CHARACTERIZATION OF THE MICROENVIRONMENT OF SALMONELLA-TYPHIMURIUM-CONTAINING VACUOLES WITHIN MDCK EPITHELIAL-CELLS
CHARACTERIZATION OF THE MICROENVIRONMENT OF SALMONELLA-TYPHIMURIUM-CONTAINING VACUOLES WITHIN MDCK EPITHELIAL-CELLS
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DOI：
10.1111/j.1365-2958.1992.tb02197.x
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发表时间：
1992-11-01
影响因子：
3.6
通讯作者：
FINLAY, BB
中科院分区：
文献类型：
--
作者：
GARCIADELPORTILLO, F;FOSTER, JW;FINLAY, BB
鼠伤寒沙门氏菌具有进入上皮细胞并在上皮细胞内繁殖的能力。在整个胞内阶段，细菌被包裹在液泡中。为了表征含细菌液泡的微环境，我们采用了一种新的方法来测量Madin-Darby犬肾（MDCK）上皮细胞内细菌中几种鼠伤寒沙门氏菌基因的表达水平。我们的研究基于使用高敏感底物荧光素-二- β -d -半乳糖苷（FDG）测定lacZ转录融合物衍生的β -半乳糖苷酶活性。iroA和mgtB基因（分别受Fe2+和Mg2+限制诱导）和cadA基因（赖氨酸存在下pH 6.0诱导，厌氧条件下表达增强）在不同感染后时间的表达特征。iroA和mgtB基因在细胞内高表达，提示液泡中游离Fe2+和Mg2+的浓度可能较低。cadA活性仅在感染后早期（4小时）检测到，这表明液泡可能具有温和的酸性，此时存在氧和赖氨酸。在全球范围内，报告的结果表明，使用高度敏感的β -半乳糖苷酶底物可以提供关于细胞内病原体（如鼠伤寒沙门氏菌）居住的微环境的信息。
Salmonella typhimurium has the capacity to enter into and multiply within epithelial cells. During the entire intracellular stage, bacteria are enclosed within a vacuole. To characterize the micro-environment of the bacteria-containing vacuoles, we have used a new method to measure the expression levels of several S. typhimurium genes in intracellular bacteria within Madin-Darby canine kidney (MDCK) epithelial cells. Our study was based on the determination of beta-galactosidase activity derived from lacZ transcriptional fusions using the highly sensitive substrate fluorescein-di-beta-D-galactoside (FDG). Expression of the iroA and mgtB genes (induced by Fe2+ and Mg2+ limitation respectively), and cadA (induced by pH 6.0 in the presence of lysine, with enhanced expression under anaerobiosis) were characterized at different post-infection times. High intracellular expression levels were detected for the iroA and mgtB genes, suggesting that the concentrations of free Fe2+ and Mg2+ in the vacuole may be low. cadA actitvity was detected only at early post-infection times (4 h), suggesting that the vacuole may have a mild-acidic pH, and oxygen and lysine present at this time. Globally, the results reported indicate that the use of a highly sensitive beta-galactosidase substrate can provide information about the micro-enviroment within which an intracellular pathogen, such as S. typhimurium, resides.