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.

The occurrence of highly conserved amyloid-forming sequences in Candida albicansProtein amyloids are characteristic of pathological conditions, including neurodegenerative diseases (4,11,17,38). These protein aggregates can also occur naturally in adhesive bacterial curli (3), melanosomes (14), condensed peptide hormone arrays (24), as regulatory prions in yeast (2, 5), and fungal hydrophobins, which are nonantigenic coats to some fungi (1,33,39). Nevertheless, such natural occurrences are relatively few, considering the negative free energy for amyloid formation (28).We have recently discovered that there are amyloid-forming sequences in the cell surface Als adhesins of Candida albicans. Cells that express these adhesins aggregate readily, and the aggregation has amyloid-like properties, including protein conformational shifting, surface birefringence, and ability to bind the amyloid-active dyes Congo red and amino-naphthalene sulfonic acid (ANS) (29). A five-to seven-residue sequence in Als1p, Als3p, and Als5p has extremely high potential for formation of ␤-aggregates, according to the protein state prediction program TANGO (13,27,31). Such ␤-aggregates include amyloids, which are ordered structures with paracrystalline regions of stacked parallel ␤-strands that are perpendicular to the long axis of micrometer-long fibrils. The strands are stabilized by interaction of identical sequences from many protein molecules (31, 32). Where TANGO analyses have shown that specific sequences have ␤-aggregate potentials greater than 20%, an insoluble ␤-aggregate state is likely to form. These ␤-aggregates nucleate formation of amyloids if the proteins can associate to form fibers (13,27,31). Sequences in the conserved 127-residue T region of Als1p, Als3p, and Als5p have ␤-aggregation potentials of Ͼ90% (27). An oligopeptide with this sequence, as well as 412-and 645-residue fragments of Als5p formed authentic amyloids, as determined by characteristic dye binding and fiber morphology. The amyloid-forming sequences were rich in the ␤-branched amino acids Thr, Val, and Ile. This amino acid composition is unusual among proteins in general, but is common in the Thr-rich mid-piece domains of yeast adhesins.Yeasts display many cell-wall-bound adhesins that mediate colonial and biofilm interactions as well as host-pathogen binding (9,21,41). Such adhesins have a common mosaic structure. In general, the adhesins have N-terminal globular binding domains (often immunoglobulin-like or lectin-like), Thr-rich mid-piece sequences including tandem repeats, and 300-to

Budding yeasts adhere to biotic or abiotic surfaces and aggregate to form biofilms, using wall-anchored glycoprotein adhesins. The process is paradoxical: adhesins often show weak binding to specific ligands, yet mediate remarkably strong adherence. Single-molecule atomic force microscopy, genomics, biochemistry, and cell biology have recently explained the puzzle, with Candida albicans Als adhesins as the paradigm. The strength of adhesion results partly from force-activated amyloid-like clustering of hundreds of adhesin molecules to form arrays of ordered multimeric binding sites. The various protein domains of eukaryotic adhesins cooperate to facilitate this fascinating new mechanism of activation.

Dental caries is a common infectious disease associated with acidogenic and aciduric bacteria, including Streptococcus mutans. Organisms that cause cavities form recalcitrant biofilms, generate acids from dietary sugars and tolerate acid end products. It has recently been recognized that micro-organisms can produce functional amyloids that are integral to biofilm development. We now show that the S. mutans cell-surface-localized adhesin P1 (antigen I/II, PAc) is an amyloid-forming protein. This conclusion is based on the defining properties of amyloids, including binding by the amyloidophilic dyes Congo red (CR) and Thioflavin T (ThT), visualization of amyloid fibres by transmission electron microscopy and the green birefringent properties of CR-stained protein aggregates when viewed under cross-polarized light. We provide evidence that amyloid is present in human dental plaque and is produced by both laboratory strains and clinical isolates of S. mutans. We provide further evidence that amyloid formation is not limited to P1, since bacterial colonies without this adhesin demonstrate residual green birefringence. However, S. mutans lacking sortase, the transpeptidase enzyme that mediates the covalent linkage of its substrates to the cell-wall peptidoglycan, including P1 and five other proteins, is not birefringent when stained with CR and does not form biofilms. Biofilm formation is inhibited when S. mutans is cultured in the presence of known inhibitors of amyloid fibrillization, including CR, Thioflavin S and epigallocatechin-3-gallate, which also inhibited ThT uptake by S. mutans extracellular proteins. Taken together, these results indicate that S. mutans is an amyloid-forming organism and suggest that amyloidogenesis contributes to biofilm formation by this oral microbe. INTRODUCTIONAmyloid represents a fibrous cross b-sheet quaternary structure comprised of ordered aggregates of peptides or proteins that demonstrate common biophysical properties (Nilsson, 2004). While amyloid formation has been extensively studied in the context of pathological states, for example Alzheimer's disease, the concept of functional amyloid has only recently emerged (Epstein & Chapman, 2008;Fowler et al., 2007;Gebbink et al., 2005;Maury, 2009a;. In bacteria, amyloid formation may be the rule rather than the exception . There are now numerous examples of surface-localized microbial proteins that aggregate and assemble into functional amyloid fibrils.Amyloid fibres have a tensile strength comparable to steel (Smith et al., 2006). The unique physical and morphological properties of amyloid and the fact that it can be formed in a regulated manner suggest that it probably represents a common quaternary structure that is widespread in biology. Therefore, it does not always represent a misfolded protein structure, but instead represents a lowenergy quaternary structure that can occur in the context of function or disease (Fowler et al., 2007). Amyloid is a non-covalent oligomer of extended intermolecular hydrogen-bonded b-sheets that s...

