Institut Pasteur, Groupe Dynamique des interactions hôte-pathogène, 75724, Paris, France.
Cell Microbiol. 2010 Apr 1;12(4):545-56. doi: 10.1111/j.1462-5822.2010.01428.x. Epub 2010 Jan 11.
Escape into the host cell cytosol following invasion of mammalian cells is a common strategy used by invasive pathogens. This requires membrane rupture of the vesicular or vacuolar compartment formed around the bacteria after uptake into the host cell. The mechanism of pathogen-induced disassembly of the vacuolar membrane is poorly understood. We established a novel, robust and sensitive fluorescence microscopy method that tracks the precise time point of vacuole rupture upon uptake of Gram-negative bacteria. This revealed that the enteroinvasive pathogen Shigella flexneri escapes rapidly, in less than 10 min, from the vacuole. Our method demonstrated the recruitment of host factors, such as RhoA, to the bacterial entry site and their continued presence at the point of vacuole rupture. We found a novel host marker for ruptured vacuoles, galectin-3, which appears instantly in the proximity of bacteria after escape into the cytosol. Furthermore, we show that the Salmonella effector proteins, SifA and PipB2, stabilize the vacuole membrane inhibiting bacterial escape from the vacuole. Our novel approach to track vacuole rupture is ideally suited for high-content and high-throughput approaches to identify the molecular and cellular mechanisms of membrane rupture during invasion by pathogens such as viruses, bacteria and parasites.
入侵哺乳动物细胞后逃入宿主细胞胞质溶胶是侵袭性病原体常用的策略。这需要在细菌被宿主细胞摄取后，围绕细菌形成的囊泡或空泡腔内的膜破裂。病原体诱导的空泡膜解体的机制还不太清楚。我们建立了一种新颖、稳健和敏感的荧光显微镜方法，可跟踪革兰氏阴性菌摄取后空泡破裂的确切时间点。这表明侵袭性肠道病原体福氏志贺菌能迅速从空泡中逃逸，不到 10 分钟。我们的方法显示了宿主因子（如 RhoA）被招募到细菌进入部位，并在空泡破裂点持续存在。我们发现了一种新的宿主标记物用于破裂的空泡，即半乳糖凝集素-3，它在逃入胞质溶胶后立即出现在细菌附近。此外，我们发现沙门氏菌效应蛋白 SifA 和 PipB2 稳定了空泡膜，抑制了细菌从空泡中的逃逸。我们追踪空泡破裂的新方法非常适合用于高通量和高内涵方法，以鉴定病毒、细菌和寄生虫等病原体入侵过程中空泡膜破裂的分子和细胞机制。
