| Design of a Transcription-based Secretion Activity Reporter (TSAR) for the Type III Secretion Apparatus of Shigella flexneri and Uses Thereof
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Author:
Date:
2014-10-20
[Abstract] Many gram-negative bacterial pathogens, including Shigella flexneri, are able to translocate bacterial proteins, dubbed effectors, across the host cell plasma membrane into the host cell cytosol using a syringe-like structure, the type three secretion apparatus (T3SA). While some bacteria use their T3SA to modulate their phagosomal environment (Salmonella spp.), establish pedestal structure to form microcolonies on the plasma membrane (Enteropathogenic Escherichi coli) or lyse their entry vacuole (Shigella spp.), they all have in common a tightly regulated activity of their T3SA. However, the tracking of the activity of the T3SA in infected cells and tissue has been difficult to perform. Using the property of MxiE-dependent promoters that are ...
[摘要] 包括灵芝氏菌在内的许多革兰氏阴性细菌病原体能够使用注射器样结构将类型三分泌物穿过宿主细胞质膜转运细菌蛋白质（配体效应子）到宿主细胞胞质溶胶中装置（T3SA）。虽然一些细菌使用它们的T3SA调节它们的吞噬体环境（沙门氏菌 spp 。），建立基质结构以在质膜上形成微集落（Enteropathogenic < em="">）或溶解它们的进入泡（志贺氏菌属。），它们都具有共同的严格调节的他们的T3SA的活性。然而，T3SA在感染的细胞和组织中的活性的跟踪一直难以进行。使用在T3SA是活性时上调的MxiE依赖性启动子的性质，我们最近设计了基于转录的分泌活性报道分子（TSAR），其允许以下的S的活性。使用快速成熟的GFP内在荧光，在组织培养细胞中实时地和体内实时分析灵敏度。在这里我们描述TSAR的设计及其应用于固定和活体样品的显微镜和流式细胞仪在结肠上皮细胞模型使用TC7组织培养细胞。
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| Detection of the Secreted and Cytoplasmic Fractions of IpaB, IpaC and IpaD by Lysozyme Permeabilization
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Author:
Date:
2014-10-20
[Abstract] Gram negative bacterial pathogens, such as Shigella flexneri, which possess a Type Three Secretion System (T3SS), are able to transfer bacterial proteins, dubbed translocators and effectors, from their cytoplasm into the cytoplasm of their host cells using a syringe like needle complex. For Shigella, it has been shown that during cellular invasion, the intrabacterial pool of translocators and effectors is completely depleted upon activation of the TTS Apparatus and is then progressively replenished while bacteria remain inside host cells. Replenishment of effectors allows for cell-to-cell spreading events, which also necessitate reactivation of the T3SA, and lead to another round of depletion of intrabacterial effector stores. To understand the state of individual ...
[摘要] 具有三型分泌系统（T3SS）的革兰氏阴性细菌病原体如灵芝氏菌能够将细菌蛋白质，配位的易位蛋白和效应子从其细胞质转移到其宿主细胞的细胞质中使用注射器如针复合物。对于志贺氏菌（Shigella），已经表明在细胞侵袭过程中，转位子和效应子的细菌池在TTS装置激活时完全耗尽，然后逐渐补充，同时细菌保留在宿主细胞内。补充效应物允许细胞间的扩散事件，这也需要T3SA的再活化，并导致细胞内效应子库的另一轮消耗。为了理解感染期间单个细胞内细菌的状态，因此有兴趣的是能够定位和评估细菌和分泌的易位蛋白和效应子池的相对数量。我们最近改编了基于EDTA和溶菌酶的方法以透化宿主细胞内存在的细菌的细胞壁，以标记尖端蛋白IpaD和易位蛋白IpaB和IpaC的细菌池。在这里，我们详细描述执行连续标记细菌和分泌池的协议。这种方法理论上可扩展到由其他分泌系统和其他细菌病原体分泌的毒力因子。
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| Fluorescence Microscopy Analysis of Drug Effect on Autophagosome Formation
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Author:
Date:
2014-04-05
[Abstract] The autophagy protein, LC3 represents a reliable characteristic marker for autophagosomal structures. The initial LC3 is processed by the cysteine protease autophagy-related gene 4 (Atg4) at its C terminus in order to create LC3-I generally localized in the cytoplasm. Afterwards LC3-I is conjugated with phosphatidylethanolamine (PE) to become LC3-PE or LC3-II predominantly localised on the autophagosomal membranes (outer and inner). Autolysosomal content of LC3-II is very low as upon autophago/lysosomal fusion it is either cleaved off from the outer membrane by Atg4 or degraded together with the inner membrane by the lysosomal activity. Therefore GFP-LC3 and mCherry-GFP-LC3 might be visualized by conventional or confocal fluorescence microscopy (FM). In this situation mCherry-GFP-LC3 or ...
[摘要] 自噬蛋白，LC3代表自噬体结构的可靠特征标记。最初的LC3由半胱氨酸蛋白酶自噬相关基因4（Atg4）在其C末端处理，以产生通常位于细胞质中的LC3-I。之后LC3-I与磷脂酰乙醇胺（PE）缀合以变成主要定位在自噬体膜（外部和内部）上的LC3-PE或LC3-II。 LC3-II的自溶酶体内含物非常低，因为在自噬/溶酶体融合时，其被Atg4从外膜切割或与内膜一起通过溶酶体活性降解。因此，GFP-LC3和mCherry-GFP-LC3可能通过常规或共聚焦荧光显微镜（FM）可视化。在这种情况下，mCherry-GFP-LC3或GFP-LC3细胞质池被可视化为均匀分散的信号，并且含有mCherry-GFP-LC3-II或GFP-LC3-II的自噬体被检测为点状形成。斑点数可以用作自噬体丰度的标志物。一般来说，我们建议计数每个细胞的GFP-LC3斑点的平均数。
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