Bacterial and fungal species produce some of the best-characterized functional amyloids, i.e. extracellular fibres that play key roles in mediating adhesion and biofilm formation. Yet, the molecular details underlying their mechanical strength remain poorly understood. Here, we use single-molecule atomic force microscopy to measure the mechanical properties of amyloids formed by Als cell adhesion proteins from the pathogen Candida albicans. We show that stretching Als proteins through their amyloid sequence yields characteristic force signatures corresponding to the mechanical unzipping of β-sheet interactions formed between surfacearrayed Als proteins. The unzipping probability increases with contact time, reflecting the time necessary for optimal inter β-strand associations. These results demonstrate that amyloid interactions provide cohesive strength to a major adhesion protein from a microbial pathogen, thereby strengthening cell adhesion. We suggest that such functional amyloids may represent a generic mechanism for providing mechanical strength to cell adhesion proteins. In nanotechnology, these single-molecule manipulation experiments provide new opportunities to understand the molecular mechanisms driving the cohesion of functional amyloid-based nanostructures.

No abstract

The Saccharomyces cerevisiae flocculins mediate the formation of cellular aggregates and biofilm-like mats, useful in clearing yeast from fermentations. An important property of fungal adhesion proteins, including flocculins, is the ability to form catch bonds, i.e., bonds that strengthen under tension. This strengthening is based, at least in part, on increased avidity of binding due to clustering of adhesins in cell surface nanodomains. This clustering depends on amyloid-like β-aggregation of short amino acid sequences in the adhesins. In Candida albicans adhesin Als5, shear stress from vortex mixing can unfold part of the protein to expose aggregation-prone sequences, and then adhesins aggregate into nanodomains. We therefore tested whether shear stress from mixing can increase flocculation activity by potentiating similar protein remodeling and aggregation in the flocculins. The results demonstrate the applicability of the Als adhesin model and provide a rational framework for the enhancement or inhibition of flocculation in industrial applications.

We tell of a journey that led to discovery of amyloids formed by yeast cell adhesins and their importance in biofilms and host immunity. We begin with the identification of the adhesin functional amyloid-forming sequences that mediate fiber formation in vitro. Atomic force microscopy and confocal microscopy show 2-dimensional amyloid “nanodomains” on the surface of cells that are activated for adhesion. These nanodomains are arrays of adhesin molecules that bind multivalent ligands with high avidity. Nanodomains form when adhesin molecules are stretched in the AFM or under laminar flow. Treatment with antiamyloid perturbants or mutation of the amyloid sequence prevents adhesion nanodomain formation and activation. We are now discovering biological consequences. Adhesin nanodomains promote formation and maintenance of biofilms, which are microbial communities. Also, in abscesses within candidiasis patients, we find adhesin amyloids on the surface of the fungi. In both human infection and a Caenorhabditis elegans infection model, the presence of fungal surface amyloids elicits anti-inflammatory responses. Thus, this is a story of how fungal adhesins respond to extension forces through formation of cell surface amyloid nanodomains, with key consequences for biofilm formation and host responses.