The target cell plasma membrane is a critical interface for Salmonella cell entry effector-host interplay
The target cell plasma membrane is a critical interface for Salmonella cell entry effector-host interplay
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DOI：
10.1111/j.1365-2958.2004.04336.x
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发表时间：
2004-11-01
影响因子：
3.6
通讯作者：
Koronakis, V
中科院分区：
文献类型：
--
作者：
Cain, RJ;Hayward, RD;Koronakis, V
沙门氏菌种类触发宿主膜褶皱，迫使其内化到非吞噬性肠上皮细胞中。这需要通过 III 型分泌系统将细菌效应蛋白递送到靶细胞中。六个易位效应器操纵细胞肌动蛋白动力学，但它们的直接和间接活动如何在空间和时间上协调以促进有效的细胞骨架重排基本上仍未被探索。为了进一步了解这一过程，我们应用机械细胞分级分离和免疫荧光显微镜来系统地研究瞬时转染和沙门氏菌感染的培养细胞中表位标记效应子的亚细胞定位。尽管五个效应器不包含明显的膜靶向结构域，但所有六个效应器都专门位于靶细胞质膜部分，并且相应地在细胞外围可见，从那里它们对肌动蛋白细胞骨架产生了不同的影响。出乎意料的是，在细胞质中没有检测到易位效应池。使用平行体外测定，我们证明异戊二烯化的细胞 GTPase Cdc42 对于沙门氏菌 GTP 交换因子和 GTPase 激活蛋白模拟 SopE 和 SptP 的膜结合是必要且充分的，它们没有内在的脂质亲和力。数据表明，宿主质膜是效应器-靶标相互作用的关键界面，并建立通用系统来进一步剖析效应器相互作用。
Salmonella species trigger host membrane ruffling to force their internalization into non-phagocytic intestinal epithelial cells. This requires bacterial effector protein delivery into the target cell via a type III secretion system. Six translocated effectors manipulate cellular actin dynamics, but how their direct and indirect activities are spatially and temporally co-ordinated to promote productive cytoskeletal rearrangements remains essentially unexplored. To gain further insight into this process, we applied mechanical cell fractionation and immunofluorescence microscopy to systematically investigate the subcellular localization of epitope-tagged effectors in transiently transfected and Salmonella-infected cultured cells. Although five effectors contain no apparent membrane-targeting domains, all six localized exclusively in the target cell plasma membrane fraction and correspondingly were visualized at the cell periphery, from where they induced distinct effects on the actin cytoskeleton. Unexpectedly, no translocated effector pool was detectable in the cell cytosol. Using parallel in vitro assays, we demonstrate that the prenylated cellular GTPase Cdc42 is necessary and sufficient for membrane association of the Salmonella GTP exchange factor and GTPase-activating protein mimics SopE and SptP, which have no intrinsic lipid affinity. The data show that the host plasma membrane is a critical interface for effector-target interaction, and establish versatile systems to further dissect effector interplay.