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The fungal pathogen Candida albicans frequently forms drug-resistant biofilms in hospital settings and in chronic disease patients. Cell adhesion and biofilm formation involve a family of cell surface Als (agglutinin-like sequence) proteins. It is now well documented that amyloid-like clusters of laterally arranged Als proteins activate cell-cell adhesion under mechanical stress, but whether amyloid-like bonds form between aggregating cells is not known. To address this issue, we measure the forces driving Als5-mediated intercellular adhesion using an innovative fluidic force microscopy platform. Strong cell-cell adhesion is dependent on expression of amyloid-forming Als5 at high cell surface density and is inhibited by a short antiamyloid peptide. Furthermore, there is greatly attenuated binding between cells expressing amyloid-forming Als5 and cells with a nonamyloid form of Als5. Thus, homophilic bonding between Als5 proteins on adhering cells is the major mode of fungal aggregation, rather than protein-ligand interactions. These results point to a model whereby amyloid-like β-sheet interactions play a dual role in cell-cell adhesion, that is, in formation of adhesin nanoclusters ( cis-interactions) and in homophilic bonding between amyloid sequences on opposing cells ( trans-interactions). Because potential amyloid-forming sequences are found in many microbial adhesins, we speculate that this novel mechanism of amyloid-based homophilic adhesion might be widespread and could represent an interesting target for treating biofilm-associated infections.

Owing to its ability to form biofilms on implanted medical devices, the fungal pathogen Candida albicans causes frequent infections in humans. A hallmark of C. albicans biofilms is the presence of two types of cells, budding yeast cells and growing hyphae, which are bound together and embedded in extracellular matrix material. Although cell-cell adhesion is critical to biofilm formation, architecture, and cohesion, we know little about the fundamental forces behind this interaction. Here, we use single-cell force spectroscopy to quantify the forces engaged in yeast-hyphae adhesion, focusing on the role of Als (agglutinin-like sequence) proteins as prototypes of cell adhesion molecules. We show that adhesion between individual yeast and hyphal cells involves strong, short-range cohesive interactions (1.1 ± 0.2 nN; 86 ± 33 nm) and weak, long-range tether interactions (0.4 ± 0.2 nN; 234 ± 81 nm). Control experiments demonstrate that these interactions originate from cell surface proteins that are specific to C. albicans. Using mutant strains deficient for Als expression, we find that Als3 proteins, primarily expressed on the germ tube, play a key role in establishing strong cohesive adhesion. We suggest a model in which cohesive adhesion during biofilm formation originates from tight hydrophobic interactions between Als tandem repeat domains on adjacent cells. When subjected to force, the two interacting cell surfaces detach, but the cell bodies remain tethered through macromolecular extensions. Our results represent the first direct, noninvasive measurement of adhesion forces between interacting fungal cells and provide novel insights into the molecular origin of the cohesive strength of fungal biofilms. © 2013 American Chemical Society.

Elucidating the molecular mechanisms behind the strength and mechanics of cell adhesion proteins is of central importance in cell biology and offers exciting avenues for the identification of potential drug targets. Here we use single-molecule force spectroscopy to investigate the adhesive and mechanical properties of the widely expressed Als5p cell adhesion protein from the opportunistic pathogen Candida albicans . We show that the forces required to unfold individual tandem repeats of the protein are in the 150-250 pN range, both on isolated molecules and on live cells. We also find that the unfolding probability increases with the number of tandem repeats and correlates with the level of cell adherence. We suggest that the modular and flexible nature of Als5p conveys both strength and toughness to the protein, making it ideally suited for cell adhesion. The single-molecule measurements presented here open new avenues for understanding the mechanical properties of adhesion molecules from mammalian and microbial cells and may help us to elucidate their potential implications in diseases such as inflammation, cancer, and infection.