Candida albicans stimulates Streptococcus mutans microcolony development via cross-kingdom biofilm-derived metabolites

Dongyeop Kim, Arjun Sengupta, Tagbo H R Niepa, Byung-Hoo Lee, Aalim Weljie, Veronica S Freitas-Blanco, Ramiro M Murata, Kathleen J Stebe, Daeyeon Lee, Hyun Koo, Dongyeop Kim, Arjun Sengupta, Tagbo H R Niepa, Byung-Hoo Lee, Aalim Weljie, Veronica S Freitas-Blanco, Ramiro M Murata, Kathleen J Stebe, Daeyeon Lee, Hyun Koo

Abstract

Candida albicans is frequently detected with heavy infection of Streptococcus mutans in plaque-biofilms from children affected with early-childhood caries, a prevalent and costly oral disease. The presence of C. albicans enhances S. mutans growth within biofilms, yet the chemical interactions associated with bacterial accumulation remain unclear. Thus, this study was conducted to investigate how microbial products from this cross-kingdom association modulate S. mutans build-up in biofilms. Our data revealed that bacterial-fungal derived conditioned medium (BF-CM) significantly increased the growth of S. mutans and altered biofilm 3D-architecture in a dose-dependent manner, resulting in enlarged and densely packed bacterial cell-clusters (microcolonies). Intriguingly, BF-CM induced S. mutans gtfBC expression (responsible for Gtf exoenzymes production), enhancing Gtf activity essential for microcolony development. Using a recently developed nanoculture system, the data demonstrated simultaneous microcolony growth and gtfB activation in situ by BF-CM. Further metabolites/chromatographic analyses of BF-CM revealed elevated amounts of formate and the presence of Candida-derived farnesol, which is commonly known to exhibit antibacterial activity. Unexpectedly, at the levels detected (25-50 μM), farnesol enhanced S. mutans-biofilm cell growth, microcolony development, and Gtf activity akin to BF-CM bioactivity. Altogether, the data provide new insights on how extracellular microbial products from cross-kingdom interactions stimulate the accumulation of a bacterial pathogen within biofilms.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1. Influences of conditioned medium on…
Figure 1. Influences of conditioned medium on the growth of S. mutans biofilm cells and microcolony development.
(A) Conditioned medium (CM) was collected and prepared from single-species bacterial (B-CM), fungal (F-CM) or bacterial-fungal (BF-CM) biofilms at different time points. S. mutans biofilms were grown on saliva-coated hydroxyapatite (sHA) disc surface in each of the CM preparations (1:5 vol/vol, CM:UFTYE). The viable cells number (colony forming units (CFU)/biofilm) was normalized by dry weight (mg) (n = 8). (B) Dose-response effects of BF-CM on the growth of S. mutans biofilm cells (n = 4). (C) Representative 3D rendering images of biofilms formed with different dilutions of BF-CM and analyzed via multi-photon confocal laser scanning microscopy. S. mutans cells stained with SYTO 9 are depicted in green, while EPS labelled with Alexa Fluor 647 is shown in red. Data represent mean ± SD. The quantitative data were subjected to analysis of variance (ANOVA) in the Tukey’s HSD test for a multiple comparison. Values are significantly different from each other at *P < 0.05 or **P < 0.01.
Figure 2. Three-dimensional (3D) architecture and quantitative…
Figure 2. Three-dimensional (3D) architecture and quantitative computational analysis of biofilm formed in BF-CM.
(A) Representative confocal images of S. mutans biofilms grown in BF-CM (1:5vol/vol, CM:UFTYE) and without supplementation (control). The bacterial microcolonies are depicted in green (SYTO 9), while the EPS-matrix is depicted in red (Alexa Fluor 647). (B) Quantitative analysis of total biovolume (biomass), cell/EPS ratio and microcolony volume (size) was performed using COMSTAT. Data represent mean ± SD (n = 8). A pairwise comparison between control and BF-CM was conducted using student’s t-test. Values are significantly different from each other at *P < 0.05 or **P < 0.01.
Figure 3. Dynamics of glucosyltransferases (Gtfs) activity…
Figure 3. Dynamics of glucosyltransferases (Gtfs) activity and gtfBC expression profiles in biofilms formed in BF-CM.
