Quantitative assessment of cytosolic Salmonella in epithelial cells.
Quantitative assessment of cytosolic Salmonella in epithelial cells.
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DOI：
10.1371/journal.pone.0084681
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Steele-Mortimer O
中科院分区：
文献类型：
--
作者：
Knodler LA;Nair V;Steele-Mortimer O
在哺乳动物细胞内，肠沙门氏菌血清型鼠伤寒沙门氏菌（S. Typhimurium）栖息在一个被称为含沙门氏菌液泡（SCV）的膜结合液泡中。我们最近发现野生型鼠伤寒沙门氏菌也在上皮细胞的细胞质中定植。在这里，我们试图量化细胞质沙门氏菌在不同上皮细胞系感染的一段时间内对总群体的贡献，并在改变液泡逃逸的条件下。我们发现溶酶促缩剂氯喹对空泡沙门氏菌起作用，而对胞质沙门氏菌不起作用。在氯喹处理后，液泡细菌不再具有转录活性或复制能力，出现降解。通过氯喹耐药试验，除了洋地黄苷渗透外，我们发现鼠伤寒沙门氏菌在许多上皮细胞系中裂解其新生液泡，尽管频率不同，并且胞浆中的超复制也很普遍。在感染后的后期，在一些上皮细胞系中，胞质细菌占总数的一半，即HeLa和Caco-2 C2Bbe1。这两种技术都精确地测量了渥曼宁处理后上皮细胞液泡溶解的增加。通过氯喹耐药试验，我们还确定了沙门氏菌致病性岛-1 (SPI-1)，而不是SPI-2，毒力质粒和鞭毛装置，是上皮细胞液泡逃逸和细胞质复制所必需的。洋地黄苷渗透和氯喹耐药试验将是有用的、互补的工具，用于破译SCV裂解和沙门氏菌在上皮细胞细胞质中复制的机制。
Within mammalian cells, Salmonella enterica serovar Typhimurium (S. Typhimurium) inhabits a membrane-bound vacuole known as the Salmonella-containing vacuole (SCV). We have recently shown that wild type S. Typhimurium also colonizes the cytosol of epithelial cells. Here we sought to quantify the contribution of cytosolic Salmonella to the total population over a time course of infection in different epithelial cell lines and under conditions of altered vacuolar escape. We found that the lysosomotropic agent, chloroquine, acts on vacuolar, but not cytosolic, Salmonella. After chloroquine treatment, vacuolar bacteria are not transcriptionally active or replicative and appear degraded. Using a chloroquine resistance assay, in addition to digitonin permeabilization, we found that S. Typhimurium lyses its nascent vacuole in numerous epithelial cell lines, albeit with different frequencies, and hyper-replication in the cytosol is also widespread. At later times post-infection, cytosolic bacteria account for half of the total population in some epithelial cell lines, namely HeLa and Caco-2 C2Bbe1. Both techniques accurately measured increased vacuole lysis in epithelial cells upon treatment with wortmannin. By chloroquine resistance assay, we also determined that Salmonella pathogenicity island-1 (SPI-1), but not SPI-2, the virulence plasmid nor the flagellar apparatus, was required for vacuolar escape and cytosolic replication in epithelial cells. Together, digitonin permeabilization and the chloroquine resistance assay will be useful, complementary tools for deciphering the mechanisms of SCV lysis and Salmonella replication in the epithelial cell cytosol.
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影响因子：
3.1
作者：
Brumell, JH;Tang, P;Finlay, BB
通讯作者：
Finlay, BB
影响因子：
13.3
作者：
Cemma, Marija;Kim, Peter Kijun;Brumell, John Hunter
通讯作者：
Brumell, John Hunter
影响因子：
56.9
作者：
CIAK, J;HAHN, FE
通讯作者：
HAHN, FE
影响因子：
6.4
作者：
Fernandez, IM;Silva, M;McCormick, BA
通讯作者：
McCormick, BA
DOI：
10.1084/jem.20030088
发表时间：
2003-08-18
期刊：
The Journal of experimental medicine
影响因子：
--
作者：
Celli J;de Chastellier C;Franchini DM;Pizarro-Cerda J;Moreno E;Gorvel JP
通讯作者：
Gorvel JP