Abstract
Cell adhesion molecules in Saccharomyces cerevisiae and Candida albicans contain amyloid-forming sequences that are highly conserved. We have now used site-specific mutagenesis and specific peptide perturbants to explore amyloid-dependent activity in the Candida albicans adhesin Als5p. A V326N substitution in the amyloid-forming region conserved secondary structure and ligand binding, but abrogated formation of amyloid fibrils in soluble Als5p and reduced cell surface thioflavin T fluorescence. When displayed on the cell surface, Als5p with this substitution prevented formation of adhesion nanodomains and formation of large cellular aggregates and model biofilms. In addition, amyloid nanodomains were regulated by exogenous peptides. An amyloid-forming homologous peptide rescued aggregation and biofilm activity of Als5pV326N cells, and V326N substitution peptide inhibited aggregation and biofilm activity in Als5pWT cells. Therefore, specific site mutation, inhibition by anti-amyloid peturbants, and sequence-specificity of pro-amyloid and anti-amyloid peptides showed that amyloid formation is essential for nanodomain formation and activation.
Highlights
Candida albicans is a human commensal fungus that is pathogenic when its growth becomes uncontrolled, especially in imunocompromised individuals
S. cerevisiae cells expressing Als5p form large aggregates, similar to those seen in C. albicans [8,39]
The peptide had no effect on the fluorescence of the empty vector and Als5pV326N aggregates (Figure 4U and 4V). These results show that the V326N peptide blocked amyloid formation and aggregation mediated by Als5p in S. cerevisiae, as well as aggregation in C. albicans
Summary
Candida albicans is a human commensal fungus that is pathogenic when its growth becomes uncontrolled, especially in imunocompromised individuals. Under such conditions these eukaryotes can form biofilms that are resistant to a variety of environmental assaults, including antimicrobials [1,2,3]. Cell wall proteins called adhesins are critical for biofilm formation, and mediate adhesion of C. albicans to various substrates and each other. A 103-residue Thr-rich T domain is highly conserved among paralogs, and contains a 7-residue sequence that forms amyloids under native–like conditions [10,11]. The overlapping binding specificities and variable expression of Als proteins make them difficult to study in C. albicans [6,7,9,15]. Expression in Saccharomyces cerevisiae has shown that Als1p, Als3p and Als5p bind to a variety of substrates and are involved in endothelial cell adhesion, fungal aggregation, and tissue invasion [7,16,17,18,19]
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