(A) Dose-response effects of BF-CM (diluted in UFTYE, vol/vol) on Gtfs activity. The data were subjected to linear regression analysis and analysis of variance (ANOVA) in the Tukey’s HSD test for a multiple comparison. Correlation coefficient between increasing amounts of BF-CM and Gtfs activity was r2 = 0.97. (B) Temporal effects of BF-CM on Gtfs activity at different stages of biofilm development; early-stage (18–28 h) and matured biofilms (28–42 h). (C) Influence of BF-CM (collected at 18 h) on gtfB and gtfC gene expression by S. mutans biofilms was analyzed at 18, 22, 28, and 32 h of development. A pairwise comparison between control and BF-CM was conducted using student’s t-test. Data represent relative ratio to control (defined as 1); mean ± SD (n = 4). Values are significantly different from each other at *P < 0.05, **P < 0.01 or ***P < 0.001.
Figure 4. Microcolony assembly and gtfB expression…
Figure 4. Microcolony assembly and gtfB expression in situ using a microfluidics-generated nanoculture system.
(A) Defined cell population of S. mutans (~30 cells per nanoculture) was encapsulated in a semipermeable polydimethylsiloxane (PDMS)-based nanoculture (microcapsules). (B) S. mutans cells were inoculated in PDMS nanocultures with culture medium (UFTYE) and seeded in the medium (outside) containing either BF-CM (1:5 vol/vol, CM:UFTYE) or without supplementation (control). The surface area of cell occupying the interior of the capsule was measured by Image J. Data represent mean ± SD (n = 12). (C) Microcolony formation and gtfB expression via S. mutans strain expressing GFP under the control of gtfB promoter; PgtfB::gfp were determined using optical and confocal microscopy. The GFP fluorescent intensity was measured by using Image J. Data represent mean ± SD (n = 20). A pairwise comparison between control and BF-CM was conducted using student’s t-test. Values are significantly different from each other at **P < 0.01, ***P < 0.001.
Figure 5. The composition of carbohydrates and…
Figure 5. The composition of carbohydrates and metabolites in the conditioned medium (CM).
CM was collected and prepared from single-species bacterial (B-CM), fungal (F-CM) or bacterial-fungal (BF-CM) biofilms as described in Materials and Methods. (A) HPAEC chromatograms of carbohydrate profile and 1H-NMR analysis for metabolites (organic acids shown). (B) The concentrations of glucose, fructose and sucrose as the main carbohydrates in undiluted CM. (C) The concentrations of formate, fumarate and lactate as the main metabolites in undiluted CM. Data represent mean ± SD (n = 3). The data were subjected to analysis of variance (ANOVA) in the Tukey’s HSD test for a multiple comparison. The letters (a and b) denote significate differences (P < 0.01).
Figure 6. Quantification of farnesol in the…
Figure 6. Quantification of farnesol in the conditioned medium (CM).
(A) HPLC profile of farnesol was obtained from CM extracted with EtOAc. The peak at 30.5 min is of farnesol (red arrows). Conditioned medium (CM) was collected and prepared from single-species bacterial (B-CM), fungal (F-CM) or bacterial-fungal (BF-CM) biofilms at 18 h. (B) Biological concentration of farnesol detected in the CM.
Figure 7. Influences of farnesol at levels…
Figure 7. Influences of farnesol at levels found in BF-CM on Gtfs activity and S. mutans biofilm formation.
(A) Influence of farnesol at different concentrations (0–200 μM) on the S. mutans biofilm cell growth. Data represent mean ± SD (n = 4). The data were subjected to analysis of variance (ANOVA) in the Tukey’s HSD test for a multiple comparison. §Indicates non-detected. (B) Gtfs activity in S. mutans biofilms grown in the presence of farnesol at a range of concentrations (12.5, 25 and 50 μM). Data represent mean ± SD (n = 6). The data were subjected to analysis of variance (ANOVA) in the Tukey’s HSD test for a multiple comparison. (C1) Microcolony development and gtfB expression via PgtfB::gfp S. mutans in biofilms formed on sHA surface in the presence of farnesol at 25 μM. Fluorescence images were obtained using confocal laser scanning microscopy. (C2) The microcolony size and GFP fluorescence intensity were measured by COMSTAT and Image J. Data represent mean ± SD (n = 4). A pairwise comparison between control and farnesol was conducted using student’s t-test. Values are significantly different from each other at **P < 0.01 or ***P < 0.001.

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