Exopolysaccharides regulate calcium flow in cariogenic biofilms.

Colin Johnson,Monika Astasov-Frauenhoffer,Muth M. Varenganayil,Olivier Braissant,Tuomas Waltimo,Alan W. Decho

doi:10.1371/journal.pone.0186256

Colin Johnson, Monika Astasov-Frauenhoffer + Show 4 more

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https://doi.org/10.1371/journal.pone.0186256

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Abstract

Caries-associated biofilms induce loss of calcium from tooth surfaces in the presence of dietary carbohydrates. Exopolysaccharides (EPS) provide a matrix scaffold and an abundance of primary binding sites within biofilms. The role of EPS in binding calcium in cariogenic biofilms is only partially understood. Thus, the aim of the present study is to investigate the relationship between the calcium dissolution rates and calcium tolerance of caries-associated bacteria and yeast as well as to examine the properties of EPS to quantify its binding affinity for dissolved calcium. Calcium dissolution was measured by dissolution zones on Pikovskaya’s agar. Calcium tolerance was assessed by isothermal microcalorimetry (IMC) by adding CaCl2 to the bacterial cultures. Acid-base titration and Fourier transform infrared (FTIR) spectroscopy were used to identify possible functional groups responsible for calcium binding, which was assessed by isothermal titration calorimetry (ITC). Lactobacillus spp. and mutans streptococci demonstrated calcium dissolution in the presence of different carbohydrates. All strains that demonstrated high dissolution rates also revealed higher rates of calcium tolerance by IMC. In addition, acidic functional groups were predominantly identified as possible binding sites for calcium ions by acid-base titration and FTIR. Finally, ITC revealed EPS to have a higher binding affinity for calcium compared, for example, to lactic acid. In conclusion, this study illustrates the role of EPS in terms of the calcium tolerance of cariogenic microbiota by determining the ability of EPS to control free calcium concentrations within the biofilms as a self-regulating mode of action in the pathogenesis of dental caries.

Highlights

Caries-associated biofilms cause loss of calcium and phosphate from tooth surfaces as bacteria living within such structures convert dietary carbohydrates into organic acids that diffuse into the tooth matrix and dissolve calcium phosphate at susceptible sites
Type strains of the following caries-associated bacteria and yeast were used in this study: Streptococcus sobrinus (ATCC 33402), Streprococcus mutans (ATCC 25175), Lactobacillus paracasei (DSM 20020), Lactobacillus casei (DSM 20011), Actinomyces viscosus (ATCC 43146), Aggregatibacter actinomycetemcomitans (DSM 8324) and Candida albicans (ATCC 90028)
All bacterial strains except for A. actinomycetemcomitans and Candida spp. showed dissolution of at least one of the three forms of calcium phosphate added to the medium (Figs 1 and 2)

Summary

Introduction

Caries-associated biofilms cause loss of calcium and phosphate from tooth surfaces as bacteria living within such structures convert dietary carbohydrates into organic acids that diffuse into the tooth matrix and dissolve calcium phosphate at susceptible sites. Exopolysaccharides regulate calcium flow in cariogenic biofilms fermentable carbohydrate leads to demineralization of dental hard tissues and to compositional changes within the microbial community of the oral biofilm [1]. The vast majority of the early colonizers on dental surfaces belong to the “mitis group” of commensal streptococci (Streptococcus sanguinis, Streptococcus oralis, and Streptococcus mitis), whereas the pathogenic “mutans group” (Streptococcus mutans, Streptococcus sobrinus) comprises only 2% or less of the initial streptococcal population, regardless of the history of caries. Acidic conditions on the demineralized/decayed tooth surface serve as a selective factor enhancing virulent multispecies biofilm formation that is predominated by S. mutans, Lactobacillus species, aciduric strains of Actinomyces and oral yeasts [3]

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