Single-molecule imaging and functional analysis of Als adhesins and mannans during Candida albicans morphogenesis

Abstract

Cellular morphogenesis in the fungal pathogen Candida albicans is associated with changes in cell wall composition that play important roles in biofilm formation and immune responses. Yet, how fungal morphogenesis modulates the biophysical properties and interactions of the cell surface molecules is poorly understood, mainly owing to the paucity of high-resolution imaging techniques. Here, we use single-molecule atomic force microscopy to localize and analyze the key components of the surface of living C. albicans cells during morphogenesis. We show that the yeast-to-hypha transition leads to a major increase in the distribution, adhesion, unfolding, and extension of Als adhesins and their associated mannans on the cell surface. We also find that morphogenesis dramatically increases cell surface hydrophobicity. These molecular changes are critical for microbe-host interactions, including adhesion, colonization, and biofilm formation. The single-molecule experiments presented here offer promising prospects for understanding how microbial pathogens use cell surface molecules to modulate biofilm and immune interactions.

Figure 1. Candida albicans

morphogenesis, cell adhesion and biofilm formation. Phase contrast (top) and fluorescence (bottom) images of Calcofluor White stained C. albicans cells adhering on plastic surfaces after 5 min (a), 90 min (b) and 48 h (c). The cartoons underneath the images emphasize the different steps leading to biofilm formation and the role of yeast and hyphal cells in the process. Cells were grown overnight in YPD medium, inoculated in RPMI to induce germination, incubated at 37°C with the plastic coverslips, and then rinsed with buffer solution.

Figure 2

AFM imaging of living yeast cells and hyphae. Two different approaches were used to immobilize C. albicans yeast cells and hyphae non-destructively: (a, b) spherical yeast cells were immobilized mechanically into porous polymer membranes, while (c, d) germinating cells were attached onto solid substrata functionalized with hydrophobic groups. (b, d) Three-dimensional AFM images of a yeast-form cell (b; 5 μm × 5 μm) and of a germinating cell (d; 15 μm × 15 μm). The two methods are simple and do not involve drying or chemical treatments that could lead to denaturation of the cell surface molecules.

Figure 3

Fluorescence imaging of Als3 proteins. Fluorescence (a, b) and phase contrast (d, e) images of WT germinating (a, d) and yeast (b, e; see on the right) cells grown for 90 min in RPMI medium, stained with anti-Als3 antibodies followed by FITC-conjugated secondary antibodies. Unlike yeast cells, germ tubes were massively immunolabelled. Fluorescence (c) and phase contrast (f) images of a germ tube from an als3Δ/als3Δ (Δals3) mutant strain, revealing a lack of fluorescence. Similar data were obtained in independent experiments using different tips and cultures.

Figure 4

Single-molecule detection of Als3 proteins on yeast cells. (a) The cell wall in C. albicans is made of an inner layer of chitin and β1,3-glucan polysaccharides that confer strength and cell shape, and an outer layer of mannose polymers, mannans (blue), covalently associated with cell wall proteins such as Als adhesins (green). To probe single Als3 proteins, C. albicans yeast cells were imaged in buffer using AFM tips terminated with an anti-Als3 antibody. (b–d) Adhesion force map (b; 1 μm × 1 μm, color scale: 300 pN), adhesion force histogram (n = 1024 force curves) together with representative force curves (c), and rupture length histogram (d) obtained by recording force curves across the surface of a C. albicans yeast-form cell using an antibody-labelled tip. The inset in (b) is a deflection image of the cell in which the * symbol indicates where the force map was recorded. The cartoons in (c) emphasize the dual detection of Als3: while some curves showed single weak adhesion peaks reflecting Als epitope recognition (top curves), others featured sawtooth patterns with multiple force peaks documenting Als Ig-to-ligand multi-point binding followed by the unfolding of the entire protein (bottom curves). (e) 3-D reconstructed map obtained by combining adhesion force values (false colors, adhesion forces are shown in green) and rupture distances (expressed as z level) measured at different x, y locations. Unless stated otherwise, all curves were obtained at 20°C using a contact time of 100 ms and a loading rate of 10,000 pN s⁻¹. Similar data were obtained in several independent experiments using different tip preparations and cell cultures.

