Mutant bacteriophage with non-catalytic endosialidase binds to both bacterial and eukaryotic polysialic acid and can be used as probe for its detection
Mutant bacteriophage with non-catalytic endosialidase binds to both bacterial and eukaryotic polysialic acid and can be used as probe for its detection
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DOI：
10.1023/a:1021147316647
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发表时间：
2001-10-01
影响因子：
3
通讯作者：
Finne, J
中科院分区：
文献类型：
--
作者：
Aalto, J;Pelkonen, S;Finne, J
在引起败血症和脑膜炎的细菌病原体的聚唾液酸多糖与神经细胞粘附分子NCAM的碳水化合物单元之间存在分子模拟。我们研究了具有催化失活的内切唾液酸酶的噬菌体突变体是否可以识别并结合细菌和真核生物的聚唾液酸，该突变体结合但不裂解聚唾液酸。在硝酸纤维素斑点印迹试验中，突变噬菌体（而不是野生型噬菌体）仍然与含聚唾液酸的细菌特异性结合，包括大肠杆菌K1和K92、B群脑膜炎球菌、溶血性曼氏杆菌（巴斯德菌）A2和非液化莫拉氏菌。最低结合要求确定为聚唾液酸链中的10个唾液酸残基。在蛋白质印迹中，突变的NCAM特异性地结合到NCAM的胚胎多唾液酸化形式，但不结合到NCAM的成人少唾液酸化形式。随后，突变体p53与二抗噬菌体抗体一起成功地用于培养细胞的荧光显微镜和石蜡包埋组织切片的光学显微镜作为真核聚唾液酸的探针。因此，引起脑膜炎的细菌的突变噬菌体结合并检测宿主组织中神经细胞粘附分子的分子模拟聚唾液酸。
There is a molecular mimicry between the polysialic acid polysaccharide of bacterial pathogens causing sepsis and meningitis, and the carbohydrate units of the neural cell adhesion molecule NCAM. We investigated whether bacteriophage mutants with catalytically disabled endosialidase, which bind but do not cleave polysialic acid, could recognise and bind to bacterial and eukaryotic polysialic acid. In nitrocellulose dot blot assay the mutant bacteriophages, but not the wild-type phages, remained specifically bound to polysialic acid-containing bacteria including Escherichia coli K1 and K92, group B meningococci, Mannheimia (Pasteurella) haemolytica A2, and Moraxella nonliquefaciens. A minimum binding requirement was determined to be 10 sialyl residues in the polysialic acid chain. In Western blots the mutant phages specifically bound to the embryonic polysialylated form of NCAM, but not to the adult less sialylated form of the molecule. The mutant phages together with secondary anti-phage antibodies were subsequently successfully used in fluorescence microscopy of cultured cells and light microscopy of paraffin-embedded tissue sections as a probe for the eukaryotic polysialic acid. Thus, mutant bacteriophages of meningitis causing bacteria bind to and detect the molecularly mimicked polysialic acid of the neural cell adhesion molecule in host tissues.