| Candida albicans maintains both commensal and pathogenic states in humans. Both states are dependent on cell-surface-expressed adhesins including those of the Als family. Heterologous expression of Als5p at the surface of Saccharomyces cerevisiae results in Als5p-mediated adhesion to various ligands, followed by formation of multicellular aggregates. Following adhesion of one region of the cell to fibronectin-coated beads, the entire surface of the cells became competent to mediate cell-cell aggregation. Aggregates formed in the presence of metabolic inhibitors or signal transduction inhibitors, but were reduced in the presence of 8-anilino-1-naphthalene-sulfonic acid (ANS) or Congo Red (CR), perturbants that inhibit protein structural transitions. These perturbants also inhibited aggregation of C. albicans . An increase in ANS fluorescence, which accompanied Als-dependent cellular adhesion, indicated an increase in cell surface hydrophobicity. In addition, C. albicans and Als5p-expressing S. cerevisiae showed an aggregation-induced birefringence indicative of order on the cell surface. The increase in birefringence did not occur in the presence of aggregation disruptants ANS or CR. These results suggest a model for Als5p-mediated aggregation in which an adhesion-triggered change in the conformation of Als5p propagates around the cell surface, forming ordered aggregation-competent regions.; The ALS Tandem repeat (TR) region has not been well characterized in Als proteins. ALS5 TR deletion fragments were constructed in order to determine the structural characteristics and to identify the regions in Als5p required for adhesion to fibronectin. Cellular adhesion assays showed the Immunoglobulinlike (IG) domain mediated adherence to fibronectin. This result was consistent with previous studies for Als1p. The Threonine conserved (TC) region was important for secretion of cell surface anchored or soluble protein. SDS-PAGE and dot blot analyses showed the TR region is O-glycoslylated. Circular Dichroism (CD) spectroscopy indicated that the TR region increases the overall amount of alpha-helix structures 6-fold. Deletion of the TR region reduced binding to fibronectin in bioassays using soluble Als5p constructs. A reduction in cell-cell aggregation was observed in assays using cell-surface TR deletion constructs. Our findings demonstrate a structural and functional role for the TR region. |
