Identification of major glucan-associated cell wall proteins of Candida albicans and their role in fluconazole resistance

Abstract

Identification of major glucan-associated proteins (GAPs) of the cell wall of a number of Candida albicans isolates susceptible or resistant to fluconazole (FLC) was addressed by direct sequencing of the protein bands resolved by unidimensional gel electrophoresis. Changes in the GAP compositions of the different strains grown in the presence of the drug were also investigated. In the FLC-susceptible strains, the major (more abundant) GAPs were enolase (46 kDa), two isoforms of phosphoglyceromutase (32 and 29 kDa), and two beta-(1-3)-exoglucanases (44 and 34 kDa), one of which (the 34-kDa component) was glycosylated. When these strains were grown in the presence of FLC there were substantial decreases in the intensities of the two enzymes of the glycolytic pathway (enolase and the phosphoglyceromutases), which were apparently replaced by enhancement of the exoglucanase constituents, particularly the 44-kDa one. This GAP pattern closely mimicked that observed in the FLC-resistant strains whether they were grown in the presence or in the absence of the drug. Both the enolase and the exoglucanase constituents were detected in the culture supernatants of FLC-treated cells, together with substantial amounts of highly glycosylated, probably mannoprotein secretory material, suggesting that FLC may cause marked alterations of GAP incorporation into the cell wall. Altogether, we were able to identify all major GAP constituents and monitor their distributions in the cell wall of C. albicans during treatment with FLC. The near equivalence of the GAP profile for the FLC-susceptible strain grown in the presence of FLC to that for the FLC-resistant strain suggests that the effects of the drug on GAPs may be stably incorporated into the cell wall of the fungus upon acquisition of resistance.

FIG. 1.

Cell wall GAPs of C. albicans strains CA2 (a) and 3153 (b). Lanes 1, controls; lanes 2, samples after treatment with FLC (5 μg/ml). The samples (7 μg of protein) were run on an SDS-10% polyacrylamide gel and stained with Coomassie brilliant blue. Molecular mass standards are expressed on the right of each gel (in kilodaltons). For technical details, see Materials and Methods.

FIG. 2.

Cell wall GAPs of FLC-sensitive strain C. albicans CA2 after staining of the gel with different strains. (a) Electrophoresis on an SDS-10% polyacrylamide gel stained with Coomassie brilliant blue; (b) transblotting onto nitrocellulose paper and reaction with antienolase immune serum (dilution, 1:100); (c) transblotting onto nitrocellulose paper and reaction with ConA-digoxigenin. In all cases, the gels were loaded with 7 μg of protein. Lanes 1, GAP patterns of control cells not treated with FLC; lanes 2, GAP patterns of FLC-treated cells. Molecular mass standards are expressed on the right of each gel (in kilodaltons). For technical details, see Materials and Methods.

FIG. 3.

Cell wall GAPs of FLC-sensitive clinical strain CO23_s of C. albicans. Lane 1, controls; lane 2, samples after treatment with FLC (1 μg/ml). Electrophoresis on an SDS-10% polyacrylamide gel loaded with 0.5 μg of protein and stained with silver. Molecular mass standards are expressed on the right (in kilodaltons). For technical details, see Materials and Methods and the legends to Fig. 1 and 2.

FIG. 4.

Cell wall GAPs of FLC-resistant strain AIDS 126 of C. albicans. Lanes 1, control; lanes 2, samples after FLC treatment. (a) SDS-10% polyacrylamide gel stained by Coomassie brilliant blue; (b) transblotting onto nitrocellulose paper and reaction with antienolase immune serum (dilution, 1:100); (c) transblotting onto nitrocellulose paper and reaction with ConA-digoxigenin. In all cases the gels were loaded with 7 μg of protein. Lanes 1, GAP patterns of control, FLC-untreated cells; lanes 2, GAP patterns of FLC-treated cells. Molecular mass standards are expressed on the right of each gel (in kilodaltons).

FIG. 5.

Proteins released by FLC-resistant strain AIDS 126 of C. albicans in the culture medium in the absence and in the presence of FLC. (a) SDS-10% polyacrylamide gel stained by use of the Silver Stain Plus kit; (b) transblotting onto nitrocellulose paper and reaction with antienolase immune serum; (c) transblotting onto nitrocellulose paper and reaction with ConA-digoxigenin. In all cases the gels were loaded with 1 μg of protein. Lanes 1, control supernatants of FLC-untreated cells; lanes 2, supernatants of FLC-treated cells. Molecular mass standards are expressed on the right of each gel (in kilodaltons). For other technical details, see Materials and Methods.