Identification of wall-specific antigens synthesized during germ tube formation by Candida albicans

Walls of the two cellular forms (blastoconidia and mycelia) of Candida albicans ATCC 26555 were obtained from cells metabolically labeled (6-h pulse) with 14C-protein hydrolysate and [3H]threonine. Walls were purified by thorough washings with buffered and sodium dodecyl sulfate solutions and digested with Zymolyase 20T. The enzymatic treatment released four major high-molecular-weight mannoproteins (HMWM), with apparent molecular masses of 650, 500, 340, and 200 kilodaltons (HMWM-650, HMWM-500, HMWM-340, and HMWM-200, respectively), from yeast cells, whereas two high-molecular-mass mannoproteins (HMWM-260 and HMWM-180) were solubilized from mycelial cells. Some additional minor low-molecular-weight species were also detected in the enzymatic digests of walls from both types of cell. Single and dual pulse-chase experiments indicated that the HMWM-260 and HMWM-180 species reflect de novo synthesis of new proteins specific for the mycelia and do not represent a topological rearrangement of blastoconidium wall components. Monoclonal antibodies were raised against the HMWM-260 species (quantitatively the predominant component in the mycelial walls), and polyclonal rabbit antibodies were obtained against yeast or mycelial cell walls. Anti-mycelial cell wall polyclonal antibodies were adsorbed to whole killed blastoconidia to remove antibodies against common blastoconidium and mycelial wall antigens. Titration by enzyme-linked immunosorbent assay revealed that the monoclonal antibodies could recognize an epitope of the protein moiety of the HMWM-260 mannoprotein. Immunoblotting and immunofluorescence techniques using these monoclonal and polyclonal antibodies confirmed that the HMWM-260 and HMWM-180 species are specific components of the envelope of the mycelial cell walls.