Yeast cell adhesion molecules have functional amyloid-forming sequences

Abstract

The occurrence of highly conserved amyloid-forming sequences in Candida albicans Als proteins (H. N. Otoo et al., Eukaryot. Cell 7:776-782, 2008) led us to search for similar sequences in other adhesins from C. albicans and Saccharomyces cerevisiae. The beta-aggregation predictor TANGO found highly beta-aggregation-prone sequences in almost all yeast adhesins. These sequences had an unusual amino acid composition: 77% of their residues were beta-branched aliphatic amino acids Ile, Thr, and Val, which is more than 4-fold greater than their prevalence in the S. cerevisiae proteome. High beta-aggregation potential peptides from S. cerevisiae Flo1p and C. albicans Eap1p rapidly formed insoluble amyloids, as determined by Congo red absorbance, thioflavin T fluorescence, and fiber morphology. As examples of the amyloid-forming ability of the native proteins, soluble glycosylphosphatidylinositol (GPI)-less fragments of C. albicans Als5p and S. cerevisiae Muc1p also formed amyloids within a few days under native conditions at nM concentrations. There was also evidence of amyloid formation in vivo: the surfaces of cells expressing wall-bound Als1p, Als5p, Muc1p, or Flo1p were birefringent and bound the fluorescent amyloid-reporting dye thioflavin T. Both of these properties increased upon aggregation of the cells. In addition, amyloid binding dyes strongly inhibited aggregation and flocculation. The results imply that amyloid formation is an intrinsic property of yeast cell adhesion proteins from many gene families and that amyloid formation is an important component of cellular aggregation mediated by these proteins.

Fig. 1.

Congo red absorbance (left column) and thioflavin T fluorescence (right column) of suspended adhesion proteins and peptides. Control spectra are solid lines, and spectra taken in the presence of aggregates are dashed. Congo red spectra show aggregate-dependent enhancement and red-shifting. Thioflavin T fluorescence in increased 2- to 30-fold in the presence of the aggregates: CaAls5p^1–1351 (A), ScMuc1p^1–1331 (B), ScFlo1p^305–315 (C), and Eap1p^117–133 (D).

Fig. 2.

Negative-stain transmission electron microscopy of fibers. Bars are 100 nm in length. (A) CaAls5p^1–1351. The arrowhead shows a less-structured aggregate, and apparent protofibrils are seen in the lower region of the long fiber. (B) ScMuc1p^1–1331. Individual fibrils are visible in the upper part of the fiber. (C) ScFlo1p^305–315. Note many smaller wavy fibrils in the background. (D) Eap1p^117–133. There are both fibers (arrow) and ribbons (arrowhead) visible.

Fig. 3.

Birefringence of cells expressing flocculins. S. cerevisiae cells were analyzed between polarizing filters with a 20× objective under bright-field conditions. The paired micrographs show identical fields between parallel (outer images) and crossed polarizing filters (central images) in the absence (left) or presence (right) of 0.67 mM Ca²⁺. The strains were BX24-2B (A and B), YIY345/pHis-PGK1-MUC1 (C and D), and W303-1B (E and F).

Fig. 4.

Thioflavin T (ThT) staining of cells expressing CaAls proteins. Shown are paired bright-field and fluorescence micrographs of S. cerevisiae W303-1B transformed with the empty vector (EV; no insert) or vectors encoding CaAls5p or CaAls1p. (A to C) Designated cells were aggregated with BSA-coated beads. Bright-field micrographs in the top row show dark spherical 2.8-μm beads interspersed with gray-colored yeast cells, which are spheroidal and larger. The bottom row shows fluorescence of the same field. (D to F) The indicated cells were aggregated with beads and then stained with thioflavin T. Bright-field micrographs are in the top row, and fluorescence of the corresponding field is shown below. (G to I) The indicated cells were concentrated by centrifugation and stained with thioflavin T. The fluorescence micrographs are on top, with the corresponding bright-field images shown below.

Fig. 5.

Thioflavin T staining of S. cerevisiae cells expressing ScFlo1p or ScMuc1p. Indicated strains were stained and visualized under bright-field (top and bottom rows), with matched fields for thioflavin T fluorescence in the middle two rows. Flocculation was induced with added Ca²⁺ for the images in the bottom two rows: left column, W303-1B; middle column, strain BX24-2B; right column, strain YIY345.

Fig. 6.

Aggregation assays with S. cerevisiae cells expressing CaAls proteins. Strain W303-1B cells carrying an empty vector or expressing the designated protein were aggregated with heat-denatured BSA-coated magnetic beads, and the beads and adherent cells were separated and examined by light microscopy (×40 magnification). Dark spherical 2.8-μm beads are interspersed with the gray-colored cells, which are spheroidal and slightly larger. Assays were carried out in the presence of amyloid-binding dyes as indicated: CR, Congo red; ThT, thioflavin T; and ThS, thioflavin S.

Fig. 7.

Effects of thioflavin S on flocculent strains of S. cerevisiae. (A) Flocculation assays in the presence of increasing concentrations of thioflavin S. (A) Strain BX24-2B expressing ScFlo1p flocculating in the presence of CaCl₂ (667 μM); (B) strain YIY345 expressing ScMuc1p flocculating in the presence of CaCl₂ (667 μM); (C) dose-response analysis of effect of thioflavin S on rates of ScMuc1p-mediated flocculation; (D) dose-response analysis of effect of thioflavin S on rates of ScFlo1p-mediated flocculation; (E) growth inhibition assay. Serial dilutions of the indicated strains were grown on the indicated media.

Fig. 8.

Model for forming multimers of flocculins through amyloid formation. Each monomeric flocculin is covalently anchored into the cell wall. The monomers are clustered on the cell surface through the interactions of amyloid-forming sequences.