Candida albicans mannans mediate Streptococcus mutans exoenzyme GtfB binding to modulate cross-kingdom biofilm development in vivo

Abstract

Candida albicans is frequently detected with heavy infection by Streptococcus mutans in plaque-biofilms from children with early-childhood caries (ECC). This cross-kingdom biofilm contains an extensive matrix of extracellular α-glucans that is produced by an exoenzyme (GtfB) secreted by S. mutans. Here, we report that mannans located on the outer surface of C. albicans cell-wall mediates GtfB binding, enhancing glucan-matrix production and modulating bacterial-fungal association within biofilms formed in vivo. Using single-molecule atomic force microscopy, we determined that GtfB binds with remarkable affinity to mannans and to the C. albicans surface, forming a highly stable and strong bond (1-2 nN). However, GtfB binding properties to C. albicans was compromised in strains defective in O-mannan (pmt4ΔΔ) or N-mannan outer chain (och1ΔΔ). In particular, the binding strength of GtfB on och1ΔΔ strain was severely disrupted (>3-fold reduction vs. parental strain). In turn, the GtfB amount on the fungal surface was significantly reduced, and the ability of C. albicans mutant strains to develop mixed-species biofilms with S. mutans was impaired. This phenotype was independent of hyphae or established fungal-biofilm regulators (EFG1, BCR1). Notably, the mechanical stability of the defective biofilms was weakened, resulting in near complete biomass removal by shear forces. In addition, these in vitro findings were confirmed in vivo using a rodent biofilm model. Specifically, we observed that C. albicans och1ΔΔ was unable to form cross-kingdom biofilms on the tooth surface of rats co-infected with S. mutans. Likewise, co-infection with S. mutans defective in GtfB was also incapable of forming mixed-species biofilms. Taken together, the data support a mechanism whereby S. mutans-secreted GtfB binds to the mannan layer of C. albicans to promote extracellular matrix formation and their co-existence within biofilms. Enhanced understanding of GtfB-Candida interactions may provide new perspectives for devising effective therapies to disrupt this cross-kingdom relationship associated with an important childhood oral disease.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1. Cell wall mannoproteins structure of Candida albicans strains.

Boxes with red and blue dotted lines describe the truncation of O- and N-linked mannans, respectively. Adapted from “Mannosylation in Candida albicans: role in cell wall function and immune recognition,” by Rebecca A. Hall and Neil A. R. Gow, 2013, Molecular Microbiology, 90(6), p. 1148 [26]. Copyright 2013 by the John Wiley & Sons Ltd. Adapted with permission.

Fig 2. GtfB binding to C. albicans wild type and its mannosylation mutant strains.

(A) Distribution and average of binding forces, (B) representative AFM scanning and force map images of wild type, pmt4ΔΔ and och1ΔΔ mutant strains (Red Hot lookup table color scheme was used to differentiate binding forces; black-to-yellow colors indicate 0–1.5 nN), (C) amount of GtfB bound on Candida surface, and (D) comparison of GtfB binding force between C. albicans wild type and purified mannans. The force-distance curves were obtained from at least 10 individual microbial cells from at least 3 distinct culture preparations per strain. Asterisk indicates that the values are significantly different from the C. albicans WT (P < 0.05).

Fig 3. Microbiological and biochemical properties of mixed-species biofilms.

(A) CFU of C. albicans, (B) CFU of S. mutans, and (C) total insoluble EPS glucan in mixed-species biofilms at early (18 h) and later (42 h) phases. Mixed-species biofilms formed with ΔgtfB S. mutans and C. albicans WT with and without GtfB supplementation (15U) were also tested. Asterisk indicates that the values are significantly different from the mixed-species biofilm formed with S. mutans and C. albicans WT (P < 0.05).

Fig 4. Cross-sectional and orthogonal confocal images of mixed-species biofilms.

(A) S.m-C.a WT, (B) S.m-C.a pmt4ΔΔ, and (C) S.m-C.a och1ΔΔ. (A1-C1) Top view of biofilms, (A2-C2) magnified images of representative areas, (A3-C3) orthogonal views of biofilms, and (A4-C4) orthogonal views of EPS glucan-matrix. S.m-C.a WT biofilm displays numerous hyphal (mostly located in the outer layer; see white arrows) and yeast form of C. albicans with abundant amount of bacteria and densely-packed EPS glucans. In contrast, S.m-C.a pmt4ΔΔ and S.m-C.a och1ΔΔ display only a few of C. albicans with significant reduction of bacterial cells and disruption of the matrix.

Fig 5. Microbiological and biochemical properties of mixed-species biofilms with other C. albicans mutant strains (efg1ΔΔ or bcr1ΔΔ) and GtfB binding strength.

CFU of (A1) C. albicans, (A2) S. mutans, and (A3) total insoluble EPS in mixed-species biofilms; (B1-3) representative confocal images of mixed-species biofilms; distribution of GtfB binding forces to (C1) C. a SN152, (C2) C. a efg1ΔΔ, (C3) C. a bcr1ΔΔ, C. a efg1ΔΔ was selected due to slow growth and hyphal defect, comparable to pmt4ΔΔ and och1ΔΔ, while bcr1ΔΔ was selected due to lack of key biofilm adhesion regulator BCR1 in C. albicans. In our model, reduced growth rate and hyphal defect or lack of BCR1 of C. albicans did not affect significantly the ability of the fungal strains to develop mixed-species biofilm with S. mutans despite some morphological differences. The force-distance curves were obtained from at least 10 individual microbial cells from at least 3 distinct culture preparations per strain.

Fig 6. Mechanical stability of mixed-species biofilms.

(A) Overview and close-up view of shear-induced biofilm mechanical strength tester, (B) schematic diagram of biofilm removal by shear stress, and (C) biofilm removal profile after application of increased shear stress. Asterisk indicates that the values are significantly different from the mixed-species biofilm formed with S. mutans and C. albicans WT (P < 0.05).

Fig 7. Scanning electron microscopy images of the in vivo plaque biofilms.

Co-infected by (A) S. mutans and C. albicans WT, (B) S. mutans and C. albicans och1ΔΔ, and (C) S. mutans and revertant of C. albicans och1ΔΔ. Boxes with red dotted lines show numerous hyphal form of C. albicans, while box with blue dotted line in (B) show reduced amount of plaque without any C. albicans.

Fig 8. Microbiological analysis of in vivo plaque biofilms.

CFU of (A) C. albicans and (B) S. mutans. CFU of C. albicans and S. mutans were substantially decreased when rats were coinfected by S. mutans and C. albicans och1ΔΔ or gtfBΔ S. mutans and C. albicans WT. Asterisk indicates that the values are significantly different from the mixed-species biofilm formed with S. mutans and C. albicans WT or the one with S. mutans and C. albicans och1ΔΔ revertant strain (P < 0.05).