Evidence for Considerable Demineralization Potential of Pyruvate Formate-Lyase Generated Acids

Objective: Caries is thought to be caused by lactic acid produced by oral streptococci, in particular Streptococcus mutans, during periods of high supply with fermentable carbohydrates. However, when carbohydrates are limited in the oral cavity (many hours during the day and at night) S. mutans lactate dehydrogenase activity is down- and pyruvate formate-lyase (PFL) activity is up-regulated resulting in the predominant production of formic acid, acetic acid, and ethanol. Interestingly, little is known on demineralization resulting from the activity of PFL. We hypothesized that a PFL-deficient strain of S. mutans will result in diminished enamel demineralization. Methods: A PFL knockout mutant was constructed by standard procedures. Biofilms consisting of five supragingival species ("Zurich feeding model") and either the PFL-mutant or the wild-type strain of S. mutans were grown on bovine enamel disks (anaerobic, limited glucose/sucrose supply, 64.5 h, 37°C). Enamel demineralization was measured by quantitative light-induced fluorescence (QLF). In a complementary animal experiment the PFL-mutant and the wild-type strain were associated with rats. During 28 days the animals received a high sucrose diet for six hours every 2nd day while during the remaining time they were fed a glucose/sucrose-free, protein-rich diet. Maxillary and mandibular molars were evaluated for plaque extent and caries incidence. Results: In vitro biofilms with the wild-type strain led to high enamel demineralization (mean ΔF=-48%±3), whereas biofilms with the PFL-mutant showed significantly less mineral loss (ΔF=-25%±3). Rats associated with the PFL-mutant had significantly less initial and advanced fissure caries than with the wild-type (p<0.05), but there was no significant difference concerning plaque extent and smooth surface caries. Conclusions: These results suggest that acids produced by up-regulated PFL activity could play a role in the development of dental caries and prove a cariogenic potential of S. mutans even under conditions of limited carbohydrate supply.