Mutant bacteriophage with non-catalytic endosialidase binds to both bacterial and eukaryotic polysialic acid and can be used as probe for its detection.
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.
Aalto
J
J
Department of Medical Biochemistry and Molecular Biology, University of Turku, Kiinamyllynkatu 10, FIN-20520 Turku, Finland.
Pelkonen
S
S
Kalimo
H
H
Finne
J
J
D000818:Animals; D001435:Bacteriophages / Q000201:enzymology* / Q000235:genetics* / Q000276:immunology; D015153:Blotting, Western; D001921:Brain / Q000166:cytology / Q000648:ultrastructure; D002460:Cell Line; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology; D005453:Fluorescence; D007106:Immune Sera / Q000378:metabolism; D007668:Kidney / Q000166:cytology; D008297:Male; D008567:Membranes, Artificial; D008853:Microscopy / Q000379:methods; D009154:Mutation; D009439:Neuraminidase / Q000378:metabolism*; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D012794:Sialic Acids / Q000378:metabolism*; D013379:Substrate Specificity
D007106:Immune Sera; D008567:Membranes, Artificial; D009844:Oligosaccharides; D012794:Sialic Acids; C021319:polysialic acid; C016735:sialooligosaccharides; C035355:endo-N-acetylneuraminidase; D009439:Neuraminidase