Nitrite Inhibition on Streptococcal Acid-production and Its Biochemical Mechanism

Objectives: The nitrate secreted into saliva is partly reduced to nitrite by oral bacteria. It was reported that nitrite inhibits the acid production from glucose by Streptococcus mutans (Sm) (Radcliffe et al., 2002) and human plaque (Yamamoto et al, 2012). However, the inhibitory mechanism is still unknown. Therefore, we attempted to elucidate it by metabolome analysis.
Methods: The cell suspensions of Sm NCTC 10449 and Streptococcus sanguinis (Ss) ATCC 10556 were mixed with 10 mM glucose in the presence or absence of 10 mM potassium nitrite and incubated for 10 min. Metabolic intermediates related to glycolysis, pentose phosphate pathway (PPP) and TCA cycle (TCAC) in the cell and lactate excreted as an end-product were analyzed metabolomically using the capillary electrophoresis and time-of-flight mass-spectrometer, before and after incubation.
Results: In the presence of nitrite, lactate production decreased significantly (0.31-fold in Sm and 0.80-fold in Ss). Among the glycolytic intermediates, glucose-6-phosphate increased (2.30-fold and 1.25-fold), while 3-phosphoglycerate (0.15-fold and 0.71-fold) and phosphoenolpyruvate (0.04-fold and 0.70-fold) decreased. Ribulose-5-phosphorate (3.15-fold and 1.71-fold) increased among the PPP intermediates, and acetyl-CoA decreased (0.01-fold and 0.31-fold) among the TCAC intermediates.
Conclusions: It was confirmed that nitrite inhibits streptococcal acid production. These metabolomic data suggest that nitrite suppresses glycolytic enzyme activities responsible for the conversion of glucose-6-phosphate to fructose-6-phosphate and for the conversion of glyceraldehyde-3-phosphate to 3-phosphoglycerate. Furthermore, it was suggested that the accumulated glucose-6-phosphate could flow into PPP, resulting in the increase of ribulose-5-phosphate. The decrease of acetyl-CoA could be due to the decrease of glycolytic flow which provides pyruvate, a precursor for acetyl-CoA production. Overall, the inhibitory mechanism of nitrite on Sm and Ss was similar, although Sm seems to be inhibited by nitrite more efficiently than Ss. Supported by JSPS (no. 23792498, 26293429, 26463154).