Abstract：
The facultative intracellular pathogen Shigella flexneri invades non-phagocytic epithelial gut cells. Through a syringe-like apparatus called type 3 secretion system, it injects effector proteins into the host cell triggering actin rearrangements leading to its uptake within a tight vacuole, termed the bacterial-containing vacuole (BCV). Simultaneously, Shigella induces the formation of large vesicles around the entry site, which we refer to as infection-associated macropinosomes (IAMs). After entry, Shigella ruptures the BCV and escapes into the host cytosol by disassembling the BCV remnants. Previously, IAM formation has been shown to be required for efficient BCV escape, but the molecular events associated with BCV disassembly have remained unclear. To identify host components required for BCV disassembly, we performed a microscopy-based screen to monitor the recruitment of BAR domain-containing proteins, which are a family of host proteins involved in membrane shaping and sensing (e.g. endocytosis and recycling) during Shigella epithelial cell invasion. We identified endosomal recycling BAR protein Sorting Nexin-8 (SNX8) localized to IAMs in a PI(3)P-dependent manner before BCV disassembly. At least two distinct IAM subpopulations around the BCV were found, either being recycled back to cellular compartments such as the plasma membrane or transitioning to become RAB11A positive \"contact-IAMs\" involved in promoting BCV rupture. The IAM subpopulation duality was marked by the exclusive recruitment of either SNX8 or RAB11A. Hindering PI(3)P production at the IAMs led to an inhibition of SNX8 recruitment at these compartments and delayed both, the step of BCV rupture time and successful BCV disassembly. Finally, siRNA depletion of SNX8 accelerated BCV rupture and unpeeling of BCV remnants, indicating that SNX8 is involved in controlling the timing of the cytosolic release. Overall, our work sheds light on how Shigella establishes its intracellular niche through the subversion of a specific set of IAMs.
摘要：
兼性细胞内病原体福氏志贺氏菌侵入非吞噬性上消化道细胞。通过一个叫做3型分泌系统的类似注射器的装置，它将效应蛋白注入宿主细胞，触发肌动蛋白重排，导致其在紧密的液泡中吸收，称为含细菌液泡（BCV）。同时,志贺氏菌诱导在进入位点周围形成大囊泡，我们将其称为感染相关的大黄体（IAMs）。进入后,志贺氏菌破坏BCV并通过分解BCV残留物而逃逸到宿主胞质溶胶中。以前,IAM形成已被证明是有效的BCV逃逸所必需的。但与BCV拆解相关的分子事件仍不清楚.要识别BCV拆解所需的主机组件，我们进行了基于显微镜的屏幕来监测含有BAR结构域的蛋白质的募集，它们是在志贺氏菌上皮细胞侵袭期间参与膜成形和感知(例如胞吞和再循环)的宿主蛋白家族。我们确定了内体再循环BAR蛋白SortingNexin-8（SNX8）在BCV分解之前以PI（3）P依赖性方式定位到IAM。在BCV周围发现了至少两个不同的IAM亚群，要么循环回细胞区室,如质膜,要么转变为RAB11A阳性\"接触IAMs\"参与促进BCV破裂。IAM亚群的双重性以SNX8或RAB11A的独家招募为标志。阻碍IAM的PI（3）P产生导致这些隔室的SNX8募集受到抑制，并延迟了两者，BCV破裂时间和BCV拆卸成功的步骤。最后,SNX8的siRNA消耗加速了BCV破裂和BCV残留物的剥离，表明SNX8参与控制胞质释放的时间。总的来说,我们的工作揭示了志贺氏菌如何通过颠覆一组特定的IAM来建立其细胞内生态位。
