Single-Cell Force Spectroscopy of Als-Mediated Fungal Adhesion

Abstract

Macroscopic assays that are traditionally used to investigate the adhesion behaviour of microbial cells provide averaged information obtained on large populations of cells and do not measure the fundamental forces driving single-cell adhesion. Here, we use single-cell force spectroscopy (SCFS) to quantify the specific and non-specific forces engaged in the adhesion of the human fungal pathogen Candida albicans. Saccharomyces cerevisiae cells expressing the C. albicans adhesion protein Als5p were attached on atomic force microscopy tipless cantilevers using a bioinspired polydopamine wet polymer, and force-distance curves were recorded between the obtained cell probes and various solid surfaces. Force signatures obtained on hydrophobic substrates exhibited large adhesion forces (1.25 ± 0.2 nN) with extended rupture lengths (up to 400 nm), attributed to the binding and stretching of the hydrophobic tandem repeats of Als5p. Data collected on fibronectin (Fn) -coated substrates featured strong adhesion forces (2.8 ± 0.6 nN), reflecting specific binding between Fn and the N-terminal immunoglobulin-like regions of Als5p, followed by weakly adhesive macromolecular bonds. Both hydrophobic and Fn adhesion forces increased with contact time, emphasizing the important role that time plays in strengthening adhesion. Our SCFS methodology provides a versatile platform in biomedicine for understanding the fundamental forces driving adhesion and biofilm formation in fungal pathogens.

Keywords: Als proteins; Candida albicans; adhesion; atomic force microscopy; force spectroscopy; fungal pathogens; single-cells.

Fig. 1

Non-destructive method for the single-cell force spectroscopy of fungal adhesion. (A, B) Preparation of the cell probes involves coating tipless cantilevers with polydopamine (adapted from Dreyer et al.) (A), followed by the controlled immobilization of a single S. cerevisiae cell (B). (C, D) Cell attachment with polydopamine does not alter cell viability: (C) phase contrast (left) and fluorescence (right) images of live (top) and heat-killed (60°C, 30 min) (bottom) yeast cells labelled with LIVE/DEAD yeast viability kit; (D) fluorescence image (overlayed with DIC) of a single yeast cell attached on a polydopamine-coated cantilever.

Fig. 2

Measuring Als5p-mediated hydrophobic forces. (A, D, G) Schemes of the experimental set-ups in which the TR and Ig regions of Als5p are shown in green and red, respectively. (B, E, H) Adhesion force histograms (n = 1024 force curves) and (C, F, I) representative retraction force curves recorded in deionized water between single S. cerevisiae cells expressing Als5p proteins and hydrophobic (CH₃) (A–C) or hydrophilic (OH) (D–F) substrates, and between single S. cerevisiae cells lacking Als5p (EV) and hydrophobic substrates (G–I). Adhesion force values used to build the histograms correspond to the largest adhesion events seen in the curves. Arrows in Fig. 2c indicate that large adhesion signatures were followed by weaker events rupturing at ~500 nm. The contact time was 350 ms. For each condition, similar data were obtained using at least 3 different cells from independent cultures and 3 different substrates.

Fig. 3

Als5p-mediated hydrophobic forces strengthen with time. Variation of the Als5p-CH₃, EV- CH₃ and Als5p-OH adhesion forces with contact time, using the same conditions as in Fig. 2. The data represent the mean ± standard deviation (S.D.; n = 256) and are fitted with an exponential decay function. Similar data were obtained using at least 3 different cells from independent cultures and 3 different substrates.

Fig. 4

Measuring specific Als5p-fibronectin (Fn) forces. Representative retraction force curves recorded in PBS buffer between single S. cerevisiae cells expressing (top) or not (bottom) Als5p proteins and Fn substrates (see text for details).

Fig. 5

The Als5p-fibronectin (Fn) interaction is characterized by a detachment force peak followed by a tether force peak. (A, C) Adhesion force histograms (n = 1024) and (B, D) histograms of rupture distances of the maximum detachment force peaks (A, B) and of the tether peaks (C, D) measured in PBS buffer between Als5p cells and Fn substrates. The contact time was 350 ms. Similar data were obtained using at least 3 different cells from independent cultures and 3 different substrates.

Fig. 6

Als5p-fibronectin (Fn) interactions strengthen with time. Variation of the Als5p-Fn maximum detachment force with contact time, using the same conditions as in Fig. 5 (closed symbols). As a control, data obtained for the EV strain lacking Als5p are also shown (open symbols). The data represent the mean ± standard deviation (S.D.; n = 256). Similar data were obtained using at least 3 different cells from independent cultures and 3 different substrates.