Westrop G D, Campbell G, Kazi Y, Billcliffe B, Coote J G, Parton R, Freer J H, Edwards J G
Department of Microbiology, University of Glasgow, UK.
Microbiology (Reading). 1994 Feb;140 ( Pt 2):245-53. doi: 10.1099/13500872-140-2-245.
When baby hamster kidney (BHK) cells are allowed to spread on fibronectin-coated substrata in the absence of serum and the presence of agents which elevate intracellular 3':5'-cyclic AMP (cAMP) levels they adopt an abnormal, stellated morphology. To determine whether the invasive adenylate cyclase (AC) toxin of Bordetella pertussis induced the same response, cell extracts were prepared from several B. pertussis strains. They were characterized for AC toxin production by enzymic assay and by immunoblotting with an AC-toxin-specific monoclonal antibody. Extracts of strains producing AC toxin induced elevated levels of intracellular cAMP in BHK cells and promoted a stellation response during cell spreading. Extracts prepared from strains defective in AC toxin production showed no effect. Using image analysis to quantify the morphological change, we have demonstrated that the effect of AC toxin on cell spreading is dose dependent. This technique is a rapid and sensitive assay for the invasive AC toxin.
当幼仓鼠肾（BHK）细胞在无血清且存在能提高细胞内3':5'-环磷酸腺苷（cAMP）水平的试剂的情况下，在纤连蛋白包被的基质上展开时，它们会呈现出异常的星状形态。为了确定百日咳博德特氏菌的侵袭性腺苷酸环化酶（AC）毒素是否会引发相同的反应，我们从几种百日咳博德特氏菌菌株中制备了细胞提取物。通过酶促测定和用AC毒素特异性单克隆抗体进行免疫印迹来对它们产生AC毒素的情况进行表征。产生AC毒素的菌株提取物可诱导BHK细胞内cAMP水平升高，并在细胞展开过程中促进星状化反应。从AC毒素产生缺陷的菌株制备的提取物则没有效果。利用图像分析对形态变化进行量化，我们证明了AC毒素对细胞展开的影响是剂量依赖性的。该技术是一种用于侵袭性AC毒素的快速且灵敏的检测方法。
