Exopolysaccharides Produced by Streptococcus mutans Glucosyltransferases Modulate the Establishment of Microcolonies within Multispecies Biofilms

Abstract

Streptococcus mutans is a key contributor to the formation of the extracellular polysaccharide (EPS) matrix in dental biofilms. The exopolysaccharides, which are mostly glucans synthesized by streptococcal glucosyltransferases (Gtfs), provide binding sites that promote accumulation of microorganisms on the tooth surface and further establishment of pathogenic biofilms. This study explored (i) the role of S. mutans Gtfs in the development of the EPS matrix and microcolonies in biofilms, (ii) the influence of exopolysaccharides on formation of microcolonies, and (iii) establishment of S. mutans in a multispecies biofilm in vitro using a novel fluorescence labeling technique. Our data show that the ability of S. mutans strains defective in the gtfB gene or the gtfB and gtfC genes to form microcolonies on saliva-coated hydroxyapatite surfaces was markedly disrupted. However, deletion of both gtfB (associated with insoluble glucan synthesis) and gtfC (associated with insoluble and soluble glucan synthesis) is required for the maximum reduction in EPS matrix and biofilm formation. S. mutans grown with sucrose in the presence of Streptococcus oralis and Actinomyces naeslundii steadily formed exopolysaccharides, which allowed the initial clustering of bacterial cells and further development into highly structured microcolonies. Concomitantly, S. mutans became the major species in the mature biofilm. Neither the EPS matrix nor microcolonies were formed in the presence of glucose in the multispecies biofilm. Our data show that GtfB and GtfC are essential for establishment of the EPS matrix, but GtfB appears to be responsible for formation of microcolonies by S. mutans; these Gtf-mediated processes may enhance the competitiveness of S. mutans in the multispecies environment in biofilms on tooth surfaces.Oral diseases related to dental biofilms afflict the majority of the world's population, and dental caries is still the single most prevalent and costly oral infectious disease (12, 32). Dental caries results from the interaction of specific bacteria with constituents of the diet within a biofilm formed on the tooth surface known as plaque (5,36). Streptococcus mutans is a key contributor to the formation of biofilms associated with dental caries disease, although other microorganisms may also be involved (3); S. mutans (i) effectively utilizes dietary sucrose (and possibly starch) to rapidly synthesize exopolysaccharides (EPS) using glucosyltransferases and a fructosyltransferase that adsorb to surfaces, (ii) adheres tenaciously to glucan-coated surfaces, and (iii) is acidogenic and acid tolerant (5, 30).In general, biofilms develop after initial attachment of microbes to a surface, followed by formation of highly structured cell clusters (or microcolonies) and further development and stabilization of the microcolonies, which are in a complex extracellular matrix (6, 49). The majority of biofilm matrices contain exopolysaccharides, and dental biofilms are no exception; up to 40% of the dry weight of dental plaque is compos...

Comparison of biofilm formation and migration of Streptococcus mutans on tooth roots and titanium miniscrews

“…Streptococcus mutans cultures were grown anaerobically on a brain heart infusion (BHI: ThermoFisher SCIENTIFIC Ltd., Waltham, USA) agar plates at 37 °C (Koo et al, 2010). Immortalized human periodontal ligament cells (ihPDL cells) were grown on a 10‐cm culture dishes with high‐glucose Dulbecco's Modified Eagle Medium (ThermoFisher SCIENTIFIC Ltd., Waltham, USA) and supplemented with 1% penicillin/streptomycin (P/S) and 10% fetal bovine serum at 37 °C until the cells were confluent (Marchesan, Scanlon, Soehren, Matsuo, & Kapila, 2011).…”

“…Periodontal biofilm microbiota has been thoroughly investigated and characterized, and Streptococcus mutans ( S . mutans ) has been found to be an important microorganism during initial biofilm formation (Koo, Xiao, Klein, & Jeon, 2010). …”

“…It is the bacteria in the primary bacterial species associated with the early stage of bacterial biofilm formation (Koo et al, 2010). In addition, recent studies found that S .…”

“…S . mutans expresses glucosyltransferases (Gtfs), which translates sucrose molecules into glucan during biofilm formation (Koo et al, 2010). …”

“…As the biofilm matures, the proportion of S . mutans decreases and the microbiota shifts to late colonizer bacterial assemblages (Koo et al, 2010; Mattos‐Graner, Smith, King, & Mayer, 2000). …”