The Role of Candida albicans Secreted Polysaccharides in Augmenting Streptococcus mutans Adherence and Mixed Biofilm Formation: In vitro and in vivo Studies

Abstract

The oral cavity is a complex environment harboring diverse microbial species that often co-exist within biofilms formed on oral surfaces. Within a biofilm, inter-species interactions can be synergistic in that the presence of one organism generates a niche for another enhancing colonization. Among these species are the opportunistic fungal pathogen Candida albicans and the bacterial species Streptococcus mutans, the etiologic agents of oral candidiasis and dental caries, respectively. Recent studies have reported enhanced prevalence of C. albicans in children with caries indicating potential clinical implications for this fungal-bacterial interaction. In this study, we aimed to specifically elucidate the role of C. albicans-derived polysaccharide biofilm matrix components in augmenting S. mutans colonization and mixed biofilm formation. Comparative evaluations of single and mixed species biofilms demonstrated significantly enhanced S. mutans retention in mixed biofilms with C. albicans. Further, S. mutans single species biofilms were enhanced upon exogenous supplementation with purified matrix material derived from C. albicans biofilms. Similarly, growth in C. albicans cell-free spent biofilm culture media enhanced S. mutans single species biofilm formation, however, the observed increase in S. mutans biofilms was significantly affected upon enzymatic digestion of polysaccharides in spent media, identifying C. albicans secreted polysaccharides as a key factor in mediating mixed biofilm formation. The enhanced S. mutans biofilms mediated by the various C. albicans effectors was also demonstrated using confocal laser scanning microscopy. Importantly, a clinically relevant mouse model of oral co-infection was adapted to demonstrate the C. albicans-mediated enhanced S. mutans colonization in a host. Analyses of harvested tissue and scanning electron microscopy demonstrated significantly higher S. mutans retention on teeth and tongues of co-infected mice compared to mice infected only with S. mutans. Collectively, the findings from this study strongly indicate that the secretion of polysacharides from C. albicans in the oral environment may impact the development of S. mutans biofilms, ultimately increasing dental caries and, therefore, Candida oral colonization should be considered as a factor in evaluating the risk of caries.

Keywords: Candida albicans; Streptococcus mutans; dental caries; fungal-bacteria interactions; matrix; mixed-biofilms; polysaccharide.

FIGURE 1

Significantly higher S. mutans recovery from mixed biofilms with C. albicans. (A) Based on CFU (cells/ml) counts, significantly higher level of S. mutans is recovered from mixed biofilms compared to single biofilm. (B) Similar results were seen when biofilms were evaluated based on GFP fluorescence intensity (arbitrary units) of S. mutans-GFP cells. Unpaired t-test; **p = 0.0053, ****p ≤ 0.0001.

FIGURE 2

Comparative CLSM analysis of S. mutans single and mixed 24-hr biofilms with C. albicans. (A,D) GFP-S. mutans single-species biofilms demonstrating a sparse biofilm with small cell aggregates. (B,C,E,F) In contrast, growth of GFP-S. mutans with C. albicans resulted in the formation of a dense and complex biofilm. (A–C) Representative 3D images showing biofilm distribution for (A) GFP-S. mutans alone, (B) GFP-S. mutans channel in a mixed biofilm with C. albicans and (C) GFP-S. mutans and C. albicans in a mixed biofilm. (E,F) Representative Z-stacks showing adherence and clumping of bacterial cells around the hyphae. GFP-S. mutans (green); C. albicans cell wall chitin stained with Calcofluor White (blue); Extracellular matrix of biofilm stained with Concanavalin-A-Alexa Fluor 647 (magenta). Images were acquired using Spinning disk confocal Nikon Ti2 inverted microscope and processed with Imaris software.

FIGURE 3

Representative Scanning Electron Microscopy (SEM) micrographs of in vitro-grown dual-species biofilms on slabs of human teeth. (A) Dentin and enamel slabs were obtained from intact 3rd molar teeth using a water-cooled diamond saw and a cutting machine. Mixed biofilms were grown on slabs for 24 h at 37°C. (B–E) SEM micrographs demonstrating a robust and thick mixed biofilm formed on the slabs consisting of intertwined hyphal matrix with aggregates of bacteria (blue arrows) formed around the hyphae (yellow arrow).

