Cross-Kingdom Cell-to-Cell Interactions in Cariogenic Biofilm Initiation

Abstract

Candida albicans is known to form polymicrobial biofilms with various Streptococcus spp., including mitis and mutans group streptococci. Streptococcus gordonii (mitis group) has been shown to bind avidly to C. albicans hyphae via direct cell-to-cell interaction, while the cariogenic pathogen Streptococcus mutans (mutans group) interacts with the fungal cells via extracellular glucans. However, the biophysical properties of these cross-kingdom interactions at the single-cell level during the early stage of biofilm formation remain understudied. Here, we examined the binding forces between S. mutans (or S. gordonii) and C. albicans in the presence and absence of in situ glucans on the fungal surface using single-cell atomic force microscopy and their influence on biofilm initiation and subsequent development under cariogenic conditions. The data show that S. gordonii binding force to the C. albicans surface is significantly higher than that ofS. mutans to the fungal surface (~2-fold). However, S. mutans binding forces are dramatically enhanced when the C. albicans cell surface is locally coated with extracellular glucans (~6-fold vs. uncoated C. albicans), which vastly exceeds the forces between S. gordonii andC. albicans. The enhanced binding affinity of S. mutans to glucan-coated C. albicans resulted in a larger structure during early biofilm initiation compared to S. gordonii-C. albicans biofilms. Ultimately, this resulted in S. mutans dominance composition in the 3-species biofilm model under cariogenic conditions. This study provides a novel biophysical aspect of Candida-streptococcal interaction whereby extracellular glucans may selectively favor S. mutans binding interactions with C. albicans during cariogenic biofilm development.

Keywords: Candida albicans; Streptococcus gordonii; Streptococcus mutans; biophysics; caries; glucans.

Figure 1.

Single-cell atomic force microscopy (sc-AFM) analysis of the adhesive interactions between Streptococcus and Candida albicans. (a) Schematic diagram of sc-AFM. AFM tipless cantilever probes were functionalized with Streptococcus mutans (or Streptococcus gordonii), and C. albicans cells were immobilized on a coverslip. Fluorescent image of S. mutans (b1) or S. gordonii (b2) functionalized AFM probes. (c1) Confocal image of immobilized C. albicans on the poly-L-lysine-coated coverslip. (c2) Total Internal Reflection Fluorescence-AFM image of glucans formed on immobilized C. albicans.

Figure 2.

Binding forces and rupture lengths of Streptococcus to yeast Candida albicans surfaces. (a) Adhesion force histogram and (b) rupture length histogram of Streptococcus mutans to uncoated (light-red columns) or glucan-coated yeast C. albicans (dark-red columns). (c) Adhesion force histogram and (d) rupture length histogram of Streptococcus gordonii to uncoated (light-green columns) or glucan-coated yeast C. albicans (dark-green columns).

Figure 3.

Binding force and rupture length of Streptococcus to hyphal Candida albicans surfaces. (a) Adhesion force histogram and (b) rupture length histogram of Streptococcus mutans to uncoated (light-red columns) or glucan-coated hyphal C. albicans (dark-red columns). (c) Adhesion force histogram and (d) rupture length histogram of Streptococcus gordonii to uncoated (light-green columns) or glucan-coated hyphal C. albicans (dark-green columns).

Figure 4.

Cross-kingdom interactions of Streptococcus mutans–glucan-coated Candida albicans and Streptococcus gordonii–glucan-coated C. albicans. Time-lapse images of (a) S. mutans–glucan-coated C. albicans (Sm-gCa) and (b) S. gordonii–glucan-coated C. albicans (Sg-gCa) biofilm initiation in the presence of 1% sucrose. Red color on the far-left panels of (a) and (b) showed glucans were formed on C. albicans surfaces. Extracellular polysaccharides channel was removed in time-lapse images (0 to 180 min) to clearly show Streptococcus–C. albicans direct cell-to-cell interactions. (c) The number of coadhesions between S. mutans (or S. gordonii) and glucan-coated C. albicans per area (319.45 × 319.45 µm) in each biofilm after 60 min of incubation (n = 20). (d) Orthogonal views of each biofilm at 180 min (left: Sm-gCa; right: Sg-gCa). (e) Biovolumes and the maximum thickness of each biofilm at 180 min (n = 3). Scale bars indicate 10 µm. Asterisk indicates that the values are significantly different between Sm-gCa and Sg-gCa. (P < 0.05).

Figure 5.

Properties of dual- and 3-species biofilms cultured in 1% sucrose. (a) Colony-forming units (CFU) and (b) dry weight of Streptococcus mutans–Candida albicans, Streptococcus gordonii–C. albicans, and S. mutans–S. gordonii–C. albicans biofilms. Representative confocal images of (c) S. mutans–C. albicans, (d) S. gordonii–C. albicans, and (e) S. mutans–S. gordonii–C. albicans biofilms. Green, red, blue, and gray colors indicate S. mutans, S. gordonii, Extracellular polysaccharides-matrix, and C. albicans, respectively. (f) Biovolumes of each strain in each biofilm. Asterisk indicates that the values are significantly different from dual-species biofilms. Hash indicates that the values are significantly different among groups. (P < 0.05) (n = 8).