Karjalainen T, Barc M C, Collignon A, Trollé S, Boureau H, Cotte-Laffitte J, Bourlioux P
Département de Microbiologie et Immunologie, Faculté de Pharmacie, Université de Paris-Sud, Châtenay-Malabry, France.
Infect Immun. 1994 Oct;62(10):4347-55. doi: 10.1128/iai.62.10.4347-4355.1994.
Our laboratory has previously shown that Clostridium difficile adherence to Caco-2 cells is greatly enhanced after heat shock at 60 degrees C and that it is mediated by a proteinaceous surface component. The experiments described here show that C. difficile could adhere to several types of tissue culture cells (Vero, HeLa, and KB) after heat shock. The type of culture medium (liquid or solid, with or without blood) had little effect on adhesion. To clone the adhesin gene, polyclonal antibodies against C. difficile heated at 60 degrees C were used to screen a genomic library of C. difficile constructed in lambda ZapII. Ten positive clones were identified in the library, one of which (pCL6) agglutinated several types of erythrocytes in the presence of mannose. In Western blots (immunoblots), this clone expressed in Escherichia coli a 40- and a 27-kDa protein; a 27-kDa protein has been previously identified in the surface extracts of heat-shocked C. difficile as a possible adhesin. The clone adhered to Vero, Caco-2, KB, and HeLa cells; the adherence was blocked by anti-C. difficile antibodies, by a surface extract of C. difficile, and by mucus isolated from axenic mice. Furthermore, the clone could attach ex vivo to intestinal mucus isolated from axenic mice. Preliminary studies on the receptor moieties implicated in C. difficile adhesion revealed that glucose and galactose could partially block adhesion to tissue culture cells, as did di- or trisaccharides containing these sugars, suggesting that the adhesin is a lectin. In addition, N-acetylgalactosamine, a component of mucus, and gelatin partially impeded cell attachment.
我们实验室先前已表明，艰难梭菌在60摄氏度热休克后对Caco - 2细胞的黏附力显著增强，且这种黏附由一种蛋白质表面成分介导。此处所描述的实验表明，热休克后的艰难梭菌能够黏附于几种类型的组织培养细胞（Vero细胞、HeLa细胞和KB细胞）。培养基类型（液体或固体，含或不含血液）对黏附影响不大。为克隆黏附素基因，使用针对60摄氏度加热的艰难梭菌的多克隆抗体筛选构建于λZapII中的艰难梭菌基因组文库。在该文库中鉴定出10个阳性克隆，其中一个（pCL6）在有甘露糖存在的情况下能凝集几种类型的红细胞。在蛋白质印迹法（免疫印迹）中，该克隆在大肠杆菌中表达一种40 kDa和一种27 kDa的蛋白质；先前已在热休克的艰难梭菌表面提取物中鉴定出一种27 kDa的蛋白质可能是黏附素。该克隆能黏附于Vero细胞、Caco - 2细胞、KB细胞和HeLa细胞；这种黏附被抗艰难梭菌抗体、艰难梭菌表面提取物以及从无菌小鼠分离的黏液所阻断。此外，该克隆能在体外黏附于从无菌小鼠分离的肠道黏液。对涉及艰难梭菌黏附的受体部分的初步研究表明，葡萄糖和半乳糖可部分阻断对组织培养细胞的黏附，含有这些糖的二糖或三糖也有同样作用，这表明黏附素是一种凝集素。此外，黏液成分之一的N - 乙酰半乳糖胺和明胶部分阻碍细胞黏附。
