Saidi R F, Jaeger K, Montrose M H, Wu S, Sears C L
Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Cell Motil Cytoskeleton. 1997;37(2):159-65. doi: 10.1002/(SICI)1097-0169(1997)37:2<159::AID-CM8>3.0.CO;2-3.
Enterotoxigenic strains of B. fragilis associated with childhood diarrhea produce a 20 kD zinc metalloprotease toxin (BFT). BFT is reported to cleave G-actin in vitro and also causes dramatic rounding and rearrangement of the F-actin cytoskeleton in human intestinal epithelial cell lines (HT29) and HT29/C1). To test the hypothesis that the proteolysis of cellular actin by BFT in vivo may contribute to these alterations in morphology and cytoskeletal architecture, we assessed the F-actin content and the arrangement of the F- and G-actin cytoskeleton in BFT-treated HT29/C1 cells by spectrofluorimetry, confocal microscopy, and immunoblotting. BFT-treated cells were compared to cells treated with C. difficile toxin A (CDA) or cytochalasin D. Using spectrofluorimetric quantification, the F-actin content of BFT- and cytochalasin D-treated cells was unchanged in contrast to a significant decrease in CDA-treated cells. By confocal microscopy, the arrangement of F- and G-actin in all treated cells was markedly different than control cells. There was no change in the immunoblotting pattern of actin in the Triton-soluble or -insoluble cellular fractions of BFT-treated HT29/C1 cells. We conclude that BFT alters the F- and G-actin cytoskeletal architecture of HT29/C1 cells without direct proteolysis of actin or decrease in F-actin content.
与儿童腹泻相关的脆弱拟杆菌产毒素菌株可产生一种20千道尔顿的锌金属蛋白酶毒素（BFT）。据报道，BFT在体外可切割G-肌动蛋白，还能使人类肠道上皮细胞系（HT29和HT29/C1）中的F-肌动蛋白细胞骨架发生显著的变圆和重排。为了验证BFT在体内对细胞肌动蛋白的蛋白水解作用可能导致这些形态和细胞骨架结构改变的假说，我们通过荧光光谱法、共聚焦显微镜和免疫印迹法评估了经BFT处理的HT29/C1细胞中F-肌动蛋白的含量以及F-和G-肌动蛋白细胞骨架的排列情况。将经BFT处理的细胞与经艰难梭菌毒素A（CDA）或细胞松弛素D处理的细胞进行比较。通过荧光光谱法定量分析，与CDA处理的细胞中F-肌动蛋白含量显著降低形成对比的是，经BFT和细胞松弛素D处理的细胞中F-肌动蛋白含量未发生变化。通过共聚焦显微镜观察，所有处理细胞中F-和G-肌动蛋白的排列与对照细胞明显不同。在经BFT处理的HT29/C1细胞的Triton可溶性或不可溶性细胞组分中，肌动蛋白的免疫印迹模式没有变化。我们得出结论，BFT改变了HT29/C1细胞的F-和G-肌动蛋白细胞骨架结构，但没有直接对肌动蛋白进行蛋白水解或降低F-肌动蛋白含量。
