Studying Als5p-dependent cell-cell adhesion in Candida albicans using FluidFM

Understanding the molecular interactions between fungal cells is a key to elucidate the fundamental mechanisms driving biofilm formation and may contribute to the development of new anti-biofilm strategies. The aggregation of Candida albicans cells is known to be mediated by the cell surface protein Als5p, but the molecular details of this interaction are poorly understood. In this work, we used the FluidFM technology, a recently developed pressure-controlled atomic force microscope (AFM) technique, to study the forces driving C. albicans cellular aggregation. We first improved existing protocols for measuring the forces between yeast cells using FluidFM-based single-cell force spectroscopy (SCFS). We used a coating made of poly-L-lysin-grafted-polyethylene glycol (PLL-g-PEG) to avoid undesired yeast-cell adhesion to the cantilever and to enable to rapidly change the probed cell, thus increasing the throughput of the analyses. We also improved the cleaning and storage conditions of the cantilever probe as well as the immobilization protocol used to attach the target cell on solid substrates. Using these strategies, the forces between Saccharomyces cerevisiae cells expressing Als5p adhesins (Als5pWT cells) were measured and found to be larger than between cells carrying an empty vector (EV cells), but similar to those measured between Als5pWT cells using conventional SCFS. These results validate the proof-of-concept of our FluidFM method for studying yeast-yeast adhesion. Next, the developed protocols allowed us to further explore the molecular details of Als5p-mediated intercellular adhesion. Adhesion signatures between Als5pWT cells grown in conditions favoring Als5p expression featured forces of ~600 pN and ruptures of ~600 nm. These forces are consistent with the unfolding of the tandem repeat (TR), threonine-rich (T) and immunoglobulin (Ig) domains from two adhesins on opposing cells, thus suggesting they involve homophilic bonds. In growth conditions that do not favor expression of the adhesin, much lower adhesion frequencies were observed, indicating that the density of adhesins on the cell surface plays an important role in cell aggregation. Finally, the influence of the amyloid T region was investigated by analyzing cells expressing adhesins bearing a specific single-site mutation in the amyloid sequence (Als5pV326N). Adhesion was still observed yet with lower forces and rupture distances, suggesting that the amyloid sequence is involved in aggregation, probably through the formation of adhesion nanoclusters, but is not fully responsible for Als5p-mediated interactions. In summary, this study contributes to the development of an advanced FluidFM methodology for studying yeast aggregation forces and enhances our understanding of the molecular forces guiding Als5p adhesion.