title: Complications in cell-surface labelling by biotinylation of Candida albicans due to avidin conjugate binding to cell-wall proteins.
abstract: Initial contact between the opportunistic fungal pathogen Candida albicans and host tissue occurs at the cell surface. Biotin derivatives have been used to label the cell-surface proteins of yeasts, with labelled proteins subsequently detected by avidin-reporter conjugates. Previous work has indicated that avidin can bind to C. albicans proteins in the absence of biotin, suggesting a possible host-cell-recognition mechanism by fungal cell-surface proteins. To investigate this mechanism, Western blots of proteins extracted from biotinylated and mock-treated cells were probed with avidin or modified-avidin reagents. Each avidin reagent bound to cell-wall proteins extracted from non-biotinylated cells. Binding did not appear to be due to the lectin-like activity of the cell-wall proteins of C. albicans or to the presence of biotin in the sample itself. Binding was inhibited by added biotin, by the chaotrope KSCN and by NaCl in a concentration-dependent manner, although inhibition varied among the avidin conjugates tested. Thus, the non-specific binding of avidin to the cell-wall proteins of C. albicans appears to involve hydrophobic and electrostatic interactions, depending on the particular avidin species. These observations demonstrate potential pitfalls in the use of avidin-biotin complexes to identify cell-surface molecules and could provide insights into protein-protein interactions at the C. albicans cell wall.
authors.affiliation: Departments of Pathology1 and Microbiology2, University of Virginia Health System, Charlottesville, VA 22908, USA.
authors.forename: James
authors.initials: J
authors.lastname: Masuoka
authors.affiliation: Departments of Pathology1 and Microbiology2, University of Virginia Health System, Charlottesville, VA 22908, USA.
authors.forename: Lori N
authors.initials: LN
authors.lastname: Guthrie
authors.affiliation: Departments of Pathology1 and Microbiology2, University of Virginia Health System, Charlottesville, VA 22908, USA.
authors.forename: Kevin C
authors.initials: KC
authors.lastname: Hazen
mesh_terms: D001360:Avidin / Q000493:pharmacokinetics*; D001665:Binding Sites; D019921:Biotinylation; D015153:Blotting, Western; D002176:Candida albicans / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002473:Cell Wall / Q000378:metabolism; D005656:Fungal Proteins / Q000378:metabolism*; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism*; D019809:Streptavidin / Q000494:pharmacology
chemical_list: D005656:Fungal Proteins; D008565:Membrane Proteins; D001360:Avidin; D019809:Streptavidin