Enzymatic targeting of mannans to disurupt cross-kingdom cariogenic biofilms

Kim, Hye-Eun, Dhall, Atul, Bawazir, Marwa, Hwang, Geelsu
University of Pennsylvania School of Dental Medicine, Department of Preventive and Restorative Sciences

Introduction

Early-childhood-caries is a severe oral disease that results in aggressive tooth decay. Particularly, a synergistic association between fungus, Candida albicans, and cariogenic bacterium, Streptococcus mutans, forms hard-to-remove, and highly-acidic-biofilms, thereby exacerbating this virulent disease. Specifically, we have previously shown that glucosyltransferases (GtfB) from S. mutans strongly bind to the mannans on the cell wall of C. albicans and leads to the enhanced production of EPS. We present the enzymatic approaches to target GtfB-mannan interactions in these cross-kingdom consortia using mannan-degrading exo-/endo-enzymes (MDEs).

Methods

We comprehensively assessed the activity of MDEs in appropriate buffers and human saliva and measure GtfB-C. albicans binding forces using single-molecule Atomic Force Microscopy (AFM). Biofilms with S. mutans and C. albicans were formed on HA discs or human enamel slabs in saliva supplemented with 1% sucrose. We examined i) the number of viable cells, ii) the amount of biomass using biochemical assays, iii) the demineralization of enamel surface using surface topography, and iv) 3D biofilm architecture/mechanical stability using confocal imaging combined shear-inducing device.

Results

AFM Data show MDEs drastically decreased the binding forces of GtfB-to-C. albicans. MEDs successfully diminished the cross-kingdom biofilm development without exhibiting microbicidal effects and the acidic environment induced by the cross-kingdom biofilms was alleviated. In turn, we observed MDEs treated biofilm has lower mechanical stability than untreated control. Moreover, MDEs very efficiently prevent the demineralization of the enamel surface by S. mutans-C. albicans biofilms. Finally, we confirmed that MDEs show minimal cytotoxicity against primary human gingival keratinocytes, demonstrating the broad potential of our approach in clinical applications.

Conclusion

The MDEs are highly effective in reducing biofilms biomass without killing microorganisms as well as alleviating an acidic pH environment conducive to tooth demineralization.