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.

Quantitative Analyses of Force-Induced Amyloid Formation in Candida albicans Als5p: Activation by Standard Laboratory Procedures

“…Our study of activation of aggregation in cell suspensions reinforces the idea that force alone is sufficient to induce surface nanodomains [ 4 , 7 , 24 ]. This remarkable activity takes place on cell surfaces without need for a signaling or metabolic response in the cell [ 4 , 5 ].…”

“…First, the amyloid sequence-containing T domain in Als5p WT is only marginally stable, and the domain unfolds in response to extension forces [ 5 , 7 ]. Domain unfolding exposes the amyloid core sequence, which in turn interacts with amyloid sequences in nearby Als adhesin molecules to form cell surface nanodomains within minutes [ 4 – 6 , 24 – 26 ]. These nanodomains consist of arrayed adhesin molecules, aggregated on the cell surface through amyloid-like interactions of the amino acids in amyloid core sequences [ 27 , 28 ].…”

The dynamics and pH-dependence of Ag43 adhesins’ self-association probed by atomic force spectroscopy

“…As further discussed in this section, we hypothesize that such interaction between Ag43α is faster and weaker than the trans-interaction detected under low pulling rate conditions. In literature, it is reported that some proteins (Als5p adhesin from C. albicans 59,60 , Nectin proteins 61 ) may associate side-by-side in cis-configuration. If adopting such a configuration for Ag43α dimers where the bends of the L-shaped proteins are in opposite direction (Figure 9a) and further considering the sole unfolding of one out of the two interacting molecules, it can be estimated that the maximal extension expected should be around 105 nm (corresponding to 292 amino-acids).…”

Nanoscale biophysical properties of the cell surface galactosaminogalactan from the fungal pathogen Aspergillus fumigatus

“…41,42 Briefly, triangular shaped tipless cantilevers (NP-O10, Microlevers, Bruker Corporation) were immersed for 1 h in a 10 mM Tris Buffer solution ( pH 8.5) containing 4 mg mL −1 dopamine hydrochloride (99%, Sigma), and dried with N 2 flow. Cell probes were prepared with a protocol that uses a bioinspired polydopamine wet adhesive.…”