Dytoc M, Fedorko L, Sherman P M
Division of Gastroenterology, Hospital for Sick Children, Toronto, Ontario, Canada.
Gastroenterology. 1994 May;106(5):1150-61. doi: 10.1016/0016-5085(94)90004-3.
BACKGROUND/AIMS: Human enteropathogenic Escherichia coli infection of epithelial cells is characterized by attaching and effacing adhesion. To determine if signal transduction responses are involved in this adhesion phenotype, levels of inositol 1,4,5-triphosphate and cytosolic free calcium were measured in tissue culture cells infected with enteropathogenic E. coli strain E2348 (serotype O127:H6).
Inositol triphosphate levels were measured by using a commercial binding assay, and intracellular calcium levels were determined by spectrofluorometry.
Elevated levels of both inositol triphosphate (182% +/- 52%; P < 0.05) and intracellular calcium (125% +/- 40%, mean +/- SE; P < 0.05) were seen after infection of HEp-2 cells with strain E2348. In contrast, inositol triphosphate and intracellular calcium levels were not elevated in HEp-2 cells infected with six E. coli strains that did not cause attaching and effacing lesions. Subcellular calcium localization using oxalate precipitation and electron microscopy showed calcium accumulation within the terminal web subjacent to regions of attaching and effacing adhesion. Depleting external calcium did not eliminate formation of attaching and effacing lesions, whereas treatment of HEp-2 cells with an intracellular calcium chelator prevented attaching and effacing lesions.
Enteropathogenic E. coli infection elevates both inositol triphosphate and intracellular calcium levels in cultured epithelial cells.
背景/目的：人肠道致病性大肠杆菌感染上皮细胞的特征是紧密黏附和消除黏附。为了确定信号转导反应是否参与这种黏附表型，我们检测了感染肠道致病性大肠杆菌E2348菌株（血清型O127:H6）的组织培养细胞中肌醇1,4,5-三磷酸和胞质游离钙的水平。
使用商业结合测定法测量肌醇三磷酸水平，通过荧光分光光度法测定细胞内钙水平。
用E2348菌株感染HEp-2细胞后，肌醇三磷酸（182%±52%；P<0.05）和细胞内钙（125%±40%，平均值±标准误；P<0.05）水平均升高。相比之下，感染六种未引起紧密黏附和消除性损伤的大肠杆菌菌株的HEp-2细胞中，肌醇三磷酸和细胞内钙水平未升高。使用草酸盐沉淀和电子显微镜进行亚细胞钙定位显示，钙在紧密黏附和消除性黏附区域下方的终末网内积累。耗尽细胞外钙并不能消除紧密黏附和消除性损伤的形成，而用细胞内钙螯合剂处理HEp-2细胞可防止紧密黏附和消除性损伤。
肠道致病性大肠杆菌感染可提高培养的上皮细胞中肌醇三磷酸和细胞内钙水平。