FIGURE 4

Evaluation of the various C. albicans effectors mediating mixed biofilm formation. (A) Based on measurement of fluorescence emitted from GFP-S. mutans, significantly higher fluorescence intensity was detected in mixed biofilms (Sm+Ca) compared to that in S. mutans single biofilm (Sm); similarly, matrix supplementation resulted in a matrix dose-dependent increase in fluorescence, but not supplementation with β-Glucan or α-Mannan (0.5 mg/mL, both). Matrix concentrations tested included 0.5, 1.25, and 0.125 mg/mL. (B) To evaluate the role of C. albicans secreted matrix polysaccharides in mediating S. mutans retention, S. mutans single biofilms were grown in cell-free spent media from the C. albicans strains. Based on measurement of emitted fluorescence from 24 h S. mutants biofilms, significantly higher S. mutans signal was detected from biofilms grown in spent media (Sm+Spent) compared to that from the S. mutans alone. The observed increase in emitted fluorescence from S. mutans biofilms grown in C. albicans spent media was significantly diminished upon enzymatic digestion of the secreted polysaccharides in the media by α-mannosidase (0.5, 1, and 2 U/mL) and β-glucanase (1,25, 2.5, and 5 U/mL) enzymes with α-mannosidase treatment being more impactful. Statistical comparison between Sm alone and Sm + spent is indicated with a black bar. Comparative analysis between Sm biofilm in digested-spent media versus Sm + spent is indicated with a blue bar. ns: not significant; **p ≤ 0.01; ***p ≤ 0.005; ****p ≤ 0.001.

FIGURE 5

Confocal Laser Scanning Microscopy (CLSM) analysis of S. mutans biofilms grown in C. albicans cell free spent media or media supplemented with purified extracellular matrix. (A) 24-hr single biofilms of GFP-S. mutans appeared sparse with minimal cells or cell aggregates seen. (B) Significantly more S. mutans cells was seen when S. mutans was grown in C. albicans spent media with considerable amount of bacterial clumping around secreted polysaccharide aggregates. (C) S. mutans biofilm grown in the presence of purified extracellular matrix from C. albicans biofilms showing large aggregates of polysaccharides with embedded aggregates of bacteria. Pink: polysaccharides stained with Con-A; Green: GFP-S. mutans.

FIGURE 6

Animal model of oral co-infection. (A) Infection protocol and timeline; animals were euthanized and infection assessed 4 days post-infcetion with C. albicans. (B) Representative image demonstrating mouse sublingual oral infection using calcium alginate swabs saturated with C. albicans cell suspension. (C) Representative image of a tongue from an infected animal with multiple white lesions typical of oral candidiasis (black arrows). (D,E) Representative images from histopathology analysis of PAS stained tongue tissue sections from co-infected mice demonstrating extensive presence of C. albicans around the periphery of the tongue along with hyphal invasion (black arrows) into the sub-epithelial tissue; influx of neutrophils (red arrow) indication local immune response to the infection. Bacterial cells are not visible with PAS stain. Scale bar 400 μm; Inset scale bar 100 μm.

FIGURE 7

Evaluation of S. mutans and C. albicans recovery from teeth and tongues of mice infected with S. mutans and C. albicans individually or in combination. (A) Significantly higher level of S. mutans recovery from teeth and tongues of co-infected animals compared to animals infected only with S. mutans. (B) No difference in the recovery of C. albicans from teeth and tongues from co-infected animals compared to animals infected only with C. albicans. ns: not significant; *p ≤ 0.05.

FIGURE 8

Comparative SEM analysis of teeth and tongues from S. mutans-infected and co-infected animals. Enhanced S. mutans colonization of oral samples from co-infected animals compared to S. mutans-infected animals. Teeth of S. mutans-only infected mouse showing scattered clumps of S. mutans compared to teeth of co-infected mouse demonstrating a thicker biofilm of C. albicans intermixed with S. mutans embedded in extracellular matrix material. Tongue of S. mutans-only infected mouse with minimal S. mutans adherence and biofilm formation; in contrast to tongue of co-infected mouse with oral candidiasis with massive hyphal invasion of oral tissue. A thick biofilm is seen consisting of C. albicans and S. mutans adhering avidly to the surface of C. albicans and within clumps of extracellular matrix. blue arrows: S. mutans, yellow arrows: C. albicans.

FIGURE 9

High magnification SEM micrograph of the surface of a tongue from a co-infected mouse with oral candidiasis demonstrating S. mutans adhering avidly to the surface of C. albicans hyphae (pseudo-colored in purple). Bar = 10 μm.