Candida albicans Mannoproteins Modulate Binding of Biofilm Matrix-producing Bacterial Exoenzyme

Objectives: Early childhood caries (ECC), a virulent-form of dental caries, is painful, difficult and costly to treat. Candida albicans are often detected together with Streptococcus mutans in plaque-biofilms from children with ECC. S. mutans-derived exoenzyme (GtfB) binds to C. albicans cell-surface with exceptionally avidity and high adhesion strength. The GtfB bound to C. albicans produce exopolysaccharides (EPS) in situ that promote the cross-kingdom interaction and the development of highly cariogenic mixed-species biofilms in vivo. We investigated the role of mannoproteins (located at the outer-most layer of the Candida cell-wall) in mediating the GtfB-C. albicans binding, and the development of mixed-species biofilms.
Methods: Using a combination of biophysical methods and genetics with an in vivo biofilm model, we examined i) the binding force dynamics of GtfB-Candida (wild-type and mannosylation-deficient mutants of C. albicans (Ca_Mut)) via single-molecule AFM; ii) the amounts of GtfB bound to each Candida by ELISA; iii) the viable cells, amount of insoluble EPS, and the 3D biofilm architecture/mechanical stability via biochemical/microbiological assays and multiphoton-confocal imaging combined with shear-inducing device; iv) formation of plaque-biofilms in vivo using a rodent model.
Results: The data show that GtfB binding forces to the Ca_MUT surface were remarkably disrupted (~3-fold less vs. wild-type; Ca_WT), resulting in significantly less GtfB bound on the fungal surface (vs. Ca_WT). Importantly, Ca_MUT were impaired in forming mixed-species biofilms with S. mutans in vitro and in vivo, and displayed substantially reduced fungal population and amounts of EPS-matrix. Finally, the 3D architecture and mechanical stability of defective biofilms with Ca_MUT were dramatically compromised (vs. Ca_WT).
Conclusions: Our data reveal the critical role of C. albicans mannosylation in mediating GtfB binding and cross-kingdom biofilm development in vivo. Enhanced understanding of GtfB-Candida interactions may provide new perspectives for devising efficacious-therapies to prevent or control the onset of this devastating oral disease.