Laboratory of Molecular Biology of Bacterial Pathogens, Cell and Molecular Microbiology Division, Institute of Microbiology, Academy of Sciences of the Czech Republic, 142 20 Prague 4, Czech Republic.
Institute of Analytical Chemistry, Academy of Sciences of the Czech Republic, 60200 Brno, Czech Republic.
Anal Biochem. 2014 Apr 1;450:57-62. doi: 10.1016/j.ab.2013.10.039. Epub 2014 Jan 9.
The aim of this study was to compare two methods for quantification of changes in intracellular potassium concentration (decrease from ∼140 to ∼20mM) due to the action of a pore-forming toxin, the adenylate cyclase toxin (CyaA) from the pathogenic bacterium Bordetella pertussis. CyaA was incubated with stably transfected K1 Chinese hamster ovary cells expressing the toxin receptor CD11b/CD18 and the decrease in potassium concentration in the cells was followed by inductively coupled plasma mass spectrometry (ICP-MS). It is shown that this method is superior in terms of sensitivity, accuracy, and temporal resolution over the method employing the potassium-binding benzofuran isophthalate-acetoxymethyl ester fluorescent indicator. The ICP-MS procedure was found to be a reliable and straightforward analytical approach enabling kinetic studies of CyaA action at physiologically relevant toxin concentrations (<1000ng/ml) in biological microsamples.
本研究旨在比较两种方法，用于定量研究由于一种成孔毒素（即来自致病性细菌百日咳博德特氏菌的腺苷酸环化酶毒素（CyaA））的作用而导致细胞内钾离子浓度（从约 140 降至约 20mM）的变化。CyaA 与稳定转染的表达毒素受体 CD11b/CD18 的 K1 中国仓鼠卵巢细胞孵育，并通过电感耦合等离子体质谱法（ICP-MS）来检测细胞内钾离子浓度的降低。结果表明，与使用钾结合苯并呋喃异邻苯二甲酸酯-乙酰氧甲酯荧光指示剂的方法相比，该方法在灵敏度、准确性和时间分辨率方面具有优势。ICP-MS 程序被发现是一种可靠且直接的分析方法，能够在生理相关的毒素浓度（<1000ng/ml）下对生物微样本中的 CyaA 作用进行动力学研究。
