Bacterial adherence on replicas of sodium dodecyl sulfate-polyacrylamide gels.
A method for determining which molecules in a complex mixture of proteins can function as bacterial receptors was devised. Salivary proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose. Bacteria that were metabolically labeled with 3H or externally labeled with 125I were incubated on the nitrocellulose replicas. After 18 h at 4 degrees C, the unbound cells were removed by repeated washing of the replicas, and the bands to which the radiolabeled bacteria bound were visualized by autoradiography. By this technique, Fusobacterium nucleatum, which adheres via carbohydrate residues on receptor molecules, and Staphylococcus aureus, which recognizes the peptide portion of fibronectin, were shown to bind specifically to their respective receptors. These results suggest that this method can be useful for profiling bacterial binding to either the carbohydrate or the protein portions of molecules present in complex mixtures, such as those composing biological fluids or tissue substrates. Structural specificities, such as recognition sequences formed by certain oligosaccharides, could be further investigated by adding the appropriate simple sugars, as well as oligosaccharide inhibitors, to the incubation medium. The latter approach is particularly important since most glycoproteins carry multiple N- and O-linked carbohydrate substituents that could serve as bacterial receptors.
Prakobphol
A
A
Division of Oral Biology, School of Pharmacy, University of California, San Francisco 94143.
Murray
P A
PA
Fisher
S J
SJ
D001422:Bacterial Adhesion; D003101:Collodion; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000502:physiology; D005353:Fibronectins / Q000378:metabolism; D005673:Fusobacterium / Q000502:physiology; D006023:Glycoproteins / Q000378:metabolism; D006801:Humans; D008970:Molecular Weight; D010306:Parotid Gland / Q000378:metabolism; D012471:Salivary Proteins and Peptides / Q000302:isolation & purification / Q000378:metabolism*; D012967:Sodium Dodecyl Sulfate; D013211:Staphylococcus aureus / Q000502:physiology; D013361:Sublingual Gland / Q000378:metabolism; D013363:Submandibular Gland / Q000378:metabolism
D005353:Fibronectins; D006023:Glycoproteins; D012471:Salivary Proteins and Peptides; D012967:Sodium Dodecyl Sulfate; D003101:Collodion