Figure 5

Cellular morphogenesis leads to a major increase in the distribution and extension of Als3 proteins. (a, d) Adhesion force maps (1 μm × 1 μm, color scale: 300 pN) recorded in buffer on the yeast (a) and germ tube (d) of a germinating cell using an anti-Als3 tip. Insets: deflection images in which the * symbols indicate where the force maps were recorded. The dashed lines in (a) emphasize Als3 clusters. (b, e) Corresponding adhesion force histograms (n = 1024) together with representative force curves. (c, f) Histograms of rupture distances (n = 1024), and 3-D reconstructed polymer maps (false colors, adhesion forces in green). Similar data were obtained in several independent experiments using different tip preparations and cell cultures.

Figure 6

Control experiments demonstrate the specificity of Als3 detection. (a, d, g, j) Adhesion force maps (1 μm × 1 μm, color scale: 300 pN) recorded in buffer with anti-Als3 tips on WT germ tubes after treatment with free anti-Als3 antibodies (0.1 mg/mL) (a) or goat serum (d), on a als3Δ/als3Δ (Δals3) germ tube blocked with goat serum (g), and on a non-blocked als3Δals3Δ/als1Δals1Δ (Δals3Δals1) germ tube (j). (b, e, h, k) Corresponding adhesion force histograms (n = 1024) together with representative force curves. (c, f, i, l) Histograms of rupture distances (n = 1024), and 3-D reconstructed polymer maps (false colors, adhesion forces in green).

Figure 7

Cell surface hydrophobicity dramatically increases during cellular morphogenesis. (a, d, g) Adhesion force maps (1 μm × 1 μm, color scale: 3000 pN) recorded in buffer on a non-germinating WT yeast cell (a), a WT germ tube (d), and an als3Δals3Δ/als1Δals1Δ (Δals3Δals1) germ tube (g) using hydrophobic tips. (b, e, h) Corresponding adhesion force histograms (n = 1024) together with representative force curves. (c, f, i) Histograms of rupture distances (n = 1024), and 3-D reconstructed hydrophobicity maps (false colors, adhesion forces in orange). Similar data were obtained in independent experiments using different tips and cultures.

Figure 8

Imaging and stretching single mannan glycoconjugates during cellular morphogenesis. (a, d, g) Adhesion force maps (1 μm × 1 μm, color scale: 300 pN) recorded in buffer on a non germinating yeast (a), on a germinating yeast (d) and on a germ tube (g) using an AFM tip labelled with ConA lectins. Insets: deflection images in which the * symbol indicates where the force maps were recorded. (b, e, h) Corresponding adhesion force histograms together with representative force curves. (c, f, i) Histograms of rupture distances (n = 1024), and 3-D reconstructed polymer maps (false colors, adhesion forces in blue). Similar data were obtained in independent experiments using different tips and cultures.

Figure 9

Probing mannans on cell wall mutants. (a, d, g) Adhesion force maps (1 μm × 1 μm, color scale: 300 pN) recorded in buffer with a Con A tip on a mnt1Δ/mnt1Δ mnt2Δ/mnt2Δ mnt3Δ/mnt3Δ mnt4Δ/mnt4Δ mnt5Δ/mnt5Δ (Δmnt1-5) germ tube altered in O-linked and N-linked mannosylations (a), on a als3Δals3Δ/als1Δals1Δ (Δals3Δals1) germ tube (d), and on a als3Δals3Δ/als1Δals1Δ (Δals3Δals1) germ tube blocked with 200 mM methyl α-D-mannopyranoside (g). (b, e, h) Corresponding adhesion force histograms together with representative force curves. (c, f, i) Histograms of rupture distances (n = 1024), and 3-D reconstructed polymer maps (false colors, adhesion forces in blue). Similar data were obtained in independent experiments using different tips and cultures.

Figure 10

Towards a molecular and functional view of Als3 and mannans during Candida albicans morphogenesis. Germination of C. albicans leads to a major increase in the distribution and extension of Als3 proteins (green) and their associated mannans (blue) on the hyphae surface, a phenomenon which promotes adhesion to various surfaces (see text for details).