The facultative intracellular pathogen 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, induces the formation of large vesicles around the entry site, which we refer to as infection-associated macropinosomes (IAMs). After entry, 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 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 establishes its intracellular niche through the subversion of a specific set of IAMs.

中文翻译：

---

志贺氏菌在上皮细胞侵袭过程中产生独特的 IAM 亚群，促进有效的细胞内生态位形成

兼性细胞内病原体侵入非吞噬性上皮肠道细胞。通过称为 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（称为含细菌液泡 (BCV)）内被吸收。同时，诱导入口位点周围形成大囊泡，我们将其称为感染相关巨胞质体 (IAM)。进入后，BCV 破裂并通过分解 BCV 残余物逃逸到宿主细胞质中。此前，IAM 的形成已被证明是 BCV 有效逃逸所必需的，但与 BCV 分解相关的分子事件仍不清楚。为了确定 BCV 分解所需的宿主成分，我们进行了基于显微镜的筛选，以监测含有 BAR 结构域的蛋白质的招募，这些蛋白质是参与上皮细胞侵袭过程中膜成形和传感（例如内吞作用和再循环）的宿主蛋白质家族。我们在 BCV 解体之前鉴定出以 PI(3)P 依赖性方式定位于 IAM 的内体回收 BAR 蛋白分选 Nexin-8 (SNX8)。在 BCV 周围发现了至少两个不同的 IAM 亚群，它们要么被回收回质膜等细胞区室，要么转变为参与促进 BCV 破裂的 RAB11A 阳性“接触 IAM”。 IAM 亚群二元性的特点是仅招募 SNX8 或 RAB11A。阻碍 IAM 的 PI(3)P 产生会抑制这些区室中 SNX8 的募集，并延迟 BCV 破裂时间和成功 BCV 分解的步骤。最后，SNX8 的 siRNA 消耗加速了 BCV 破裂和 BCV 残余物的剥落，表明 SNX8 参与控制胞质释放的时间。总的来说，我们的工作揭示了如何通过颠覆一组特定的 IAM 来建立其细胞内生态位。