Figure 3

Sap6 amyloid regions induce fungal cell aggregation proportionally with the degree of amyloid structure shown by Congo red binding, and zinc further increases amyloid structure. (A) Amyloid structure of peptides P1–P4 and a scrambled P2 peptide (sP2) was evaluated by their affinity for Congo red that specifically binds to amyloid structures. Absorbance spectra (400–700 nm) of Congo red alone (solid black lines) or with peptides P1–P4 (dashed red lines) were measured. Autoaggregation of germinated C. albicans was observed microscopically following 15 min incubation with each peptide. Cell aggregation was the largest for P2, with moderate autoaggregation with P4 and P1, while P3 induced very little aggregation. Amyloid structure (as shown by amount of Condo red absorbance) was largest for P2, followed by P1 and P4, while P3 showed the least absorbance. The scrambled peptide sP2 did not form aggregates nor show Congo red absorbance. (B) Thioflavin T (ThT) binding with amyloid structures was measured (440 nm–520 nm) with peptide P2 and sP2 (10 µM) (left panel) or rSap6 (right panel) following pre-incubation with ZnCl₂ (0–0.5 mM) for 4 h. ThT fluorescence was maximal at 480 nm (showing the presence of amyloid structure) for both P2 and full length rSap6, but the scrambled P2 peptide had no evidence of amyloid structure as shown by the lack of ThT absorbance. Addition of zinc to both P2 and rSap6 increased amyloid structure as shown by the increased fluorescence. Data are the mean ±SD for three independent experiments.