Rolland Steven J, Lifschin Zachary J, Weddle Erin A, Yum Lauren K, Miyake Tsuyoshi, Engel Daniel A, Agaisse Hervé F
Department of Microbiology, Immunology, and Cancer Biology, University of Virginia School of Medicine, Charlottesville, Virginia, United States of America.
PLoS Pathog. 2025 May 16;21(5):e1012707. doi: 10.1371/journal.ppat.1012707. eCollection 2025 May.
Shigella flexneri is a human intracellular pathogen responsible for bacillary dysentery (bloody diarrhea). S. flexneri invades colonic epithelial cells and spreads from cell to cell, leading to massive epithelial cell fenestration, a critical determinant of pathogenesis. Cell-to-cell spread relies on actin-based motility, which leads to formation of membrane protrusions, as bacteria project into adjacent cells. Membrane protrusions resolve into intermediate structures termed vacuole-like protrusions (VLPs), which remain attached to the primary infected cell by a membranous tether. The resolution of the membranous tether leads to formation of double-membrane vacuoles (DMVs), from which S. flexneri escapes to gain access to the cytosol of adjacent cells. Here, we identify the class III PI3K family member PIK3C3 as a critical determinant of S. flexneri cell-to-cell spread. Inhibition of PIK3C3 decreased the size of infection foci formed by S. flexneri in HT-29 cells. Tracking experiments using live-fluorescence confocal microscopy showed that PIK3C3 is required for efficient resolution of VLPs into DMVs. PIK3C3-dependent accumulation of PtdIns(3)P at the VLP membrane in adjacent cells correlated with the transient recruitment of the membrane scission machinery component Dynamin 2 at the neck of VLPs at the time of DMV formation. By contrast, Listeria monocytogenes did not form VLPs and protrusions resolved directly into DMVs. However, PIK3C3 was also required for L. monocytogenes dissemination, but at the stage of vacuole escape. Finally, we showed that PIK3C3 inhibition decreased S. flexneri dissemination in the infant rabbit model of shigellosis. We propose a model of Shigella dissemination in which vacuole formation relies on the PIK3C3-dependent accumulation of PtdIns(3)P at the VLP stage of cell-to-cell spread, thereby supporting the resolution of VLPs into DMVs through recruitment of the membrane scission machinery component, DNM2.
福氏志贺菌是一种导致细菌性痢疾（血性腹泻）的人类细胞内病原体。福氏志贺菌侵入结肠上皮细胞并在细胞间传播，导致大量上皮细胞穿孔，这是发病机制的一个关键决定因素。细胞间传播依赖于基于肌动蛋白的运动性，随着细菌突入相邻细胞，会导致膜突起的形成。膜突起会分解为称为液泡样突起（VLP）的中间结构，这些结构通过膜性系链与初始感染细胞相连。膜性系链的分解导致双膜空泡（DMV）的形成，福氏志贺菌从DMV中逸出，从而进入相邻细胞的细胞质。在此，我们确定III类PI3K家族成员PIK3C3是福氏志贺菌细胞间传播的关键决定因素。抑制PIK3C3会减小福氏志贺菌在HT-29细胞中形成的感染灶的大小。使用实时荧光共聚焦显微镜进行的追踪实验表明，PIK3C3是VLP有效分解为DMV所必需的。PIK3C3依赖的PtdIns(3)P在相邻细胞的VLP膜上的积累与在DMV形成时VLP颈部膜分裂机制成分发动蛋白2的短暂募集相关。相比之下，单核细胞增生李斯特菌不会形成VLP，突起会直接分解为DMV。然而，PIK3C3对于单核细胞增生李斯特菌的传播也是必需的，但作用于液泡逃逸阶段。最后，我们表明抑制PIK3C3会减少福氏志贺菌在志贺菌病幼兔模型中的传播。我们提出了一种志贺菌传播模型，其中空泡形成依赖于PIK3C3依赖的PtdIns(3)P在细胞间传播的VLP阶段的积累，从而通过募集膜分裂机制成分DNM2来支持VLP分解为DMV。
