Live-Cell Imaging of Phosphoinositide Dynamics and Membrane Architecture during Legionella Infection
Live-Cell Imaging of Phosphoinositide Dynamics and Membrane Architecture during Legionella Infection
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军团菌感染期间磷酸肌醇动力学和膜结构的活细胞成像
作者：
Wagner
军团菌病的病原体嗜肺军团菌在阿米巴和巨噬细胞中的一个独特的膜结合区室（含军团菌空泡（LCV））中复制。LCV的形成受细菌Icm/Dot IV型分泌系统控制，该系统将约300种不同的“效应”蛋白易位到宿主细胞中。一些移位的效应物通过磷酸肌醇（PI）脂质锚到LCV膜。在这里，我们使用土壤变形虫Dictyosteelium discoideum，产生荧光PI探针，分析LCV PI动态活细胞成像。在摄取野生型或Icm/Dot缺陷型L. PtdIns（3，4，5）P3在早期吞噬体上瞬时积累平均40 s，并在摄取后1 min内获得PtdIns（3）P3。尽管含有Δ icmT突变体细菌的吞噬体仍然被PtdIns（3）P修饰，但超过80%的野生型LCV在2 h内逐渐失去这种PI。该过程伴随着PtdIns（3）P阳性膜浓缩到细胞中心的主要重排。PtdIns（4）P瞬时定位于携带野生型或ΔicmT嗜肺军团菌的早期吞噬体，并在摄取后数分钟内被清除。在随后的2 h内，PtdIns（4）P仅在野生型LCV上稳定积累，而野生型LCV在空间上与钙连接蛋白阳性内质网（ER）分离至少8 h，保持离散的PtdIns（4）P身份。对于携带ER募集缺陷的Δ sidC-sdcA突变细菌的LCV，PtdIns（4）P阳性和ER膜的分离更加明显，而不影响病原体液泡中的初始细菌复制。这些研究结果阐明了时间和空间的动态PI脂质牵连LCV的形成，并提供深入了解宿主细胞膜和效应蛋白interactions.IMPORTANCEThe环境细菌嗜肺军团菌是军团菌肺炎的病原体。这种细菌在自由生活的阿米巴原虫和哺乳动物免疫细胞中形成一个允许复制的隔室，即含军团菌的空泡（LCV）。为了破坏宿主细胞的过程，细菌分泌数量惊人的~300种不同的蛋白质进入宿主细胞。这些蛋白质中的一些结合磷脂酰肌醇（PI）脂质以装饰LCV。PI脂质是参与宿主细胞膜动力学和LCV形成的关键因素。使用Dictyosteliumamoeacetylate产生一个或两个不同的荧光探针，我们阐明了高时空分辨率的动态LCV PI模式。值得注意的是，内吞PI脂质PtdIns（3）P从LCV中缓慢清除，从而使宿主细胞的消化机制丧失能力，而PtdIns（4）P在LCV上逐渐积累，从而能够与宿主细胞器进行关键的相互作用。LCV PI模式是细菌效应蛋白的时空构型的基础，因此代表了LCV形成的关键方面。
The causative agent of Legionnaires’ disease, Legionella pneumophila, replicates in amoebae and macrophages in a distinct membrane-bound compartment, theLegionella-containing vacuole (LCV). LCV formation is governed by the bacterial Icm/Dot type IV secretion system that translocates ~300 different “effector” proteins into host cells. Some of the translocated effectors anchor to the LCV membrane via phosphoinositide (PI) lipids. Here, we use the soil amoeba Dictyostelium discoideum, producing fluorescent PI probes, to analyze the LCV PI dynamics by live-cell imaging. Upon uptake of wild-type or Icm/Dot-deficient L. pneumophila, PtdIns(3,4,5)P3transiently accumulated for an average of 40 s on early phagosomes, which acquired PtdIns(3)Pwithin 1 min after uptake. Whereas phagosomes containing ΔicmTmutant bacteria remained decorated with PtdIns(3)P, more than 80% of wild-type LCVs gradually lost this PI within 2 h. The process was accompanied by a major rearrangement of PtdIns(3)P-positive membranes condensing to the cell center. PtdIns(4)Ptransiently localized to early phagosomes harboring wild-type or ΔicmT L. pneumophilaand was cleared within minutes after uptake. During the following 2 h, PtdIns(4)Psteadily accumulated only on wild-type LCVs, which maintained a discrete PtdIns(4)Pidentity spatially separated from calnexin-positive endoplasmic reticulum (ER) for at least 8 h. The separation of PtdIns(4)P-positive and ER membranes was even more pronounced for LCVs harboring ΔsidC-sdcAmutant bacteria defective for ER recruitment, without affecting initial bacterial replication in the pathogen vacuole. These findings elucidate the temporal and spatial dynamics of PI lipids implicated in LCV formation and provide insight into host cell membrane and effector protein interactions.IMPORTANCEThe environmental bacterium Legionella pneumophila is the causative agent of Legionnaires’ pneumonia. The bacteria form in free-living amoebae and mammalian immune cells a replication-permissive compartment, theLegionella-containing vacuole (LCV). To subvert host cell processes, the bacteria secrete the amazing number of ~300 different proteins into host cells. Some of these proteins bind phosphoinositide (PI) lipids to decorate the LCV. PI lipids are crucial factors involved in host cell membrane dynamics and LCV formation. UsingDictyosteliumamoebae producing one or two distinct fluorescent probes, we elucidated the dynamic LCV PI pattern in high temporal and spatial resolution. Notably, the endocytic PI lipid PtdIns(3)Pwas slowly cleared from LCVs, thus incapacitating the host cell’s digestive machinery, while PtdIns(4)Pgradually accumulated on the LCV, enabling critical interactions with host organelles. The LCV PI pattern underlies the spatiotemporal configuration of bacterial effector proteins and therefore represents a crucial aspect of LCV formation.
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影响因子：
3.4
作者：
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通讯作者：
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影响因子：
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作者：
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通讯作者：
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影响因子：
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作者：
G. Gerisch;A. Müller
通讯作者：
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影响因子：
4.4
作者：
Mampel, J;Spirig, T;Hilbi, H
通讯作者：
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