic Gram-negative bacteria use a type three secretion apparatus (T3SA) to translocate effector proteins into host cells. Detecting and monitoring the T3SA of intracellular bacteria within intact host cells has been challenging. Taking advantage of the tight coupling between T3S effector-gene transcription and T3SA activity in Shigella flexneri, together with a fast-maturing green fluorescent protein, we developed reporters to monitor T3SA activity in real time. These reporters reveal a dynamic temporal regulation of the T3SA during the course of infection. T3SA is activated initially during bacterial entry and downregulated subsequently when bacteria gain access to the host cell cytoplasm, allowing replenishment of the bacterial stores of T3S substrates necessary for invading neighboring cells. Reactivation of the T3SA was strictly dependent on actin-based motility and formation of plasma membrane protrusions during cell-to-cell spread. Thus, the T3SA is subject to a tight on/off regulation within the bacterial intracellular niche.
许多致病性革兰氏阴性菌使用 III 型分泌装置（T3SA）将效应蛋白转运到宿主细胞中。在完整的宿主细胞内检测和监测胞内细菌的 T3SA 一直具有挑战性。利用福氏志贺菌中 T3S 效应基因转录与 T3SA 活性之间的紧密偶联，以及快速成熟的绿色荧光蛋白，我们开发了报告基因来实时监测 T3SA 活性。这些报告基因揭示了感染过程中 T3SA 的动态时间调节。T3SA 在细菌进入时最初被激活，随后当细菌进入宿主细胞质时被下调，从而补充了入侵邻近细胞所需的 T3S 底物的细菌储存。T3SA 的再激活严格依赖于细胞间传播过程中的肌动蛋白依赖性运动和质膜突起的形成。因此，T3SA 在细菌细胞内环境中受到严格的开/关调节。
