Khan A S, Johnston N C, Goldfine H, Schifferli D M
Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia 19104, USA.
Infect Immun. 1996 Sep;64(9):3688-93. doi: 10.1128/iai.64.9.3688-3693.1996.
Certain strains of enterotoxigenic Escherichia coli adhere to piglet intestinal epithelial cells by means of the 987P fimbriae. The 987P fimbrial structure consists of a helical arrangement of three fimbrial proteins, namely, the major subunit FasA and two minor subunits, FasF and FasG. FasG, which is located at the fimbrial tip and at various positions along the fimbriae, mediates 987P binding to glycoprotein receptors. In this study, we isolated and analyzed the structure of piglet glycolipid brush border receptors and characterized their cognate ligands on the 987P fimbriae. Two major glycolipid bands recognized by 987P fimbrial probes in thin-layer chromatography overlay assays were further purified by high-performance thin-layer chromatography and shown to comigrate with control galactosylceramide containing hydroxylated fatty acids and with sulfatide. Their structures were confirmed by fast atom bombardment mass spectrometry, which detected homologous series of ceramide monohexoside and sulfatide with hydroxylated fatty acyl chains ranging from h16:0 to h24:0. Assembled 987P fimbriae, pre- and postassembly dissociated fimbrial subunits, and Fab fragments of specific anti-FasG, -FasF, and -FasA were used to inhibit 987P-mediated bacterial binding to the two identified piglet glycolipids and corresponding isoreceptor controls. Only assembled fimbriae and anti-FasG Fab fragments were significantly able to inhibit bacterial binding to sulfatide, indicating that in addition to glycoproteins, FasG recognizes a specific glycolipid of piglet brush borders. In contrast, only anti-FasA Fab fragments were significantly able to inhibit bacterial binding to galactosylceramide with hydroxylated fatty acids and piglet hydroxylated ceramide monohexoside, indicating that FasA may determine a third type of ligand-receptor interaction in the piglet intestines. Since these bacterial adhesins recognize their respective glycolipid receptors only after being assembled in their final fimbrial quaternary structure, adhesin binding may involve cooperative interactions and the subunits by themselves may have very low binding affinities. Alternatively, conformation-sensitive domains of these subunits present in the assembled fimbriae may be required for glycolipid binding.
某些产肠毒素大肠杆菌菌株借助987P菌毛粘附于仔猪肠上皮细胞。987P菌毛结构由三种菌毛蛋白呈螺旋排列组成，即主要亚基FasA和两个次要亚基FasF及FasG。位于菌毛尖端和沿菌毛不同位置的FasG介导987P与糖蛋白受体结合。在本研究中，我们分离并分析了仔猪糖脂刷状缘受体的结构，并鉴定了其在987P菌毛上的同源配体。在薄层色谱覆盖试验中被987P菌毛探针识别的两条主要糖脂带通过高效薄层色谱进一步纯化，并显示与含羟基脂肪酸的对照半乳糖神经酰胺和硫苷脂迁移率相同。它们的结构通过快原子轰击质谱法得以确认，该方法检测到了神经酰胺单己糖苷和硫苷脂的同源系列，其羟基化脂肪酰链范围从h16:0到h24:0。组装好的987P菌毛、组装前后解离的菌毛亚基以及抗FasG、抗FasF和抗FasA的Fab片段被用于抑制987P介导的细菌与两种已鉴定的仔猪糖脂及相应异受体对照的结合。只有组装好的菌毛和抗FasG Fab片段能够显著抑制细菌与硫苷脂的结合，这表明除了糖蛋白外，FasG还识别仔猪刷状缘的一种特定糖脂。相反，只有抗FasA Fab片段能够显著抑制细菌与含羟基脂肪酸的半乳糖神经酰胺和仔猪羟基化神经酰胺单己糖苷的结合，这表明FasA可能决定了仔猪肠道中第三种类型的配体 - 受体相互作用。由于这些细菌粘附素只有在组装成最终的菌毛四级结构后才识别其各自的糖脂受体，粘附素结合可能涉及协同相互作用，并且亚基自身的结合亲和力可能非常低。或者，组装好的菌毛中这些亚基的构象敏感结构域可能是糖脂结合所必需的。
